Suppressive effect of endogenous regucalcin on protein tyrosine phosphatase activity in the nucleus of rat brain: Attenuation with increasing age

The effect of endogenous regucalcin, which is a regulatory protein of Ca2+ signaling, on protein tyrosine phosphatase activity in the nucleus of brain tissues of young and aged rats was investigated. Phosphotyrosine was used as the substrate for assay of protein tyrosine phosphatase activity. Protein tyrosine phosphatase activity in the brain nucleus of young (5 weeks old) rats was significantly increased in the presence of calcium chloride (5-50 µM) in the enzyme reaction mixture. The increase was completely blocked by the addition of trifluoperazine (10-50 µM), an antagonist of calmodulin, indicating that the enzyme is activated by endogenous Ca2+/ calmodulin. The addition of regucalcin (10−4-10−8 M) in the enzyme reaction mixture caused a significant decrease in protein tyrosine phosphatase activity in the absence or presence of calcium chloride (20 µM). Brain nuclear protein tyrosine phosphatase activity was significantly raised in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the enzyme reaction mixture. The increase was completely prevented by the addition of regucalcin (10−6 M). In the brain nucleus of aged (50 weeks old) rats, protein tyrosine phosphatase activity was elevated significantly as compared with that of 5 weeks old rats. The effect of anti-regucalcin monoclonal antibody in increasing the enzyme activity in the brain nucleus of aged rats was seen in the presence of 50 ng/ml of the antibody. Such an effect was not found by the antibody of 10 and 25 ng/ml. Regucalcin protein in brain nucleus was detected by Western blot analysis. This level was significantly decreased by increasing age. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein tyrosine phosphatase activity in the nucleus of rat brain, and that this regulation is attenuated with increasing age.