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The Journal of membrane biology

Chemical modification reveals involvement of different sites for nucleotide analogues in the phosphatase activity of the red cell calcium pump.


PMID 9625778

Abstract

The calcium pump of plasma membranes catalyzes the hydrolysis of ATP and phosphoric esters like p-nitrophenyl phosphate (pNPP). The latter activity requires the presence of ATP and/or calmodulin, and Ca2+ [22, 25]. We have studied the effects of nucleotide-analogues and chemical modifications of nucleotide binding sites on Ca2+-pNPPase activity. Treatment with fluorescein isothiocyanate (FITC), abolished Ca2+-ATPase and ATP-dependent pNPPase, but affected only 45% of the calmodulin-dependent pNPPase activity. The nucleotide analogue eosin-Y had an inhibitory effect on calmodulin-dependent pNPPase (Kieosin-Y = 2 microM). FITC treatment increased Kieosin-Y 15 times. Acetylation of lysine residues with N-hydroxysuccinimidyl acetate inactivates Ca2+-ATPase by modifying the catalytic site, and impairs stimulation by modulators by modifying residues outside this site [9]. Acetylation suppressed the ATP-dependent pNPPase with biphasic kinetics. ATP or pNPP during acetylation cancels the fast component of inactivation. Acetylation inhibited only partially the calmodulin-dependent pNPPase, but neither ATP nor pNPP prevented this inactivation. From these results we conclude: (i) ATP-dependent pNPPase depends on binding of ATP to the catalytic site; (ii) the catalytic site plays no role in calmodulin-dependent pNPPase. The decreased affinity for eosin-Y of the FITC-modified enzyme, suggests that the sites for these two molecules are closely related but not overlapped. Acetimidation of the pump inhibited totally the calmodulin-dependent pNPPase, but only partially the ATP-pNPPase. Since calmodulin binds to E1, the E1 conformation or the E2 if E1 transition would be involved during calmodulin-dependent pNPPase activity.

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