Proteolysis of the protein inhibitor of calcium-dependent proteases produces lower molecular weight fragments that retain inhibitory activity.
The specific inhibitor of calcium-dependent proteases was purified from soluble extracts of bovine heart. The protein had a molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gels and migrated on gel filtration chromatography with an apparent molecular weight of 250,000. The inhibitor specifically blocked the action of the two calcium-dependent proteases, CDP-I and CDP-II, but did not influence a variety of other proteases including trypsin, chymotrypsin, or Staphylococcus aureus V8 protease. These latter enzymes extensively degraded the inhibitor to discrete lower molecular weight peptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by gel filtration chromatography. Under the conditions studied, proteolysis of the inhibitor had little or no effect on its inhibitory activity; isolated peptides with molecular weights as low as 17,000 retained inhibitory function. A number of various-sized inhibitor fragments were isolated by gel filtration chromatography and by SDS-PAGE. These fragments were compared with the intact inhibitor for their ability to inhibit CDPs. As suggested previously by us and others, one molecule of intact inhibitor appears to inhibit up to five molecules of calcium-dependent protease. The inhibitor fragments of decreasing size inhibited correspondingly fewer molecules of protease. These results suggest that the inhibitor protein contains multiple functional domains and may explain some of the discrepancies in reported molecular weights for this protein.
DeMartino
G N
GN
Department of Physiology, University of Texas Health Science Center, Dallas 75235.
Wachendorfer
R
R
McGuire
M J
MJ
Croall
D E
DE
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*
D006023:Glycoproteins; D010446:Peptide Fragments; C039304:calpain inhibitors