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.

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