Progress 01/01/00 to 12/31/04
Outputs
This project was to provide expertise and demonstrate fesability of a proteomic approach to research in the agricultural and food sciences. The project has been a success in that there are now several laboratories that carry out 2-D electrophoretic fractionation of proteins on a routine or semi-routine basis.
Impacts
Development of proteomic expertise in this project has changed or enhanced the research direction of several laboratories. This has, in turn, provided an impetus for adoption of these techniques by other laboratories at the University of Nebraska.
Publications
- P. Xiang, E.J. Haas, M.G. Zeece, J. Markwell and G. Sarath (2004) C-terminal 23 kDa polypeptide of soybean Gly m Bd 28 K is a potential allergen. Planta, 220: 56-63.
- R. Roychaudhuri, G. Sarath, M. Zeece and J. Markwell (2004) Stability of the allergenic soybean Kunitz trypsin inhibitor. Biochim. Biophys. Acta. 1699: 207-212
- T. Heng-Moss, G. Sarath, F. Baxendale, D. Novak, S. Bose, X. Ni and S. Quisenberry (2004) Characterization of oxidative enzyme changes in buffalograsses challenged by Blissus occiduus. J. Econ. Entomol. 97: 1086-1095
- Kothapalli, N., G. Sarath and J. Zempleni (2005) biotinylation of K12 in histone H4 decreases in response to DNA double-strand breakes in human JAr choriocarcinoma cells. J. Nutr. 135: 2337-2342
- Y.C. Chew, G. Camporeale, N. Kothapalli, G. Sarath and J. Zempleni (2005) Lysine residues in N-terminal and C-terminal regiions of human histone H2A are targets for biotinylation by biotinidase. J. Nutr. Biochem., in press.
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Progress 10/01/03 to 09/30/04
Outputs
An aspect to our research is to better understand the reversible denaturation of plant proteins. As a model system, we examined the denaturation and renaturation of soybean Kunitz trypsin inhibitor (SKTI). This is a 21.5 kD protein that belongs to the family of all antiparallel beta-sheet proteins, is a food allergen, and is unusually resistant to thermal and chemical denaturation. Spectroscopic and biochemical techniques such as circular dichroism, 8-anilino-1-napthalene sulfonate fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Thermal denaturation appears to be a two state process centered on 60 C and somewhat sensitive to ionic strength. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid and denaturation at lower temperatures than the native protein. Limited proteolysis
with chymotrypsin during renaturation after heating showed that the native structure reforms at around 60 C reversing the denaturation. Circular dichroism spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten globule structure. The existence of this intermediate was established by 8-anilino-1-napthalene sulfonate fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid denaturation may be important for its role as a food allergen and support for the molten globule as an intermediate in thermal denaturation and renaturation. An ability to decrease the stability of SKTI would presumibly decrease its potential as a food allergen.
Impacts
The potential ability of the Arabidopsis FDH to assume a conformation able to use NADP in a chloroplastic compartment is of potential physiological interest and will be the focus of future studies. The ability to engineer an NADPH-utilizing FDH could lead to a second site of carbon dioxide fixation for the plant cell.
Publications
- R. Roychaudhuri, G. Sarath, M. Zeece and J. Markwell (2004) Stability of the allergenic soybean Kunitz trypsin inhibitor. Biochim. Biophys. Acta, 1699: 207-212.
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Progress 10/01/02 to 09/30/03
Outputs
The major effort involved study of the leaf formate dehydrogenase (FDH; EC 1.2.1.2), an NAD-dependent enzyme that catalyzes the oxidation of formate to CO2 in the mitochondria of higher plants. We have noticed that the formate and NAD+ substrate had Km values for the native Arabidopsis FDH were significantly higher than previously reported for the purified enzyme. These observations were also observed with the Arabidopsis FDH expressed in tobacco plants. Heating of the Arabidopsis FDH at 60 C for five minutes resulted in a decrease in the Km values to more closely resemble the previously reported values. The heating-induced changes in FDH are consistent with the existence of the enzyme in a metastable state that can be interconverted between different forms by heating to a molten state. We have developed spectroscopic and enzymatic techniques for examining such thermally-induced reversible processes using the soy Kunitz trypsin inhibitor as a model system.
Interestingly, we have also discovered that once the FHD has been heated at 60 C for five minutes and cooled, it is able to use NADP+ as a substrate for the oxidation of formate. To be able to initiate studies on in vitro mutagenesis of the Arabidopsis FDH, it was necessary to be able to induce the production of an active enzyme in Escherichia coli or another microbial system. The FDH coding sequence, minus the signal sequence, was cloned into the pET28 expression vector and transformed into E. coli. Initial attempts at induction by IPTG resulted in specific activities of 1 to 2 mU/mg protein, approximately equal to that of Arabidopsis leaf extracts. We have subsequently discovered that if after induction, cultures are continued incubating at 15 C for two days, the amount of active FDH continues to increase and will reach values of 1000 to 5000 mU/ml. These are above the reported specific activity of the purified Arabidopsis FDH and over half of the cellular protein appears to be that
of the FDH. We will utilize the FDH expressed in E. coli to initiate in vitro mutagenic studies on cofactor specificity and stability. Our interest in the FDH originally stemmed from observations that methanol may enhance plant growth under water-limiting conditions. We have observed that spraying leaves of C3 plants will generally cause some increase in FDH specific activity within 48 h. To get a strategic sense of the changes caused by methanol treatment, we analyzed mRNA levels in control plants and plants treated with methanol 24 or 48 h prior to harvesting. There was not a pattern of large changes in genes associated with one-carbon metabolism. Surprisingly, there was a pattern of increases in the expression of proteins associated with photosynthetic electron transfer components. These results will be repeated and may help in an understanding of how methanol impacts photosynthetic efficiency in water-limited plants.
Impacts
The potential ability of the Arabidopsis FDH to assume a conformation able to use NADP in a chloroplastic compartment is of potential physiological interest and will be the focus of future studies. The ability to engineer an NADPH-utilizing FDH could lead to a second site of carbon dioxide fixation for the plant cell.
Publications
- H.A. Ramberg, B.J.S.C. Olson, J.N. Nishio, J. Markwell and J.C. Osterman 2002 The role of methanol in promoting plant growth: an update. Rev. Plant Biochem. Biotechnol., 1: 113-126.
- T. M. Heng-Moss, X. Ni, T. Macedo, J. P. Markwell, F. P. Baxendale, S. S. Quisenberry, and V. Tolmay 2003 Comparison of chlorophyll and carotenoid concentrations among Russian wheat aphid (Homoptera: Aphididae)-infested wheat isolines. J. Econ. Entomol. 96: 475-481.
- R.D. Baack, J. Markwell, P.L. Herman and J.C. Osterman 2003 Kinetic behavior of the Arabidopsis thaliana leaf formate dehydrogenase is thermally sensitive. J. Plant Physiol., 160: 445-450.
- R. Roychaudhuri, G. Sarath, M. Zeece and J. Markwell 2003 Reversible denaturation of the soybean Kunitz trypsin inhibitor. Arch. Biochem. Biophys., 412 20-26.
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Progress 10/01/01 to 09/30/02
Outputs
Formate dehydrogenase (FDH; EC 1.2.1.2) is a NAD-dependent enzyme that catalyzes the oxidation of formate to carbon dioxide in the mitochondria of higher plants. Sequence analyses and other preliminary experiments suggested that FDH might also be targeted to the chloroplasts of Arabidopsis and other plant species. In the present study, transgenic Arabidopsis and tobacco plants that overexpress Arabidopsis FDH were produced. The FDH specific activity in the leaf tissue of the transgenic plants increased an average of 4.5 fold for Arabidopsis and 31.5 fold for tobacco. Immunodetection and enzyme assays of intact chloroplasts fractionated from the leaves of transgenic tobacco plants suggested that Arabidopsis FDH is present in the chloroplast. Immunogold labeling of Arabidopsis and tobacco detected FDH in both the mitochondria and chloroplasts of the leaf cells. Two previous kinetic studies on the Arabidopsis thaliana leaf NAD-dependent formate dehydrogenase (EC 1.2.1.2)
have demonstrated two very different sets of Km values for the formate and NAD+ substrates. We examined the kinetics of the enzyme partially purified from a leaf extract by gel-filtration desalting and chromatography on DEAE-cellulose, as well as by isolation of a mitochondria-enriched fraction obtained by differential centrifugation. Both of these methods produce a formate dehydrogenase enzyme with the higher Km values of approximately 10 mM formate and 75 microM NAD+. The kinetic properties of the Arabidopsis formate dehydrogenase expressed to high levels in transgenic tobacco plants were also those of the high Km form. The high Km form of the enzyme converted to a low Km form by heating for 5 minutes at 60C. An Arrhenius plot of the activity during the heating process was linear, indicating that the heating did not cause alterations in either the active site or the thermal dependence of the catalytic reaction. We conclude that the native form of the formate dehydrogenase probably
resembles the form with the higher Km values. Heating seemingly converts this native enzyme to the molten globule state and cooling results in formation of a non-native structure with altered kinetic properties.
Impacts
Determination that the FDH enzyme exists in chloroplasts and that it has relatively high Km values for formate and NAD+ raise the possibility that it can have a previously unappreciated function. Some studies have shown that formate can be a primary product of photosynthesis. It is possible that FDH in the chloroplast may be involved in this phenomenon.
Publications
- P.A. Herman, H.A. Ramberg, R.D. Baack, J. Markwell and J.C. Osterman. 2002 Formate dehydrogenase in Arabidopsis thaliana: overexpression and subcellular localization in leaves. Plant Sci. 163: 1137-1145.
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Progress 10/01/00 to 09/30/01
Outputs
Overall, this project was successful. At the time this project was proposed there was no proteomic capability on the UNL campus and little awareness among some administrators about the potential for proteomic analyses in the post-genomic era. Drs. Markwell, Sarath and Zeece personally visited with a number of vice-chancellors, deans, directors and senior faculty to describe proteomics and its value to life science research. We feel that these efforts contributed to the present awareness of proteomics as an area in which UNL should build strength. In terms of actual research stimulated by this project, the outcome was generally successful. However, we found that the reliance upon undergraduate research assistants to perform the proteomic fractionations was inefficient. Considerable training is required to acquire expertise in 2-D electrophoretic fractionation. Most of the undergraduates employed by this project did not continue to work once trained, but either accepted
other employment opportunities or decided to instead focus on their studies. Thus, much time and resources (salary and supplies used in training) were invested in student assistants that did not actually produce scientific data on real samples. A second limitation was the interface between the front-end of protein fractionation and the back-end of mass spectrometric identification of proteins. Our initial assumption that proteins fractionated by 2-D electrophoresis could be readily identified by the UNL mass spectrometric facility was overly optimistic. In fact, the preparation of samples for the analyses was not trivial, the throughput was low and the instrument seemed to be frequently down. The 2-D fractionation systems purchased as part of this project remain in the laboratories of Drs. Madhavan, Markwell and Zeece and continue to be used for proteomic analyses. Dr. Markwells research has used proteomic analysis to identify different forms of formate dehydrogenase in the
Arabidopsis chloroplast and mitochondrion. This research is continuing to determine if these enzymes have different biological roles. This work was presented at the Plant Biology 2001 meeting this Summer. Dr. Zeece has used proteomic analysis to identify protein contaminants in soybean lecithin and established that these proteins are allergens. A manuscript entitled Protemic analysis of allergens in soy lecithin will be published shortly. In addition, Dr. Zeece and Dr Michael Meagher have initiated collaborative efforts to employ proteomic analysis for characterization of protein products developed by the PBDF.
Impacts
This project stimulated institutional interest in proteomics and was at least partially responsible for the development of the UNL core facility in proteomic analysis.
Publications
- J.E. Specht, K. Chase, M. Macrander, G.L. Graef, J. Chung, J.P. Markwell, M. Germann, J.H. Orf and K.G. Lark (2001) Soybean response to water: A QTL analysis of drought tolerance. Crop Science, 41: 493-509.
- X. Ni, S.S. Quisenberry, T. Heng-Moss, J. Markwell, G. Sarath, R. Klucas and F. Baxendale (2001) Oxidative Responses of Resistant and Susceptible Cereal Leaves to Symptomatic and Non-Symptomatic Cereal Aphid (Hemiptera: Aphididae) Feeding. J. Econ. Entomol., 94: 743-751.
- X. Gu, T. Beardslee, M. Zeece, G. Sarath and J. Markwell (2001) Identification of IgE-binding proteins in soy lecithins. Int. Arch. Allergy Immunol., in press.
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Progress 10/01/99 to 09/30/00
Outputs
Formate dehydrogenase (E.C. 1.2.1.2) is a mitochondrial-localized NAD-requiring enzyme in green plants. We are interested in this enzyme because in methanotropic microorganisms, ancestors to photosynthetic organisms, this enzyme appears to control whether one-carbon molecules are assimilated or dissimilated. The enzyme activity and corresponding mRNA in leaves of Arabidopsis thaliana are induced by treatment with one-carbon metabolites. The cDNA for the Arabidopsis formate dehydrogenase is similar to that of other plants except for the N-terminal region which is predicted to target chloroplasts as well as mitochondria. The high specific of activity of the enzyme in isolated chloroplasts suggests it is targeted to both mitochondria and chloroplasts in Arabidopsis. Formate dehydrogenase from Arabidopsis was partially purified and Km values for formate and NAD+ were determined to be 10 mM and 65 microM respectively; the Ki for NADH was 17microM. We conclude that formate
dehydrogenase is normally present in Arabidopsis chloroplasts and that sensitivity to inhibition by NADH may play a role in whether cellular formate is assimilated or dissimilated. To further explore the post-translational processing involved in organellar targeting of the formate dehydrogenase, we have developed the ability to fractionate Arabidopsis proteins by two-dimensional electrophoresis. We have also prepared polyclonal antibodies against the recombinant formate dehydrogenase apoprotein. Using a proteomic approach, we are in the process of determining how the nascent apoprotein is differentially processed by the mitochondrial and organellar compartments.
Impacts
Formate dehydrogenase is one of two enzymes metabolically positioned to be able to modulate one-carbon flux into the photorespiratory cycle and thereby impact photosynthetic efficieicy. Understanding how to regulate the expression of formate dehydrogenase differentially into chloroplasts and mitochondria may permit manipulation of photosynthetic efficiency. While the effect may be minimal under eutrophic conditions, it may markedly impact yields under stress conditions.
Publications
- Olson, B.J.S.C., Skavdahl, M., Ramberg, H., Osterman, J.C. and Markwell, J.P. 2000 Formate dehydrogenase in Arabidopsis thaliana: characterization and possible targeting to the chloroplast. Plant Sci., in press
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