INHIBITION OF CA2+ CALMODULIN-DEPENDENT PHOSPHATASE-ACTIVITY BY REGUCALCIN IN RAT-LIVER CYTOSOL - INVOLVEMENT OF CALMODULIN-BINDING/

The regulatory effect of regucalcin on Ca2+/calmodulin-dependent phosp hatase activity and the binding of regucalcin to calmodulin was invest igated. Phosphatase activity toward phosphotyrosine, phosphoserine, an d phosphothreonine in rat liver cytosol was significantly increased by the addition of Ca2+ (100 mu M) and calmodulin (0.30 mu M) These incr eases were clearly inhibited by the addition of regucalcin (0.50-1.0 m u M) into the enzyme reaction mixture. The cytosolic phosphoamino acid phosphatase activity was significantly elevated by the presence of an ti-regucalcin monoclonal antibody (0.2 mu g/ml), suggesting that endog enous regucalcin in the cytosol has an inhibitory effect on the enzyme activity. This elevation was prevented by the addition of regucalcin (0.50 mu M). Purified calcineurin phosphatase activity was significant ly increased by the addition of calmodulin (0.12 mu M) in the presence of Ca2+ (1 and 10 mu M) This increase was completely inhibited by the presence of regucalcin (0.12 CIM) The inhibitory effect of regucalcin was reversed by the addition of calmodulin with the higher concentrat ion (0.36 mu M) Regucalcin has been demonstrated to bind on calmodulin -agarose beads by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The present study demonstrates that regucalcin in hibits Ca2+/calmodulin-dependent protein phosphatase activity in rat l iver cytosol, and that regucalcin can bind to calmodulin. (C) 1998 Wil ey-Liss. Inc.