Progress 10/01/02 to 09/30/07
Outputs
We are writing manuscripts on two related projects: 1) NADH channeling between dehydrogenases, and 2) characterizing a-toxin permeabilized pores in cells in situ (micro-permeabilization). Both are concerned with characterizing 'enzyme organization' (heterologous enzyme associations) in vivo. NADH channeling is the transfer of NADH directly from the donor enzyme, Ed, to an acceptor enzyme, Ea, without the dissociation of NADH from Ed.NADH into the bulk phase. We have determined that there is no reliable evidence of a physiological NADH channeling in the solution-phase. The apparent evidence for this channeling is due to artifacts, primarily from light scattering and side reactions. The light scattering results from small amounts of denatured enzyme from the extremely high [Ed]'s that are used in the common test for this channeling. However, NADH channeling is essentially guaranteed in the solid-state enzyme complexes that we have demonstrated among dehydrogenases. A
manuscript detailing all known forms of artifacts in this test is nearing completion. A manuscript describing the evidence of dehydrogenase hetero-enzyme solid-state complexes was published in 2006. A manuscript explaining the kinetics of the enigmatic situation - enzymes with protein substrates, but without detection of equilibrium association between enzyme and protein - is in preparation. In the second project, we have: developed a method for the quantitative assay of mammalian cell micro-permeabilization; measured the molecular sieving properties and size exclusion limits of alpha-toxin pores in red blood cells; discovered critical errors in methods commonly used to determine the osmotic pressure of polymer solutions employed to measure toxin pores; designed a new membrane osmometer; and demonstrated the limitations and advantages of various methods of assessing cell micro-permeabilization. Two articles are published and 5 are in preparation from this second project.
Impacts
Catalytic and metabolic properties of enzyme organization are very different from those of unassociated enzymes (the classical view). NADH channeling is probably the most controversial topic in biochemistry. The studies described above resolve this controversy. Micro-permeabilization of cells is essential to permit direct studies of individual and coupled enzymes with similar organization to that of cells in vivo.
Publications
- 1. Lehoux, E.A., Baker, S.M., Bush, J.A., and Spivey, H.O. 2004. An assay of mammalian cell micropermeabilization based on measurements of cellular lactate production. Anal. Biochem. 334:234-238. 2. Svedruzic, Z.M. and Spivey, H.O. 2006. Interaction between mammalian glyceraldehyde-3-phosphate dehydrogenase and L-lactate dehydrogenase from heart and muscle. Proteins: Structure, Function, and Bioinformatics 63:501-511.
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Progress 10/01/05 to 09/30/06
Outputs
We are writing manuscripts on two related projects: 1) NADH channeling between dehydrogenases, and 2) characterizing a-toxin permeabilized pores in cells in situ (micro-permeabilization). Both are concerned with characterizing 'enzyme organization' (heterologous enzyme associations) in vivo. NADH channeling is the transfer of NADH directly from the donor enzyme, Ed, to an acceptor enzyme, Ea, without the dissociation of NADH from Ed.NADH into the bulk phase. We have determined that there is no reliable evidence of a physiological NADH channeling in the solution-phase. The apparent evidence for this channeling is due to artifacts, primarily from light scattering and side reactions. The light scattering results from small amounts of denatured enzyme from the extremely high [Ed]'s that are used in the common test for this channeling. However, NADH channeling is essentially guaranteed in the solid-state enzyme complexes that we have demonstrated among dehydrogenases. A
manuscript detailing all known forms of artifacts in this test is nearing completion. A manuscript describing the evidence of dehydrogenase hetero-enzyme solid-state complexes was published in 2006. A manuscript explaining the kinetics of the enigmatic situation - enzymes with protein substrates, but without detection of equilibrium association between enzyme and protein - is in preparation. In the second project, we have: developed a method for the quantitative assay of mammalian cell micro-permeabilization; measured the molecular sieving properties and size exclusion limits of alpha-toxin pores in red blood cells; discovered critical errors in methods commonly used to determine the osmotic pressure of polymer solutions employed to measure toxin pores; designed a new membrane osmometer; and demonstrated the limitations and advantages of various methods of assessing cell micro-permeabilization. Two articles are published and 5 are in preparation from this second project.
Impacts
Catalytic and metabolic properties of enzyme organization are very different from those of unassociated enzymes (the classical view). NADH channeling is probably the most controversial topic in biochemistry. The studies described above resolve this controversy. Micro-permeabilization of cells is essential to permit direct studies of individual and coupled enzymes with similar organization to that of cells in vivo.
Publications
- Lehoux, E.A., Baker, S.M., Bush, J.A., and Spivey, H.O. 2004. An assay of mammalian cell micropermeabilization based on measurements of cellular lactate production. Anal. Biochem. 334:234-238.
- Svedruzic, Z.M. and Spivey, H.O. 2006. Interaction between mammalian glyceraldehyde-3-phosphate dehydrogenase and L-lactate dehydrogenase from heart and muscle. Proteins: Structure, Function, and Bioinformatics 63:501-511.
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Progress 10/01/04 to 09/30/05
Outputs
We continue to pursue two independent, but somewhat related projects: 1) in vitro studies of the kinetic and association properties of NADH dehydrogenase pairs; and 2) in situ studies of the membrane pores of alpha-toxin micro-permeabilized cells. The common goal is to characterize the potential enzyme organization in vivo. Organization here means complexes (metabolons) of one enzyme with one or more other enzymes (hetero-enzyme associations). Recent studies reveal this organization to be far greater than previously thought. Current data on enzyme organization are compelling, but incomplete. There is considerable controversy about the existence and kinetic properties of NADH dehydrogenase complexes. Our studies appear to answer most of these questions. For lactate and glyceralde-3-phosphate dehydrogenases, we find that highly specific, hetero-associations exist between mammalian forms of both enzymes, but only in media that are molecularly crowded (MC), as is the case
in vivo. MC means extremely high concentration of macromolecules. We also extensively studied the potential of substrate channeling (SC) of the substrate NADH between soluble enzymes. SC means that the substrate is passed directly from one enzyme (E1) to another (E2) in an E1-E2 complex. This mechanism prevents the release of the substrate from E1-substrate into the bulk media, which the classical mechanism requires. In contrast to previous reports and except for one non-physiological enzyme pair, we find no evidence that this occurs in normal media (media in vitro without MC conditions). Nevertheless, the complexes found in MC media, described above, provide an excellent opportunity for SC. We also have provided a theoretical enzyme mechanism that explains the enigmatic enzyme-protein reactions that occur without detectable enzyme-protein complexes. Three articles are in preparation describing these findings. In the second project, we have: developed a method for the quantitative
assay of mammalian cell micro-permeabilization; determined the molecular sieving properties and size exclusion limits of alpha-toxin pore-complexes; found artifacts in the determination of the osmotic pressure of polymer solutions; and demonstrated the limitations and advantages of various methods of assessing micro-permeabilization. Four articles are published and 3 in preparation from this project.
Impacts
Potential catalytic and metabolic properties of organized enzymes are much different than those of unassociated enzymes (Merz and Spivey, BioEssays,1989), a fact that is not well known. Micro-permeabilization of cells is virtually essential to permit direct studies of individual and coupled enzymes in situ. Current published methods and conclusions are inadequate or incorrect on this topic.
Publications
- Murugan, E., Sherman, Jr., R. L., Spivey, H. O. and Ford, W. T. (2004). Catalysis by hydrophobically modified poly(propylenimine) dendrimers having quaternary ammonium and tertiary amine functionality. Langmuir 20, 8307-8312.
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Progress 10/01/03 to 09/30/04
Outputs
We are pursuing two independent, but related projects: 1) studies in vitro of the kinetic and association properties of NADH dehydrogenase pairs; and 2) studies in situ of the membrane pores of alpha-toxin micro-permeabilized cells. The common goal is to characterize the potential enzyme organization in vivo. Organization here means complexes (metabolons) of one enzyme with one or more other enzymes (hetero-enzyme associations). Recent studies reveal this organization to be far greater than previously thought. Current data on enzyme organization are compelling, but incomplete. There is considerable controversy about the existence and kinetic properties of NADH dehydrogenase complexes. Our studies appear to answer most of these questions. For lactate and glyceralde-3-phosphate dehydrogenases, we find that highly specific, hetero-associations exist between mammalian forms of both enzymes, but only in media that are molecularly crowded (MC), as is the case in vivo. MC
means extremely high concentration of macromolecules. We also extensively studied the potential of substrate channeling (SC) of the substrate NADH between soluble enzymes. SC means that the substrate is passed directly from one enzyme (E1) to another (E2) in an E1-E2 complex. This mechanism prevents the release of the substrate from E1-substrate into the bulk media, which the classical mechanism requires. In contrast to previous reports and except for one non-physiological enzyme pair, we find no evidence that this occurs in normal media (media in vitro without MC conditions). Nevertheless, the complexes found in MC media, described above, provide an excellent opportunity for SC. We also have provided a theoretical enzyme mechanism that explains the enigmatic enzyme-protein reactions that occur without detectable enzyme-protein complexes. Three articles are in preparation describing these findings. In the second project, we have: developed a method for the quantitative assay of
mammalian cell micro-permeabilization; determined the molecular sieving properties and size exclusion limits of alpha-toxin pore-complexes; found artifacts in the determination of the osmotic pressure of polymer solutions; and demonstrated the limitations and advantages of various methods of assessing micro-permeabilization. Three articles are published and 4 in preparation from this project.
Impacts
Potential catalytic and metabolic properties of organized enzymes are much different than those of unassociated enzymes (Merz and Spivey, BioEssays,1989), a fact that is not well known. Micro-permeabilization of cells is virtually essential to permit direct studies of individual and coupled enzymes in situ. Current published methods and conclusions are inadequate or incorrect on this topic.
Publications
- Lehoux, E. A., Baker, S. M., Bush, J. A., and Spivey, H. O. (2004). An assay of mammalian cell micropermeabilization based on measurements of cellular lactate production, Anal. Biochem. 334, 234-238.
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Progress 10/01/02 to 09/30/03
Outputs
We are studying the kinetic and association properties of dehydrogenase enzymes. The major objective this year is to elucidate the mechanism for NADH transfer between donor and acceptor pairs of dehydrogenases. In the classical mechanism, the acceptor enzyme can utilize only free NADH, i.e., NADH that is not bound to the donor enzyme. However, a possible alternative is channeled NADH, whereby NADH is transferred directly from donor enzyme to the acceptor enzyme without release of NADH into the bulk medium. If NADH channeling does occur, important metabolic properties result as we have published. The criterion of channeling from the in vitro channeling test is R = experimental reaction velocity/velocity predicted assuming the classical mechanism only. We believe that the enzymes tested (glyceraldehye-3-phosphate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, ?-glycerol-3-phosphate dehydrogenase) do not channel NADH in vitro in contrast to previous
appearances. We believe the previous appearances resulted from a light-scattering artifact, interfering with the 340nm-signal in presence of high protein concentration. We avoid the light scattering artifact by use of dual wavelength measurements. The most likely results, when artifacts are avoided, are R values not significantly higher than 1 or 2, i.e., no channeling within probable experimental errors. A former graduate student disputes our findings. We have arranged for him to return to our lab where we will repeat the experiments in question this March, 2004. In either event, we find that several mammalian dehydrogenases associate with each other in molecular crowded media such as exist in vivo. Therefore, NADH channeling in vivo is still a realistic possibility.
Impacts
The question of NADH channeling in vitro has been intensely debated for many years. We think our results will resolve this controversy. We also demonstrate the association of certain mammalian dehydrogenases. This adds further evidence of specific enzyme associations in vivo with likely metabolic consequences. It should be possible to explore these consequences with molecular genetic methods.
Publications
- Lehoux, E. A., Baker, S. M., Kovina, M. K., Hays, F. A., Spivey, H. O. (2003). Absence of Evidence for Metabolite-Modulated Association between ?-Glycerol-3-phosphate Dehydrogenase and L-Lactate Dehydrogenase, Biochemistry 42, 6259-6263.
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Progress 10/01/01 to 09/30/02
Outputs
We are studying enzyme associations with each other and the catalytic consequences of these interactions. Of special interest is substrate channeling where the substrate is passed directly from one enzyme to another. In contrast, in the classical mechanism the substrate dissociates from the first enzyme and diffuses through the bulk phase before associating with the second enzyme. In extended studies of NADH channeling we have discovered two subtle artifacts that make this channeling very unlikely. We have also found ways to circumvent these artifacts in most cases. We are attempting to redetermine NADH channeling with solid-state enzyme complexes as expected in vivo for some enzymes. In other studies we are extending our studies of permeabilized cells in order to test for substrate channeling and enzyme activities in situ.
Impacts
Our research is essential in understanding the basis of coupled enzymatic reactions. Metabolism had been interpreted as the result of independent enzymatic reactions. However, we now realize that many reactions are coupled in a process of substrate channeling and that many enzymes exist in enzyme complexes with different properties from those of the isolated enzymes. Therefore, our understanding of metabolism depends critically on an understanding of these coupled and dependent processes.
Publications
- No publications reported this period
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Progress 10/01/00 to 09/30/01
Outputs
We are studying associations of enzymes with each other and other macromolecular structures, and the catalytic consequences of these interactions. Of special interest are studies of substrate channeling, which requires enzyme associations at least transiently during the catalytic cycle of the enzyme accepting the channeled substrate. Substrate channeling provides a micro-compartmentation of enzyme-bound metabolites within an organelle. This additional level of spatial compartmentation provides the cell with numerous significant metabolic advantages relative to the conventional view of a cell with randomly mixed enzymes and metabolites(Spivey, H. O. and Merz, J. M. (1989) Metabolic Compartmentation. BioEssays, 10, 127-130). However, evidence for substrate channeling and its consequences is difficult to obtain and is missing for many metabolic sequences. Thus the goals of one of our projects are to characterize enzyme properties and to test for substrate channeling in
situ using a-toxin permeabilized cells. a-Toxin is nearly unique in providing pores permeable to only small molecules (many, but not all metabolites) so that all the macromolecular structure of the cell is retained. Literature on a-toxin permeabilization of cells is lacking in important details and misleading or conflicting in other details, e.g., the size of resulting pores. We have: 1) developed an improved method to assess cell permeabilization; 2) have found the size exclusion limit (SEL) of a-toxin pores to be very similar in a number of tested cell lines and close to that found with artificial lipid bilayers; 3) characterized the rates of transport of small molecules across the a-toxin pores of red blood cells, and 4) found that the SEL of a-toxin pores is not - contrary to widely held views - dependent on toxin concentration, except at extremely low concentrations. Knowing the SEL of a-toxin is important for: 1) understanding the pathological effects of a-toxin on host cells,
2) understanding the mechanism of this model pore-forming toxin, 3) applications such as the delivery of drugs to target cells made selectively permeable with a-toxin, and 4) successful tests of substrate channeling. Four different publications are in preparation from these studies, which we expect to submit before the new year. The goals of another project are to develop improved methods for tests of NADH channeling in vitro and to assess NADH channeling in vivo. We have obtained improved analytical ultracentrifugation (AUC) data testing enzyme associations among these enzymes and developed much better computer programs for analyzing these data. We have also demonstrated a kinetic mechanism and model equations that are compatible with the experimental kinetic data and the lack of detectable enzyme associations in the absence of the catalytic reaction. We expect to submit three manuscripts on this project before the new year. We have made progress in experiments to test for NADH
channeling in vivo. As head of the State AUC facility, I have assisted three other research groups gather and analyze AUC data.
Impacts
We expect our research to contribute to a better understanding of the molecular mechanisms of metabolism and other intracellular processes. An understanding of molecular mechanisms provides especially fruitful ways to obtain advances in nutrition, pharmaceutics, diagnostic methods, and chemotherapies. The phenomena we research are fundamental to metabolism of all organisms.
Publications
- No publications reported this period
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Progress 10/01/99 to 09/30/00
Outputs
We are studying associations of enzymes with each other and other macromolecular structures, and the catalytic consequences of these interactions. Of special interest are studies of substrate channeling in vitro and in situ - a form of micro-compartmentation within a cell compartment. Thus, substrate channeling should: isolate intermediates from competing reactions, protect labile intermediates from destruction, and circumvent unfavorable equilibria and kinetics imposed by bulk phase metabolite concentrations, to name a few of its advantages relative to the conventional view of uniformly mixed enzymes and metabolites. One of our projects attempts to utilize a-toxin permeabilized cells to permit isotope dilution tests of substrate channeling in the glycolytic pathway. This toxin is essentially unique in providing pores permeable to only small molecules (many, but unfortunately not all metabolites) so that all of the macromolecular structure of the cell is retained. We
have developed a new method with several advantages for the assessment of such cell micro-permeabilization. This will be submitted for publication soon. We have re-assessed the size-exclusion limit of a-toxin pores in cells. Our results bring a resolution to the current conflicting estimates and demonstrates that the size-exclusion limit has often been overestimated due to various causes including partial cell lysis. These results will soon be published. We have uncovered evidence of an association between a-glycerophosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. This association could have potentially important physiological consequences. Another project studies the NADH channeling between dehydrogenases (DeHs) in vitro. We have made gathered additional analytical ultracentrifuge (AUC) data on this system and have invested considerable time to improve computer programs that analyze data on these complex systems, which exhibit self-association and must be tested
for hetero-associations as well. We have gathered AUC data for three different research groups and analyzed them together with extensive data from a fourth research group. The P.I. wrote an NSF grant application that was awarded for three years, supervised completion of two Ph.D. theses, and has worked extensively on publications that we plan to submit soon. He has also supervised an undergraduate research student.
Impacts
We expect our research to contribute to a better understanding of the molecular mechanisms of metabolism and other intracellular processes. An understanding of molecular mechanisms provides especially fruitful ways to obtain advances in nutrition, pharmaceutics, diagnostic methods, and chemotherapies. The phenomena we research are fundamental to metabolism of all organisms.
Publications
- No publications reported this period
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Progress 10/01/98 to 09/30/99
Outputs
We are studying associations of enzymes with each other and other macromolecular structures, and the catalytic consequences of these interactions. Of special interest are studies of substrate channeling in vitro and in situ - a form of micro-compartmentation within a cell compartment. Thus, substrate channeling should: isolate intermediates from competing reactions, protect labile intermediates from destruction, and circumvent unfavorable equilibria and kinetics imposed by bulk phase metabolite concentrations, to name a few of its advantages relative to the conventional view of uniformly mixed enzymes and metabolites. One of our projects attempts to utilize a-toxin permeabilized cells to permit isotope dilution tests of substrate channeling in the glycolytic pathway. This toxin is essentially unique in providing pores permeable to only small molecules (many, but unfortunately not all metabolites) so that all of the macromolecular structure of the cell is retained. We
have developed a new method with several advantages for the assessment of such cell micro-permeabilization. This will be submitted for publication very soon. We have started a collaborative project with the aim of demonstrating that alpha-toxin can permeabilize nucleated cells to larger molecules. This results from the formation of lesions in the plasma membrane rather than the toxin pores themselves, which are too small for several substrates of interest. We also think that we have found two permeabilized cell systems which we can use in our upcoming substrate channeling experiments. Another project studies the NADH channeling between dehydrogenases (DeHs) in vitro. We have completed analytical ultracentrifuge measurements on one such DeH pair which prove that the enzymes do not associate at equilibrium, although they associate maximally during the catalytic reaction. A kinetic explanation of this has been lacking - it had been claimed to be impossible. However, we have shown how a
more realistic kinetic model than previously considered can entirely explain this behavior, which is common to several other enzyme systems. We have made plans that may directly demonstrate the enzyme associations by spectroscopic methods. Progress on molecular modeling of the enzyme interactions involved in NADH channeling have also been made. As supervisor of our State's analytical ultracentrifuge (AUC) facility, I invested several hundred hours in developing badly needed computer programs for data analyses. These will be made available to similar facilities throughout the world. We have made additional AUC measurements and analyses for one continuing research group and two new ones and have nearly completed an extensive web page for users. I also completed an extensive review article of substrate channeling methods and obtained NSF funds for support of undergraduate student research.
Impacts
We expect our research to contribute to a better understanding of the molecular mechanisms of metabolism and other intracellular processes. An understanding of molecular mechanisms provides especially fruitful ways to obtain advances in nutrition, pharmaceutics, diagnostic methods, and chemotherapies. The phenomena we research are fundamental to metabolism of all organisms.
Publications
- Spivey, H. O. and Ovadi, J. (1999) Substrate Channeling. Methods, Companion to Meth. Enzymol. 19, 306-321.
- Vonnahme, K. A., Malayer, J. R., Spivey, H. O., Ford, S. P., Clutter, A., and Geisert, R. D. (1999) Detection of Kallikrein Gene Expression and Enzymatic Activity in Porcine Endometrium During the Estrous Cycle and Early Pregnancy. Biol. Reprod., 61, 1235-1241.
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Progress 10/01/97 to 09/30/98
Outputs
We are studying associations of enzymes with each other and other macromolecular structures, and the catalytic consequences of these interactions. Of special interest are studies of substrate channeling in vitro and in situ - a process that should provide numerous metabolic advantages relative to the widely accepted view that these enzymes are randomly distributed in their cellular compartments. One study attempts to utilize alpha-toxin permeabilized cells to permit isotope dilution tests of substrate channeling in the glycolytic pathway. This toxin provides pores with special advantages relative to other methods of cell alpha-toxin permeabilization. In contrast to published reports, we find that permeabilization does not produce large enough pores to permit the rapid entry of glycolytic intermediates or ATP. We have established collaborations with leading groups throughout the world that are also using and experiencing this problem. By sharing different origins and
preparations of this toxin and different cell types, we have made progress in understanding this problem, and expect to have the problem understood within the next year. In another project, we have completed a thorough study of the reported NADH modulated association of glycerol-phosphate and lactate dehydrogenases, which is a potentially very significant phenomenon. Using improved methods and instrument, we demonstrate that these enzymes do not associate at any level of NADH, in contrast to the previous report. The artifact present in their study is clearly identified. The results have been submitted for publication. These results support our hypothesis that these enzyme associations, detected indirectly by kinetic data, are present only during the catalytic reaction. I supervise a modern analytical ultracentrifuge (AUC) facility for the State. Extensive AUC studies have been completed for both our research and that of others including studies of: possible association of
dehydrogenases capable of NADH channeling; association-dissociation of biliproteins (native and mutants), and oligomeric state of pyruvate dehydrogenase kinase (PDK) isozymes and effects of the lipoyl domain on their association. I have also written computer programs that improve our ability to analyze AUC data. We expect to submit several publications on projects completed this period soon. We have also assisted colleagues in the Animal Science Department with fluorescence assays of enzyme activities important in their research on reproductive physiology. A coauthored manuscript has been submitted about one month ago.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 10/01/96 to 09/30/97
Outputs
We are studying associations of enzymes with: other enzymes and macromolecular structures, and the catalytic consequences of these. Of special interest is substrate channeling, which should provide numerous metabolic advantages relative to a random mixture of enzymes. In one project, channeling in the glycolytic pathway at the phosphofructokinase and pyruvate kinase reactions is measured in a-toxin permeabilized PC12 cells using the isotopic dilution method. In this project, we: 1) finished developing a simple, specific and precise method for determining radiospecific activity of (14C)lactate in complex biological samples; 2) demonstrated the stability & integrity of permeabilized PC12 cells; 3) identified optimal glycolysing conditions for these cells; 4) discovered that glycolysis by these cells has an absolute requirement for Mg2+, which can be exploited to quantitate the permeability of cells more completely than previous methods, and 5) began cellular volume of
distribution measurements for selected molecules - measurements required for subsequent experiments. For studies on NADH channeling in vitro, we: 1) developed model equations describing the kinetic behavior of the system more completely and providing improved criteria for detecting NADH channeling; 2) purified several isozymes; 3) tested the model equation, and 4) characterized associations between several of the enzymes capable of NADH channeling.
Impacts
(N/A)
Publications
- LEHOUX, E. A., SVEDRUZIC, Z., AND SPIVEY, H. O. (1997) Determination of the Specific Radioactivity of [14C]Lactate by Enzymatic Decarboxylation and 14CO2 Collection. Anal. Biochem., vol. 253,
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Progress 10/01/95 to 09/30/96
Outputs
We are studying associations between enzymes or between enzymes and the cytoskeletal network, and the catalytic consequences of these. Of special interest is substrate channeling, which should provide numerous metabolic advantages relative to a random mixture of enzymes. This year we: 1) Characterized the purity and cytotoxicity properties of our purified alpha-toxin. 2) Found optimum conditions for permeabilizing our cells with alpha-toxin. 3) Developed an enzymatic method for measuring specific radioactivity of 14C-lactate in biological extracts containing other radiolabelled compounds. This will greatly facilitate our subsequent studies. 4) Using capilary electrophoresis and agarose gel electrophoresis, we extensively tested for associations between dehydrogenases that might correlate with NADH channeling. 5) Using primarily PEG coprecipitation and analytical ultracentrifugation, discovered and extensively characterized the association between lactate and
glyceraldehyde-3-P dehydrogenases. 6) Obtained a multi-user instrument-and a personal national grant. 7) Attended a national training course in analytical ultracentrifugation. 8) Discovered principles explaining how substrate channeling could occur without association of enzymes at equilibrium. 9) Presented these and related ideas at an international meeting. 10) Helped two colleagues, one in Animal Science and one in Chemistry complete their respective projects.
Impacts
(N/A)
Publications
- Ford, W.T., El-Hamshary, H., Stefanithis, I., Spivey, H.W., Hassanein, M., and Selim, A. 1996. Autoxidation of 2-Mercaptoethanol Catalyzed by Cobalt(II) Phthalocyaninetetrasulfonate Bound to Cationic Latexes. New Journal of Chemistry, 20: 5.
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Progress 10/01/94 to 09/30/95
Outputs
We are studying associations between enzymes or between enzymes and the cytoskeletal network, and the catalytic consequences of these. Of special interest is substrate channeling, which should provide numerous metabolic advantages relative to a random mixture of enzymes. We made substantial progress in four areas this year. 1) We overcame the problems in permeabilizing PC12 cells for small molecules, and also purified enough alpha-toxin for our experiments. This toxin is not available commercially with adequate purity and cost. 2) We have found apparent differences in the NADH channeling abilities of bacterial and mammalian malate dehydrogenases. Extensive, experimental and molecular graphic studies have been made to test for differences in association between dehydrogenase enzymes or differences in their structure that would explain their differences in channeling ability. An improved understanding of this channeling has also been achieved by extending the
theoretical analysis of this process. Two publications are nearing completion on these studies. 3) I completed a multi-user instrument grant application for an analytical ultracentrifuge that was highly rated by NSF though its funding is uncertain at this time. This involved detailed, state-of-the art research plans including those for studying complex polysaccharides. Methods for the latter are especially challenging and only partially developed. 4) I completed one stage of a cooperative study leading to the publication and abstract listed.
Impacts
(N/A)
Publications
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Progress 10/01/93 to 09/30/94
Outputs
We are studying the potential interactions between pairs of enzymes or between enzymes and the cytoskeletal network, and the catalytic consequences of these interactions. Of special interest to us is substrate channeling, which should provide numerous metabolic advantages relative to a random mixture of enzymes and their metabolites. Since our last report, we have completed several projects as indicated by the three listed publications. The study of malate dehydrogenase in reverse micelles failed to show effects of phospholipids on this enzyme, so this project will not be pursued further. Considerable progress has been made on two new projects. In the first of these we have demonstrated NADH channeling between (alpha)-glycerophosphate and lactate dehydrogenases with the rabbit muscle forms of each enzyme and revealed the problems with previous studies that disputed such channeling. We have also characterized strong effects of buffer ions on this channeling. In the
second study, we have attempted to permeabilize PC12 cells to large macromolecules and demonstrate channeling in the glycolytic pathway in situ. Although we can get good permeabilization of other cultured cells, technical difficulties still persist with the PC12 cells we wish to study. We expect alternative strategies will work soon.
Impacts
(N/A)
Publications
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Progress 10/01/92 to 09/30/93
Outputs
Our published studies have demonstrated highly efficient channeling of NADH in vitro from substrate level dehydrogenases to complex I of the electron transport chanin. Such channeling provides numerous potential advantages for metabolism in this pathway where the majority of the energy of nutrients is captured. Since the last report, we have developed an alternative test for this substrate channeling (J. Molec. Recog., in press). Its major advantage is that it requires less than 1000-fold smaller quantities of the first enzyme in the reaction than the previous test. This should allow us to extend these measurements to mutant forms of the enzyme to clarify structure/function relationships. A collaborative arrangement to obtain these mutants has been established. In another project, we have developed a generally applicable, sensitive and simple measurement of weak enzyme interactions in vitro. Such interactions are important, because higher enzyme concentrations and
excluded volume effects in vivo enhance these associations. A 1:1 molar complex of malate dehydrogenase and citrate synthase was confirmed by this method. Studies of malate dehydrogenase and citrate synthase in reverse micelles have been attempted to characterize their interactions with phospholipids and with each other. With the recent arrival of two graduate students, projects to measure the binding of glycolytic enzymes to the cytoskeletal network have begun.
Impacts
(N/A)
Publications
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Progress 10/01/91 to 09/30/92
Outputs
Our published studies have demonstrated highly efficient channeling of NADH in vitro from substrate level dehydrogenases to Complex I of the electron transport chain. Such channeling provides numerous potential advantages for metabolism in this pathway, where the majority of the energy of nutrients is captured. Since the last report, we have tried to identify the subunit of Complex I to which malate dehydrogenase (MDH) docks and initiated studies to determine the domain of MDH that is involved in this interaction. The first goal was pursued using anti-idiotopic antibodies (AIA) to MDH. Complex I was resolved on SDS-PAGE and the protein subunits transferred to membranes. The resolved subunits were exposed sequentially with AIA and enzyme coupled secondary antibody to generate stained bands of the AIA bound subunits. A single subunit of 30kDa was indicated. Proof that this is the 30kDa subunit of Complex I awaits amino acid sequencing of terminal segments of this
subunit and competitive binding assays. This project and others have been delayed due to lack of funds. The second goal was briefly initiated using limited tryptic digests of MDH and testing for their ability to bind to MDH or displace native MDH from Complex I. Manuscripts describing previous work on the Complex I system and general considerations of substrate channeling are in preparation.
Impacts
(N/A)
Publications
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Progress 10/01/90 to 09/30/91
Outputs
Recent evidence demonstrates that many of the intermediates of metabolism are passed directly from one enzyme to the next one without dissociating from the enzymes. Significantly different metabolic behavior could be achieved by this "substrate channeling" process than expected without it. The major pathways for capturing the energy of nutrients in aerobic organisms are mitochondrial oxidations and electron transport. We are studying several of these enzymes to: test for substrate channeling and reveal the molecular mechanisms and metabolic consequences. This past year, we have: 1) found conditions discriminating specific from nonspecific binding of enzymes to Complex I in submitochondrial particles, 2) characterized NADH channeling to beef heart Complex I by the competing reaction method, 3) characterized the effects of other mitochondrial enzymes on the binding of malate dehydrogenase (MDH) to Complex I, and 4) demonstrated that substrate channeling is the only
mechanism consistent with the enzyme buffering test of channeling. The first goal has been critical to the success of the second two. Although we previously demonstrate.
Impacts
(N/A)
Publications
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Progress 10/01/89 to 09/30/90
Outputs
Enzymes are the catalysts which are required for virtually all metabolic reactions. Recent evidence demonstrates that many of the intermediates of metabolism are passed directly from one enzyme to the next one without dissociating from the enzymes. Significantly different metabolic behavior could be achieved by his "substrate channeling" process than expected without it. A related finding is that many enzymes are physically associated with the next enzyme in the metabolic path. The major pathways for capturing the energy of nutrients in aerobic organisms are mitochondrial oxidations and electron transport. We are studying several of these enzymes to test for substrate channeling, reveal the molecular mechanisms and metabolic consequences, and to characterize the enzyme associations. During the past year, we characterized the binding and NADH channeling of several enzymes to Complex I within submitochondrial particles (SMP). SMP provide a more native form of
Complex I than the highly purified Complex I previously used. Attempts to adapt an alternative test of substrate channeling to Complex I have been made, but have not succeeded yet. Such a method would permit measurements on some enzymes not possible with the previous method and would also require far smaller quantities of donor enzymes. The latter would facilitate measurements planned with mutant forms of enzymes.
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Progress 10/01/88 to 09/30/89
Outputs
Enzymes are the catalysts which are required for virtually all metabolic reactions. Recent evidence demonstrates that many of the intermediates of metabolism are passed directly from one enzyme to the next one without dissociating from the enzymes. Significantly different metabolic behavior could be achieved by this "substrate channeling" process than expected without it. A related, finding is that many enzymes are physically associated with the next enzyme in the metabolic path. The major pathways for capturing the energy of nutrients in aerobic organisms are mitochondrial oxidations and electron transport. We are studying several of these enzymes to: test for substrate channeling, reveal the molecular mechanismas and metabolic consequences, and to characterize the enzyme associations. During the past year,we have: 1) extended our substrate channeling experiments in vitro, characterizing the NADH channeling to complex I from the following dehydrogenases (DeH) -
mitochondrial and cytoplasmic malate DeH, beta-hydroxyacylCoA DeH, and glutamate DeH. The mechanism and kinetic constants of this channeling were also determined. 2) Thoroughly reinvestigated and confirmed the NADH channeling from lactate DeH to alpha-glycerol-phosphate DeH, which had been disputed by another research group.
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Progress 10/01/87 to 09/30/88
Outputs
Enzymes are the catalysts which are required for virtually all metabolic rections. They are also crucial in regulation; i.e., modifying rates of metabolism in response to changing external and internal conditions. Recent evidence demonstrates that many of the intermediates of metabolism are passed directly from one enzyme to the next one without dissociating from the enzymes. Significantly different metabolic behavior could be achieved by this "substrate channeling" process than expected without it. A related, recent finding is that many enzymes are physically associated with the next enzyme in the metabolic path. The major pathways for capturing the energy of nutrients in aerobic organisms are mitochondrial oxidations and electron transport. We are studying several of these enzymes to: test for substrate channeling, reveal the molecular mechanisms and metabolic consequences, and to characterize the enzyme associations. During the past year, we have: improved
our purification of complex I; demonstrated substrate channeling of NADH from malate dehydrogenase to complex I (the first component of the electron transport pathway); shown that the NADH donor enzymes tested compete for the same receptor site on complex I; and obtained evidence identifying the receptor subunits on complex I. We have also enumerated the potential advantages of substrate channeling to metabolism in a publication.
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Progress 10/01/86 to 09/30/87
Outputs
Enzymes are the catalysts for metabolic reactions. Since many of these enzymes are soluble and unassociated when isolated, it was presumed that these enzymes are randomly distributed in the cell. Recent evidence, however, makes it more likely that many of these "soluble" enzymes exist in association with one another. Significantly different metabolic properties would be expected for such an arrangement of enzymes compared to a random arrangement. We are studying the association of several enzymes to the heart mitochondrial electron transport system components, complex I and II. Complex I has about 25 dissimilar subunits. Using chemical crosslinking and chromatographic methods, we are trying to identify the subunit(s) to which malate dehydrogenase binds. Preliminary results with a widely used crosslinker (DSP) indicated two subunits. Subsequently, a crosslinker (SASD) with improved properties (specificity and ability to isotopically label) became available, which
we have been trying to use. Its use is considerably more complex and results are not yet available. Repairs have been made to our stopped-flow instrument that should permit us to test for substrate channeling of NADH between malate dehydrogenase and complex I. Progress has been made in finding conditions to permit testing whether fumarase or succinate thiokinase binds to complex II.
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Progress 09/01/85 to 10/30/86
Outputs
Enzymes are the catalysts for metabolic reactions. Since many of these enzymes are soluble and unassociated when isolated, it was presumed that these enzymes are randomly distributed in the cell. Recent evidence, however, makes it more likely that many of these "soluble" enzymes exist in association with one another and in the solid-state in cell. Significantly different metabolic properties would be expected for such an arrangement and state of enzymes compared with the situation previously assumed. We are studying the possible association of everal enzymes to the pig heart mitochondrial electron transport system components, complex I and II. Binding of enzymes catalyzing reactions that are sequential to those of complex I and II is most likely. Based on promising recommendations, extensive attempts were made this year to improve the purification procedure for complex I, but without success. Enzyme binding and kinetic measurements were, therefore, reiniated with
available complex I, including that sent from another laboratory. In related studies we have extended our data on the specificity and nature of the association between malate dehydrogenase and citrate synthase enzymes, and data on polyethylene glycol induced protein associations.
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Progress 01/01/85 to 09/30/85
Outputs
Enzymes are the catalysts for metabolic reactions. Since many of these enzymes are soluble and unassociated when isolated, it was presumed that these enzymes are randomly distributed in the cell. Recent evidence, however, makes it more likely that many of these "soluble" enzymes exist in association with one another and in the solid-state in the cell. Significantly, different metabolic properties would be expected for such an arrangement and state of enzymes compared with the situation previously assumed. We are studying the possible association of severa enzymes to the pig heart mitochondrial electron transport system components, complex I and II is most likely. Most of the year was spent in trying to purify complex I from pig heart. A suitable purification was finally achieved. Preliminary results suggest that malate dehydrogenase binding is not specific to this complex, in contrast to an earlier report. Testing for binding to complex II is more difficult
than for complex I due to the physical state of complex II. We developed a method (incorporation of complex II into phospholipid vesicles) that permits binding measurements. The binding of fumarase and succinyl thiokinase to purified complex II appears to be weak. Conditions or factors that might increase this binding will be tested during this year.
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Progress 01/01/84 to 12/30/84
Outputs
Enzymes are the catalysts for metabolic reactions. In most cases, enzymes existwithin the cell at much higher concentration than can be used for studies in vitro of their catalytic properties with usual methods. Since many of these enzymes are soluble and unassociated when isolated, it was presumed that these enzymes are randomly distributed in the cell. Recent evidence, however, makes it more likely that many of these "soluble" enzymes exist in association with one another an in the solid-state in the cell. No kinetic properties of the complex between the important enzymes, malate dehydrogenase (MDH) and citrate synthase (CS) of the Krebs cycle, had been measured, however. Using polyethylene glycol (PEG) to generate a solid-state complex between these enzymes, we have demonstrated that the oxaloacetate (OAA) produced by MDH is utilized by CS without significant diffusion away from the solid-state compelx. This finding alters to some extent our understanding of
the way this and similar metabolic pathways are regulated. In another study, we tested the generality of the effect of PEG in causing protein associations in solution. We found this effect with all seven proteins tested, covering the molecular weight range 14,000 to 670,000, though the size of the associated species are enormous, only a very samll percent of the total enzyme associates. These findings contribute to our understanding of interactions among macromolecules in concentrated solutions as exist in vivo.
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Progress 01/01/83 to 12/30/83
Outputs
Current evidence makes it more likely that the enzymes in mitochondrial matrix exist in associated complexes with each other and in the solid-state rather than exist independently of each other in the solution phase. This is because of the very high concentration of macromolecular solutes in vivo. Polyehylene glycol (PEG) induces some of the enzyme associations in vitro, which are highly likely to exist in vivo, and is useful in producing suspensions of solid phase enzymes suitable for kinetic studies. Thus PEG solutions permit one to characterize enzymatic properties under conditions that are more physiological with regard to their molecular state than previously used. Our progress in this research area during the year is as follows. 1) PEG and other macromolecules enhanced the association of glutamate dehydrogenase (GDH), increased its inhibition by GTP, and decreased the solubility of GDH. An excluded volume mechanism, which should be very significant in vivo,
appears to be largely, but not completely responsible for these effects. 2) Kinetic measurements have been made for the firt time on solid-state complexes of malate dehydrogenase and citrate synthase. The results support the view that metabolism in such complexes, which are expected to exist in vivo, is many times more efficient than with a homogeneous solution of the same quantity of enzymes.
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Progress 01/01/82 to 12/30/82
Outputs
Current evidence makes it more likely that the enzymes in mitochondrial matrix exist in associated complexes with each other and in the solid-state rather than exist independently of each other in the solution phase. Polyethylene glycol (PEG) induces some of the enzyme associations In Vitro, which are highly likely to exist In Vivo, and is useul in producing suspensions of solid phase enzymes suitable for kinetic studies. Thus PEG solutions permit one to characterize enzymatic properties under conditions that are more physiological with regard to their molecular state than previously used. Our progress in this research area during the year is as follows. Due to large pH errors in commercial pH electrodes at low ionic strength, most of the measurements characterizing the effects of solution conditions on the citrate synthase (CS)-malate dehydrogenase (MDH) enzyme complexes in PEG solutions have been repeated. The PEG effects on the association of glutamate
dehydrogenase have been more completely analyzed. Methods permitting measurements on suspensions of solid enzyme crystals have been developed. Large changes in the specific activity of crystals of MDH relative to solution phase enzyme were found. Dynamic light scattering measurements on several protein solutions reveal that PEG induces a special type of protein association for each of them.
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Progress 01/01/81 to 12/30/81
Outputs
Cellular compartments, especially mitochondrial matrix, contain very high concentrations of macromolecules. Polyethylene glycol (PEG) appears to provide many of the general effects of polymers (e.g. excluded volume) without the experimental complications of physiological polymers. We have, therefore, tried to characterize specific enzyme associations induced by PEG. These enzyme associations may cause significant changes in enzymatic properties relative to those measured with conventional conditions in vitro. Dynamic light scattering (DLS) and electron microscopic (EM) measurements permitted us to calculate the distribution of molecular sizes in the PEG-malate dehydrogenase (MDH)-citrate synthase (CS) system by use of the newer method of analyzing DLS data referred to in the last report. Extremely large enzyme complexes were found, but they represent less than 0.03% of the total enzyme mass in solution. The majority of enzyme mass in the precipitate, however,
appears to be MDS-CS complexes. We have measured the solubilities of the MDH-CS complex as a function of ionic composition, and to a lesser extent pH, temperature and PEG concentration. The results are needed to distinguish the enzyme complexes in the solution phase from those in the precipitate, and homogeneous from heterogeneous complexes. Increasing evidence suggests that mitochondrial matrix enzymes exist in a solid suspension rather than a homogeneous solution in vivo.
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Progress 01/01/80 to 12/30/80
Outputs
Cellular compartments, especially mitochondrial matrix, contain very high concentrations of macromolecules. Polyethylene glycol (PEG) appears to provide many of the general effects of polymers (e.g. excluded volume) without the experimental complications of physiological polymers. We have, therefore, tried to characterize specific enzyme associations induced by PEG. These enzyme associations may cause significant changes in enzymatic properties relative to those measured with conventional conditions in vitro. Dynamic light scattering measurements could in principle provide many advantages for the characterizations desired. Calculations with simulated data containing random errors revealed that published methods for analyzing such data are inadequate for systems with moderately broad molecular weight (MW) distributions. We have now implemented a far better method reported at a 1980 international meeting, which appears to be adequate for the broad MW distributions
that we have. The effect of solution conditions (pH, ionic strength, etc.) on enzyme association were studied. Much greater enzyme interaction occurs at pH 6.5 than 7.5, but the majority of enzyme in solution still remains unassociated and the associated enzyme is primarily homologous at pH 6.5. The heterologous enzyme complexes are much less soluble than the other forms, however. Thus the majority of enzyme precipitates as heterologous complexes at polymer concentrations far below those in mitochondrial matrix.
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Progress 01/01/79 to 12/30/79
Outputs
Recently evidence has been gathered from several studies, which strongly suggests that soluble cellular polymers can cause several enzymes to form specific enzyme-enzyme complexes. This complex formation may greatly change the enzymes' catalytic properties. similar effects have been found with polyethylene glycol (PEG) or dextron when both have been used. Only PEG effects were reported for the polymer induced association of malate dehydrogenase with citrate synthese (MDH-CS), the enzymes we have been studying. Thus we have confined most of our polymer studies to PEG effects on these two enzymes. Dynamic and static light scattering, ultracentrifugation, affinity chromatography, and chemical cross-linking experiments have been made. PEG with either enzyme alone or their mixture causes both enzymes to partially aggregate into large complexes. Increasing the mol. wt. of PEG from 6500 to 20,000 causes a nearly 20-fold increase in the amount of enzyme in the complexed
form. Palmitoyl-CoA (20 MuM) increases the percentage of total enzyme in the associated form and the size of the complexes dramatically. Enzyme interactions without palmitoyl-CoA are primarily homologous (MDH-MDH and CS-CS) except at high PEG concentrations. Palmitoyl-CoA appears to enhance heterologous associations. Further studies of the effects of PEG, dextran and other cellular polymers on enzyme associations and activities are planned in order to better estimate the activities of enzymes in vivo.
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Progress 01/01/78 to 12/30/78
Outputs
We have pursued three projects with the enzyme porcine mitochondrial malate kehydrogenase (MDH). First, we studied the reported "anomalous kinetics" of the coupled enzyme reactions of aspartate aminotransferase and MDH reported by Bryce et al. (1976) Biochem. J. 153, 571. If valid, this anomaly would revise our estimates of the catalytic activity of several of the key enzymes in the Kreg's cycle by 8-fold or more. We found no anomaly by the first of two experimental methods they used. We found a source of experimental error in the second method, which most likely explains their results. We also uncovered an error in their theoretical predictions, which makes their conclusions invalid. Secondly, we have demonstrated with photon correlation measurements, that polymers similar to those in vivo can induce extensive complex formation between the enzymes MDH and citrate synthase. Finally having previously shown that several of the kinetic constants of MDH were
grossly different from earlier published data, we have extended these measurements to other constants and physiological conditions. The latter two projects are still in progress, while the first project will be completed upon preparation of a manuscript for publication.
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Progress 01/01/77 to 12/30/77
Outputs
1) To elucidate the mechanism of liver glucose-6-P dehydrogenase (G6PD) inhibition by palmitoyl-CoA, we attached a fluorescent label (fluoroscein) to G6PD, which did not alter its catalytic activity. This label on G6PD was found inadequate for polarization measurements to monitor induced subunit dissociations (in contrast to its success on MDH, demonstrated by others), but may still be useful for monitoring conformational changes. We plan to use other fluorescent labels or light scattering measurements to correlate subunit dissociations with enzyme activity. A potential value of the G6PD study is to clarify what factors regulate the rate of NADPH synthesis, which is required for lipogenesis. 2) Oxaloacetate (OAA) is considered a pace-setting substrate, since its low concentration in vivo is thought to greatly affect rates of aerobic metabolism, gluconeogenesis, and lipogenesis. Our studies seek to identify and quantitate the factors regulating the concentration of
OAA. Recent studies (ours and others) have indicated previously ignored factors, which greatly alter the activity of OAA producing enzymes. We have, therefore, tried to extend our measurements to OAA reduction by malate dehydrogenase and the effects of coupled enzymes. Commercially available enzyme of the last several months, however, was found aberrant, and we've started to purify our own enzyme.
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Progress 01/01/76 to 12/30/76
Outputs
1) Stopped-flow measurements have revealed that patmitoyl-CoA inhibition of rat liver glucose-6-phosphate dehydrogenase depends on enzyme concentration. Sedimentation velocity data indicate that this results from dissociation of enzyme by palmitoyl-CoA; an equilibrium between dissociated and native enzyme isestablished when the enzyme is partially inhibited. 2) Measurements have been completed demonstrating that tetraalkylammonium cations do not bind to ATP. Measurements are also in progress with MgyRG specific potential electrodes and MgATP at physiological ionic strengths. If they agree with MgATP stability constants determined by the pH titration method, as refined in our lab, the discrepancies between the results of Rechnitz et al. (1970) J. Amer. Chem. Soc.92, 5839) and older literature must be ascribed to differences in ionic strengths, contrary to their claims. 3) Our Durrum stopped-flow instrument has been interfaced to a PDP 11/40 computer with a RT-11
support system. 4) Studies on rabbit muscle glyceraldehyde 3-phosphate dehydrogenase demonstrate that the enzymatic mechanisms proposed by Orsi and Cleland for pH 8.6 (1972) Biochemistry 11, 102) or Trentham (1971) Biochem. J. 122, 71) are not adequate at phsyiological pH values.
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Progress 01/01/75 to 12/30/75
Outputs
1) Steady-state kinetic studies of the mechanism of rat liver glucose 6-phosphate dehydrogenase have been completed revealing kinetic constants and steady state patterns. 2) Further studies on pig heart mitochondrial malate dehydrogenase have been completed to evaluate the extent of its inhibition by ATP with cellular concentrations of enzyme, metabolites, and buffer components, & to determine reasons for the difference observed between the dilute & concentrated enzyme reported last year. 3) The effects of cellular concentrations of malate dehydrogenase (MDH) and citrate synthase on the + CoA +NADH) have been measured. They indicate substantial shifts in the equilibria due to binding principally of MDH reaction products to MDH. 4) Measurements of metal (Mg(2+) and Na(+)) - ATP stability constants are being pursued to test a recent challenge to the validity of the previous literature data and to some fundamental principles involved in evaluating stability constants
with cellular concentrations of competing ions and ionic strength.
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Progress 01/01/74 to 12/30/74
Outputs
In contrast to recent claims concerning the function of lactate dehydrogenase (LDH) isoenzymes, rapid kinetic (stopped-flow) measurements revealed that the pyruvate dead-end complex of heart LDH inhibits lactate oxidation as strongly aspyruvate reduction, and that lactate did not alter the dissociation of the dead-end complex. The proposed advantages of muscle isoenzyme in anaerobic muscles were substantiated, however. Rapid kinetic fluorescence measurements demonstrate three conformational transitions of pyruvate carboxylase in responseto binding ATP (substrate) and acetyl-CoA (activator). Circular dichroic spectra of the enzyme indicate 70% helix, and 0% (beta) pleated sheet secondary structures. Small pertubatrons of the dichroism of side chains is revealed whenATP and acetyl-CoA bind to the enzyme. Stopped-flow measurements on mitochondrial malate dehydrogenzse demonstrate significantly different properties of concentrated enzyme from those obtained with
dilute enzyme by conventional methods. Stopped-flow measurements of the fast CayRG uptake by sarcoplasmic reticulum from pig muscle reveals no differences between normal animals and those susceptible to malignant hyperthermia, although disruption of this uptake was considered a likely primary or secondary cause of the fatal syndrome in animals and
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Progress 01/01/73 to 12/30/73
Outputs
Rapid kinetic (stopped-flow) measurements completed on three regulatory enzymes to characterize their properties at cellular enzyme concentrations. The intrinsic steady-state properties of rat liver pyruvate kinase-1 (PK-1), rat liver glucose 6-phosphate dehydrogenase (G6PD) and bovine liver glutamate dehydrogenase (GDH) are unaltered by such high enzyme concentrations. These results contrast with reports on red blood cell G6PD and earlier impressions on GDH. Apparent changes in kinetic properties of PK-1 and GDH with increasing enzyme concentrations are explained by experimentally induced enzyme transitionsrather than by enzyme polymerizations. Experimental and mathematical methods for characterizing these transitions, which are prevalent among enzymes at high concentrations, were developed. Such characterizations are essential to distinguish alternative explanations of the experimental data and correctly assess enzyme properties at cellular concentrations. The
steady-state mechanismof rabbit muscle glyceraldehyde 3-phosphate dehydrogenase was shown to shift from a random to an ordered sequence of substrate additions and product dissociations as pH was increased from pH 7.0 to 8.6. This was correlated with an increasing rate of the redox step relative to the release of products. Acid dissociation and metal-ligand stability constants of Mg(II) 5-phosphoribosyl 1-pyrophosphate have been determined and an improved computer program developed for analysing similar data for other systems.
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Progress 01/01/72 to 12/30/72
Outputs
Fast kinetic measurements have been made on solutions with high enzyme concentrations, comparable to their cellular levels to 1) assess their properties under these conditions, and 2) to elucidate mechanisms of catalysis and regulation of their activity. Extensive data on bovine and porcine lactate dehydrogenase isozymes demonstrate that substantial pyruvate inhibition of the heart isozyme would occur under conditions of muscle tissue, in contrast to recent reports. Lactate inhibition, however, appears negligible. Rate and equilibrium constants for formation of bovine enzyme.NAD.pyruvate ternary complex are obtained and shown to be similar to properties of the muscle isozyme, indicating a mechanism in which a dissociation between tetrameric enzyme and monomer subunits precedes ternary complex formation. A study of the acetyl-CoA activation of chicken liver pyruvate carboxylase was complete. Acetyl-CoA concentrations required for activation of the enzyme to 1/2 of
V(m) increased nine fold when the enzyme was increased to physiological concentrations, although the subunit interaction energies remain constant. A mechanism of coupled enzyme association and acetyl-CoA binding equilibria is indicated. Two transitions in enzyme forms prior to achieving fully activated enzyme are observed. To elucidate the catalytic mechanism of yeast nicotinate phosphoribosyltransferase, initial velocity, product inhibition, isotope exchange, and substrate binding studies were made.
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Progress 01/01/71 to 12/30/71
Outputs
Fast kinetic measurements have been made on enzyme solutions at high concentrations comparable to their cellular levels to 1) assess their propertiesunder these conditions and 2) to elucidate their mechanisms of catalysis and regulation. When cellular levels of the substrates and coupled enzymes, malate dehydrogenase and citrate synthase are used, the specific reaction rates increase greatly as compared with dilute enzyme solutions, and ATP inhibition isdecreased. Measurements on mammalian glyceraldehyde 3-phosphate dehydrogenase demonstrate that all four sites are catalytically active, resolving this wide spread controversy. Rate constants for the oxidation and NADH release steps aredetermined and published equilibrium constants of enzyme-NADRG dissociation constants are shown to be in error due to use of partially inactivated enzyme. A new computer integration method has been applied to the staff differential equations of enzyme kinetics permitting integrations at
speeds 10y to 10DT times faster than previously used routines. Thus we have written a curve fitting program which functions with modest cost, whereas it was previously considered unfeasible. Large pH errors have been found under conditions widely used in biochemical research. The source of errors and ways to circumvent them have been found. Manuscripts on the above will be submitted for publication during the next month. Experiments are in progress to test the control properties of liver pyruvate kinase and pyruvate carboxylase at cellular enzyme levels and thephysiological significance of pyruvate control of lactate reutilization. The stability constants for Mg(2)PP-ribose-P are also being determined.
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Progress 01/01/70 to 12/30/70
Outputs
The kinetic properties of beef liver glutamate dehydrogenase, beef heart lactatedehydrogenase and pig heart malate dehydrogenase have been characterized at highenzyme concentrations (near 1 mg/ml), comparable to their cellular levels. Similar studies are in progress with glyceraldehyde phosphate dehydrogenase, liver pyruvate kinase and citrate synthase. Major changes in the inhibitions (reduced or eliminated) of glutamate dehydrogenase and lactate dehydrogenase occur when their enzymy concentrations are increased to cellular levels. Large changes in the activity of glyceraldehyde phosphate dehydrogenase are also foundin an incomplete study. On the other hand, substrate inhibition of malate dehydrogenase is unaffected by enzyme concentration. The transition in kinetic properties of glutamate dehydrogenase has been correlated with the extent of itsmolecular association. Many of the physical and enzymatic properties of yeast nicotinate phosphoribasyltransferase
have been characterized and a manuscript written. Temperature jump apparatus has been constructed and successfully tested, though several improvements in its capabilities will still be sought. Several useful computer programs have been written and used to analyze complex coupled enzymy systems and transient kinetic data.
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Progress 01/01/69 to 12/30/69
Outputs
The kinetic properties of three enzymes, which are thought to be important in the control of metabolism, have been investigated for the first time at high enzyme concentrations (N/mg/ml), comparable to their cellular levels. Rapid kinetic measurements (stop flow), which are required for these studies have revealed significant changes in the properties of beef liver glutamate dehydrogenase (GDH) and beef heart lactate dehydrogenase when they were concentrated, whereas no changes were observed for pig heart malate dehydrogenase when it was concentrated to cellular levels. The transition in GDH properties has been correlated with association of the enzyme to a tetramer at 0.8/mg/ml and alterations in a binding site separate from the catalytic site.Investigations of four other control enzymes at cellular concentration levels have been started. Substrate binding, isotope exchange and kinetic measurementshave been made on purified nicotinate phosphoribosyl transferase
from yeast. These measurements demonstrate that ATP functions to phosphorylate the enzyme; Further details of the enzyme mechanism are also indicated. Temperature jump apparatus has been assembled but further modifications are required.
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Progress 07/01/68 to 12/30/68
Outputs
Stopped flow measurements on glutamate dehydrogenase solutions at high concentrations, comparable to intracellular levels, have revealed differences inthe control properties of the enzyme, as compared with those found in dihite enzyme solutions. The results will be presented at the meetings of the Federation of American Societies for Experimental Biology in April, 1969, and a manuscript covering these results is being prepared for publication. Considerable construction of temperature jump apparatus has been completed and testing of electronic and mechanical parts is expected within a month. Researchprojects within this classification have been initiated recently by two graduatestudents. These students have made stopped flow measurements on two other control enzymes at high concentrations, comparable to their intracellular levels. Large changes in their catalytic properties were found in both cases.
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