Inhibitory effect of regucalcin on Ca 2+-dependent protein kinase activity in rat brain cytosol: involvement of endogenous regucalcin

Abstract

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 μg/ml) or dioctanoylglycerol (5 μg/ml) in the enzyme reaction mixture caused a significant increase in protein kinase activity in the presence of CaCl 2 (1 mM), indicating that Ca 2+ 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 Ca 2+/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.