Stimulatory effect of regucalcin on proteolytic activity in rat renal cortex cytosol: involvement of thiol proteases.
The effect of regucalcin, a calcium-binding protein, on neutral proteolytic activity in the cytosol of rat kidney cortex was investigated. Proteolytic activity was significantly increased in the presence of regucalcin (0.01-0.25 microM) in the enzyme reaction mixture. This increase was not significantly altered by the addition of CaCl, (0.01 and 1.0 mM) or EGTA (1.0 mM), indicating that the effect of regucalcin was independent on Ca2+. The effect of regucalcin to increase proteolytic activity was completely prevented in the presence of N-ethylmaleimide (5 mM), a modifying reagent of thiol groups. Proteolytic activity was clearly elevated by dithiothreitol (5 mM). This elevation was further enhanced by regucalcin (0.1 microM). Meanwhile, the stimulatory effect of regucalcin on proteolytic activity was not significantly altered in the presence of diisopropylfluorophosphate (2.5 mM), an inhibitor of serine proteases. Also, the regucalcin effect was not appreciably changed by the addition of EDTA (2.5 mM), a chelator of metal ions, indicating that it is not involved in metal-related proteases. These results demonstrate that regucalcin can increase proteolytic activity in the cytosol of rat kidney cortex. Regucalcin may activate thiol proteases independent on Ca2+.
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, Shizuoka City, Japan.
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Yamaguchi
D000818:Animals; D002118:Calcium / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D002265:Carboxylic Ester Hydrolases; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology*; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D006868:Hydrolysis; D047908:Intracellular Signaling Peptides and Proteins; D007672:Kidney Cortex / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D010455:Peptides / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015238:Sulfotransferases
D002135:Calcium-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D003546:Cysteine Endopeptidases; D005033:Ethylmaleimide; D002118:Calcium; D004229:Dithiothreitol