Capillary electrophoretic analysis of mu- and m-calpain using fluorescently labeled casein substrates.
Calpains are unique calcium-dependent thiol proteases that have been proposed to participate in a number of physiological processes including signal transduction and protein turnover in skeletal muscle. Calpains exist in two major forms. Interestingly, the two forms of protease show no significant difference in their action on various substrates. The only demonstrable difference in their activity involves the concentration of calcium required for activation. Both mu- and m-calpains typically achieve half maximal activation at 50 microM and 0.7 mM calcium, respectively. The focus of this study was to examine the action of both forms of calpain on casein substrates and assess whether any differences could be observed in the resulting peptide finger print using capillary electrophoresis. Purified mu- and m-calpain were incubated for various lengths of time with Oregon Green labeled alphas- and beta-casein. The reactions were stopped with sodium dodecyl sulfate (SDS) and products separated by capillary electrophoresis in micellar electrokinetic capillary chromatography (MEKC) mode using laser-induced fluorescence (LIF) detection. Comparison of the electropherograms showed no difference in the peptide profile for either enzyme. However, it was found that beta-casein was hydrolyzed more extensively than alphas-casein, by both enzymes. Capillary electrophoresis was found to be a very sensitive technique for detection of calpain activity. Using beta-casein as substrate, the CE approach was able to detect 2-3 ng of calpain activity. The results also suggest that capillary electrophoresis is a useful tool for proteolytic investigations of protein structure.
Gu
X
X
Department of Food Science and Technology, University of Nebraska-Lincoln, 68583-0919, USA.
Whipple-VanPatter
G
G
O'Dwyer
M
M
Zeece
M
M
D000818:Animals; D002154:Calpain / Q000032:analysis* / Q000378:metabolism; D002364:Caseins; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods*; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D013379:Substrate Specificity
D002364:Caseins; D005456:Fluorescent Dyes; D002154:Calpain