Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent protein phosphatase activity in rat brain cytosol.
The effect of Ca2+-binding protein regucalcin on neutral phosphatase activity in rat brain cytosol was investigated. Phosphatase activity was assayed in a reaction mixture containing the cytosolic protein in the presence of phosphotyrosine, phosphoserine, and phosphothreonine. The presence of calcium chloride (10(-5) and 10(-4) M) in the enzyme reaction mixture caused a significant increase in phosphatase activity toward three phosphoaminoacids. The enzyme activity toward phosphoserine and phosphothreonine was significantly enhanced by the addition of calmodulin (1 or 5 microg/ml) in the presence of calcium (10(-5) M). Such an effect was not seen in the presence of phosphotyrosine. Trifluoperazine (2x10(-5) M), an antagonist of calmodulin, completely inhibited calcium (10(-5) M)-increased phosphatase activity toward phosphoserine and phosphothreonine, whereas it had no effect on the enzyme activity toward phosphotyrosine. Regucalcin (10(-9) M) significantly inhibited phosphatase activity toward three phosphoaminoacids without or with Ca2+ addition. The inhibitory effect of regucalcin (10(-10) and 10(-9) M) was also seen in the presence of Ca2+ (10(-5) M) and calmodulin (5 microg/ml). The presence of anti-regucalcin monoclonal antibody (20 or 50 ng/ml) in the enzyme reaction mixture caused a significant elevation of phosphatase activity toward three phosphoaminoacids; this effect was completely abolished by addition of regucalcin (10(-9) M). The present study suggests that the endogenous regucalcin has an inhibitory effect on Ca2+/calmodulin-dependent protein phosphatase activity in rat brain cytosol.
Hamano
T
T
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, Shizuoka City 422-8526, Japan.
Yamaguchi
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015415:Biomarkers; D001921:Brain / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D064026:Calbindins; D019703:Calcineurin / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002265:Carboxylic Ester Hydrolases; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000201:enzymology; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D064030:S100 Calcium Binding Protein G / Q000494:pharmacology; D009418:S100 Proteins / Q000494:pharmacology; D015238:Sulfotransferases; D014268:Trifluoperazine / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D015415:Biomarkers; D064026:Calbindins; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D047908:Intracellular Signaling Peptides and Proteins; C030564:S-100 calcium-binding protein alpha subunit; D064030:S100 Calcium Binding Protein G; D009418:S100 Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D014268:Trifluoperazine; D019000:Phosphotyrosine; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D019703:Calcineurin; D002118:Calcium