Stimulation of cardiac sarcoplasmic reticulum calcium pump by acylphosphatase. Relationship to phospholamban phosphorylation.
Ca2+ transport by cardiac sarcoplasmic reticulum is tightly coupled with the enzymatic activity of Ca2+-dependent ATPase, which forms and decomposes an intermediate phosphoenzyme. Heart sarcoplasmic reticulum Ca2+ pump is regulated by cAMP-dependent protein kinase (PKA) phospholamban phosphorylation, which results in a stimulation of the initial rates of Ca2+ transport and Ca2+ ATPase activity. In the present studies we found that acylphosphatase from heart muscle, used at concentrations within the physiological range, actively hydrolyzes the phosphoenzyme of cardiac sarcoplasmic reticulum Ca2+ pump, with an apparent Km on the order of 10(-7) M, suggesting an high affinity of the enzyme for this special substrate. In unphosphorylated vesicles acylphosphatase enhanced the rate of ATP hydrolysis and Ca2+ uptake with a concomitant significant decrease in apparent Km for Ca2+ and ATP. In vesicles whose phospholamban was PKA-phosphorylated, acylphosphatase also stimulated the rate of Ca2+ uptake and ATP hydrolysis but to a lesser extent, and the Km values for Ca2+ and ATP were not significantly different 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 phosphorylation of phospholamban results in an increase of the rate at which 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 enzyme is less marked in PKA-phosphorylated than in unphosphorylated heart vesicles.
Nediani
C
C
Dipartimento di Scienze Biochimiche, Università di Firenze, Viale Morgagni 50, 50134 Firenze, Italy.
Fiorillo
C
C
Marchetti
E
E
Pacini
A
A
Liguri
G
G
Nassi
P
P
D017766:Acid Anhydride Hydrolases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002417:Cattle; D004789:Enzyme Activation; D006868:Hydrolysis; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D000097024:Acylphosphatase; D000099211:Phospholamban
D017766:Acid Anhydride Hydrolases; D000255:Adenosine Triphosphate; D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D000097024:Acylphosphatase; D000099211:Phospholamban