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.
Takahashi
H
H
Laboratory of Metabolism and Endocrinology, Graduate School of Nutritional Sciences, University of Shizuoka, Japan.
Yamaguchi
M
M
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