Inhibitory effect of regucalcin on Ca(2+)-dependent protein kinase activity in rat brain cytosol: involvement of endogenous regucalcin.
The effect of regucalcin, a Ca(2+)-binding protein, on Ca(2+)-dependent protein kinase activity in the brain cytosol of rats with different ages (5 and 50 weeks old) was investigated. The addition of calmodulin (10 microg/ml) or dioctanoylglycerol (5 microg/ml) in the enzyme reaction mixture caused a significant increase in protein kinase activity in the presence of CaCl2 (1 mM), indicating that Ca2+ calmodulin or protein kinase C is present in the cytosol. Such an increase was completely prevented by the addition of regucalcin (10(-7) M). Moreover, regucalcin (10(-7) M) significantly inhibited cytosolic protein kinase activity without Ca2+/calmodulin or dioctanoylglycerol addition. Meanwhile, the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the enzyme reaction mixture caused a significant elevation of protein kinase activity, suggesting an inhibitory effect of endogenous regucalcin. Brain cytosolic protein kinase activity was significantly elevated by increasing age (50-week-old rats). Also, regucalcin (10(-7) M) significantly decreased protein kinase activity without Ca(2+) addition in the brain cytosol of aged rats. However, the effect of anti-regucalcin monoclonal antibody (50 ng/ml) in elevating protein kinase activity was not seen in the brain cytosol of aged rats. These results suggest that regucalcin has an inhibitory effect on Ca(2+)-dependent protein kinase activity in rat brain cytosol, and that the effect of endogenous regucalcin may be weakened in the brain cytosol of aged rats.
Hamano
T
T
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, Shizuoka City, Japan.
Hanahisa
Y
Y
Yamaguchi
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000494:pharmacology / Q000502:physiology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases; D003600:Cytosol / Q000201:enzymology; D005260:Female; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015238:Sulfotransferases
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D002118:Calcium