“Chemical Modification Reveals Involvement of Different Sites for Nucleotide-Analogs in the Phosphatase Activity of the Red Cell Calcium Pump”

DONNET, C; CARIDE, A.J.; TALGHAM, S; ROSSI, J.P.F.C.

JOURNAL OF MEMBRANE BIOLOGY

Springer Verlag

Lugar: New York; Año: 1998 p. 217 - 224

0022-2631

Abstract. The calcium pump of plasma membranes catalyzesthe hydrolysis of ATP and phosphoric esters likep-nitrophenyl phosphate (pNPP). The latter activity requiresthe presence of ATP and/or calmodulin, and Ca2+[22, 25]. We have studied the effects of nucleotideanaloguesand chemical modifications of nucleotidebinding sites on Ca2+-pNPPase activity. Treatment withfluorescein isothiocyanate (FITC), abolished Ca2+-ATPase and ATP-dependent pNPPase, but affected only45% of the calmodulin-dependent pNPPase activity.The nucleotide analogue eosin-Y had an inhibitoryeffect on calmodulin-dependent pNPPase (Kieosin-Y 4 2mM). FITC treatment increased Kieosin-Y 15 times.Acetylation of lysine residues with N-hydroxysuccinimidylacetate inactivates Ca2+-ATPase by modifying thecatalytic site, and impairs stimulation by modulators bymodifying residues outside this site [9]. Acetylation suppressedthe ATP-dependent pNPPase with biphasic kinetics.ATP or pNPP during acetylation cancels the fastcomponent of inactivation. Acetylation inhibited onlypartially the calmodulin-dependent pNPPase, but neitherATP nor pNPP prevented this inactivation. From theseresults we conclude: (i) ATP-dependent pNPPase dependson binding of ATP to the catalytic site; (ii) thecatalytic site plays no role in calmodulin-dependentpNPPase. The decreased affinity for eosin-Y of theFITC-modified enzyme, suggests that the sites for thesetwo molecules are closely related but not overlapped.Acetimidation of the pump inhibited totally the calmodulin-dependent pNPPase, but only partially the ATPpNPPase.Since calmodulin binds to E1, the E1 conformationor the E2 Û E1 transition would be involvedduring calmodulin-dependent pNPPase activity.