Activating effect of regucalcin on (Ca(2+)-Mg2+)-ATPase in rat liver plasma membranes: relation to sulfhydryl group.
The activating mechanism of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca(2+)-Mg2+)-ATPase in the plasma membranes of rat liver was investigated. (Ca(2+)-Mg2+)-ATPase activity was markedly increased by a sulfhydryl (SH) group protecting reagent dithiothreitol (DTT; 2.5 and 5 mM as a final concentration), while the enzyme activity was significantly decreased by a SH group modifying reagent N-ethylmaleimide (NEM; 0.5-5 mM). The effect of DTT (5 mM) to increase the enzyme activity was clearly blocked by NEM (5 mM). Regucalcin (0.25-1.0 microM) significantly increased (Ca(2+)-Mg2+)-ATPase activity. This increase was completely blocked by NEM (5 mM). Meanwhile, digitonin (0.04%), which can solubilize the membranous lipids, significantly decreased (Ca(2+)-Mg2+)-ATPase activity. Digitonin did not have an effect on the DTT (5 mM)-increased enzyme activity. However, the effect of regucalcin (0.25 microM) increasing (Ca(2+)-Mg2+)-ATPase activity was entirely blocked by the presence of digitonin. The present results suggest that regucalcin activates (Ca(2+)-Mg2+)-ATPase by the binding to liver plasma membrane lipids, and that the activation is involved in the SH groups which are an active site of the enzyme.
Laboratory of Metabolism and Endocrinology, Graduate School of Nutritional Sciences, University of Shizuoka, Japan.
H
H
Takahashi
M
M
Yamaguchi
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000502:physiology*; D002265:Carboxylic Ester Hydrolases; D002462:Cell Membrane / Q000187:drug effects* / Q000201:enzymology; D004072:Digitonin / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000201:enzymology*; D008297:Male; D008563:Membrane Lipids / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D015238:Sulfotransferases
D002135:Calcium-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008563:Membrane Lipids; D013438:Sulfhydryl Compounds; D006160:Guanosine Triphosphate; D006493:Heparin; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D017301:Ca(2+) Mg(2+)-ATPase; D004072:Digitonin; D005033:Ethylmaleimide; D004229:Dithiothreitol