Effects of acylphosphatase on the activity of erythrocyte membrane Ca2+ pump.
Acylphosphatase, purified from human erythrocytes, actively hydrolyzes the acylphosphorylated intermediate of human red blood cell membrane Ca(2+)-ATPase. This effect occurred with acylphosphatase amounts (up to 10 units/mg membrane protein) that fall within the physiological range. Furthermore, a very low Km value, 3.41 +/- 1.16 (S.E.) nM, suggests a high affinity in acylphosphatase for the phosphoenzyme intermediate, which is consistent with the small number of Ca(2+)-ATPase units in human erythrocyte membrane. Acylphosphatase addition to red cell membranes resulted in a significant increase in the rate of ATP hydrolysis. Maximal stimulation (about 2-fold over basal) was obtained at 2 units/mg membrane protein, with a concomitant decrease in apparent Km values for both Ca2+ and ATP. Conversely, similar amounts of acylphosphatase significantly decreased (by about 30%) the rate of Ca2+ transport into inside-out red cell membrane vesicles, albeit that reduced apparent Km values for Ca2+ and ATP were also observed in this case. A stoichiometry of 2.04 Ca2+/ATP hydrolyzed was calculated in the absence of acylphosphatase; in the presence of acylphosphatase optimal concentration, this ratio was reduced to 0.9. Acylphosphatase activity, rather than just protein, was essential for all the above effects. Taken together these findings suggest that, because of its hydrolytic activity on the phosphoenzyme intermediate, acylphosphatase reduces the efficiency of the erythrocyte membrane Ca2+ pump. A possible mechanism for this effect is that the phosphoenzyme is hydrolyzed before its transport work can be accomplished.
Nassi
P
P
Dipartimento di Scienze Biochimiche, Università di Firenze, Florence, Italy.
Nediani
C
C
Liguri
G
G
Taddei
N
N
Ramponi
G
G
D017766:Acid Anhydride Hydrolases; D001693:Biological Transport, Active; D002118:Calcium / Q000097:blood; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000302:isolation & purification; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000097:blood* / Q000494:pharmacology; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D002118:Calcium; D000252:Calcium-Transporting ATPases; D010744:Phosphoric Monoester Hydrolases; D000097024:Acylphosphatase