Role of regucalcin as an activator of Ca(2+)-ATPase activity in rat liver microsomes.
The effect of Ca(2+)-binding protein regucalcin on Ca(2+)-ATPase activity in isolated rat liver microsomes was investigated. The presence of regucalcin (0.1-1.0 microM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the microsomes. Thapsigargin (10(-6) M), a specific inhibitor of microsomal Ca(2+) pump enzyme (Ca(2+)-ATPase), clearly inhibited regucalcin (0.5 microM)-increased microsomal Ca(2+)-ATPase activity. Liver microsomal Ca(2+)-ATPase activity was markedly decreased by N-ethylmaleimide (NEM; 2.5 mM), while the activity was clearly elevated by dithiothreitol (DTT; 2.5 mM), indicating that the sulfhydryl (SH) group of the enzyme is an active site. The effect of regucalcin (0.5 microM) in increasing Ca(2+)-ATPase activity was completely inhibited by the presence of NEM (2.5 mM) or digitonin (10(-2) %), a solubilizing reagent of membranous lipids. Moreover, the effect of regucalcin on enzyme activity was seen in the presence of Ca(2+) ionophore (A23187; 10(-7) M). The present study demonstrates that regucalcin can stimulate Ca(2+) pump activity in rat liver microsomes, and that the protein may act the SH groups of microsomal Ca(2+)-ATPase.
Takahashi
H
H
Laboratory of Endocrinology and Molecular Matabolism, Graduate School of Nutritional Sciences, University of Shizuoka, Shizuoka City 422-8526, Japan.
Yamaguchi
M
M
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015238:Sulfotransferases; D019284:Thapsigargin / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D007476:Ionophores; D000001:Calcimycin; D003994:Bucladesine; D019284:Thapsigargin; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D000252:Calcium-Transporting ATPases