Characterization of regucalcin effect on proteolytic activity in rat liver cytosol: relation to cysteinyl-proteases.
The increasing effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was characterized. The proteolytic activity was markedly elevated by the addition of regucalcin (0.1-0.5 microM) in the absence of Ca2+. This increase was not significantly altered by the presence of diisopropylfluorophosphate (DPF; 2.5 mM)--although DFP caused a significant decrease in the proteolytic activity. Regucalcin (0.25 microM) additively enhanced the dithiothreitol (DTT; 1.0 mM)--increased proteolytic activity, while the regucalcin or DTT effect was completely abolished by NEM (5 mM), indicating that regucalcin may act on the SH group in proteases. Also, regucalcin (0.25 microM) enhanced the effect of Ca2+ (10 microM) increasing liver proteolytic activity, suggesting that regucalcin does not influence on the active sites for Ca2+ in proteases. Moreover, the proteolytic activity of regucalcin (0.25 microM) was significantly decreased by the presence of calpastatin (24 micrograms/ml), an inhibitor of Ca(2+)-activated neutral protease (calpain). Now, regucalcin (0.25 microM) increased about 7-fold the activity of m-calpain isolated from rabbit skeletal muscle. These observations demonstrate that regucalcin directly activates cysteinyl-proteases. Regucalcin may have a role as a potent proteolytic activator in the cytoplasm of liver cells.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology*; D004229:Dithiothreitol / Q000494:pharmacology; D004357:Drug Synergism; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007531:Isoflurophate / Q000494:pharmacology; D008099:Liver / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D018482:Muscle, Skeletal / Q000201:enzymology; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D015238:Sulfotransferases
D002135:Calcium-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007531:Isoflurophate; C033668:calpastatin; D004492:Edetic Acid; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D002154:Calpain; D003546:Cysteine Endopeptidases; D005033:Ethylmaleimide; D002118:Calcium; D004229:Dithiothreitol