Progress 09/01/01 to 08/31/05
Outputs
The overall goal of this project was to determine how the calpain system functions in postmortem muscle to cause postmortem tenderization. The studies use bovine skeletal muscle. The project directly addressed tenderness. Three proteins in the calpain system are involved in postmortem tenderization: 1) u-calpain, a protease requiring micromolar [Ca2+] for activity; 2) m-calpain a protease requiring millimolar [Ca2+] for activity; and 3) calpastatin, a inhibitor specific for the two calpains. Elevated calpastatin activity has been shown to be highly associated with a decreased rate of postmortem tenderization. Activity of u-calpain decreases rapidly during postmortem storage and u-calpain is nearly inactive after 3 days postmortem. Calpastatin activity also decreases during postmortem storage, but less rapidly than u-calpain activity, whereas m-calpain activity is affected only slightly by postmortem storage. It is unclear whether Ca2+ concentrations would ever get high
enough in postmortem muscle to initiate m-calpain activity and u-calpain has been suggested as the calpain involved in postmortem tenderization. Yet, u-calpain is nearly completely inactive after 2-3 days. Calpastatin is colocalized with the calpains.
Impacts
Tenderness is a complex trait influenced largely by nongenomic factors. Attempts to improve tenderness by genetic selection alone, therefore, would result in slow progress, and postslaughter intervention is necessary to improve tenderness. Consequently, research on ways to increase/ensure meat tenderness during postmortem storage has considerable economic importance.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The finding that phospholipid micelles lower the Ca2+ concentration required for autolysis of the calpains led to a hypothesis suggesting that the calpains are translocated to the plasma membrane where they interact with phospholipids to initiate their autolysis. However, the effect of plasma membranes themselves on the Ca2+ concentration required for calpain autolysis has never been reported. Also, if interaction with a membrane lowers the Ca2+ required for autolysis, the membrane-bound-calpain must autolyze itself, because it would be the only calpain having the reduced Ca2+ requirement. This implies that the autolysis is an intramolecular process, although several studies have shown that autolysis of the calpains in an in vitro assay and in the absence of phospholipid is an intermolecular process. Inside-out vesicles prepared from erythrocytes had no effect on the Ca2+ concentration required for autolysis of either u- or m-calpain, although phosphatidylinositol
(PI) decreased the Ca2+ concentration required for autolysis of the same calpains. The presence of a substrate for the calpains, B-casein, reduced the rate of autolysis of both u- and m-calpain both in the presence and in the absence of PI, suggesting that u- and m-calpain autolysis is an intermolecular process in the presence of PI just as it is in the absence.
Impacts
How calpain activity is regulated in cells is still unclear, but the calpains ostensibly participate in a variety of cellular processes including remodeling of cytoskeletal/membrane attachments, different signal transduction pathways, and apoptosis. Deregulated calpain activity following loss of Ca2+ homeostasis results in tissue damage in response to events such as myocardial infarcts, stroke and brain trauma.
Publications
- Zalewska, T., Thompson, V.F., Goll, D.E. (2004). Effect of phosphatidylinositol and inside-out erythrocyte vesicles on autolysis of u- and m-calpain from bovine skeletal muscle. Biochim. et Biophy. Acta 1693, 125-133.
- Wendt, A., Thompson, V.F., Goll, D.E. (2004). Interaction of calpastatin with calpain: a review. Biol. Chem. 385, 465-472.
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Progress 01/01/03 to 12/31/03
Outputs
The calpain system originally comprised three molecules: two Ca2+-dependent proteases, u-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. Both u- and m-calpain are heterodimers containing an identical 28kDa subunit and an 80kDa subunit that shares 55-65 percent sequence homology between the two proteases. The crystallographic structure of m-calpain reveals six domains in the 80kDa subunit. 1) a 19 amino acid NH2 terminal sequence, 2) and 3) two domains that constitute the active site, IIa and IIb. 4) domain III, 5) an 18 amino acid extended sequence liking domain III to domain IV; and 6) domain IV, which resembles the penta EF hand family of polypeptides. The single calpastatin gene can produce eight or more calpastatin polypeptides ranging from 17 to 85kDa by use of different promoters and alternative splicing events. The physiological significance of these different calpastatins is unclear,
although all bind to three different places on the molecule, binding to at least two of the sites is Ca2+ dependent.
Impacts
How calpain activity is regulated in cells is still unclear, but the calpains ostensibly participate in a variety of cellular processes including remodeling of cytoskeletal/membrane attachments, different signal transduction pathways, and apoptosis. Deregulated calpain activity following loss of Ca2+ homeostasis results in tissue damage in response to events such as myocardial infarcts, stroke and brain trauma.
Publications
- Goll, D.E., Thompson, V.F., Li, H., Wei, W., Cong, J. (2003). The calpain system. Physiol. Rev. 83, 731-801.
- Thompson, V.F., Lawson, K.R., Barlow, J., Goll, D.E. (2003). Digestion of u- and m-calpain by trypsin and chymotrypsin. Biochim. et Biophy. Acta 1648, 140-153.
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Progress 01/01/02 to 12/31/02
Outputs
It has been difficult to purify calpastatin without using a step involving heating to 90-100 degrees C. Preparations of calpastatin obtained after heating often contain several polypeptides that have been ascribed to proteolytic degradation. Because calpastatin is highly susceptible to proteolytic degradation and several different calpastatin isoforms can be produced by using different start sites of transcription translation and or alternative splicing from the single calpastatin gene. It would be useful, therefore, to have a method for purifying calpasatatin that does not involve heating. At low ionic strength, calpastatin from skeletal muscle extracts binds quantitatively to an immunoaffinity column made by coupling a monoclonal antibody.
Impacts
The immunoaffinity column is especially useful for purifying calpastatin from small tissue samples in a single step.
Publications
- Wei, W., Li, H., Cong, J., Thompson, V.F., Goll, D.E. 2002. Immunoaffinity purification of calpastatin and calpastatin constructs. Biochim. Biophys. Acta 1597:97-106.
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