STIMULATION OF CARDIAC SARCOPLASMIC-RETICULUM CALCIUM-PUMP BY ACYLPHOSPHATASE - RELATIONSHIP TO PHOSPHOLAMBAN PHOSPHORYLATION

Ca2+ transport by cardiac sarcoplasmic reticulum is tightly coupled wi th the enzymatic activity of Ca2+-dependent ATPase, which forms and de composes an intermediate phosphoenzyme, Heart sarcoplasmic reticulum C a2+ pump is regulated by cAMP-dependent protein kinase (PRA) phosphola mban phosphorylation, which results in a stimulation of the initial ra tes of Ca2+ transport and Ca2+ ATPase activity. In the present studies we found that acylphosphatase from heart muscle, used at concentratio ns within the physiological range, actively hydrolyzes the phosphoenzy me of cardiac sarcoplasmic reticulum Ca2+ pump, with all apparent K-m on the order of 10(-7) M, suggesting an high affinity of the enzyme fo r this special substrate. In umphosphorylated vesicles acylphosphatase enhanced the rate of ATP hydrolysis and Ca2+ uptake with a concomitan t significant decrease in apparent K-m, for Ca2+ and ATP. In vesicles whose phospholamban was PKA-phosphorylated, acylphosphatase also stimu lated the rate of Ca2+ uptake and ATP hydrolysis but to a lesser exten t, and the K-m values for Ca2+ and ATP were not significantly differen t with respect to those found in the absence of acylphosphatase. These findings suggest that acylphosphatase, owing to its hydrolytic effect , accelerates the turnover of the phosphoenzyme intermediate with the consequence of an enhanced activity of Ca2+ pump, It is known that pho sphorylation of phospholamban results in an increase of the rate at wh ich the phosphoenzyme is decomposed. Thus, as discussed, a competition between phospholamban and acylphosphatase effect on the phosphoenzyme might be proposed to explain why the stimulation induced by this enzy me is less marked in PKA-phosphorylated than in unphosphorylated heart vesicles.