Differential reactivity of lysine residues of the red blood cell Ca2+ pump involved in the E1-E2 conformational equilibrium.
1. Modification of Lys residues of the Ca(2+)-ATPase from human red blood cells with methyl acetimidate (MA) inhibited up to 70% of the Ca(2+)-ATPase activity. Furthermore, calmodulin-activated p-nitrophenyl phosphatase activity was fully inhibited at non-limiting concentrations of MA. 2. Treatment with MA inhibited phosphorylation of the Ca(2+)-ATPase. 3. When the enzyme was treated with 7.2 mM-MA in the presence of 100 microM-Ca2+, Ca(2+)-ATPase activity was decreased by 33%, whereas when the membranes were treated with MA in the presence of 50 microM-VO4(3-), this activity was decreased by only 8%. 4. When membranes were either proteolysed or preincubated with 1 mM-Ca2+, MA quickly inactivated the Ca(2+)-ATPase (k = 1.2 min-1). On the other hand, inactivation of membranes preincubated in the absence of Ca2+ and Mg2+ was slow (k = 0.08 min-1). 5. When the activity was measured in the absence of calmodulin, MA decreased to the same extent the values of KCa (the apparent dissociation constant for Ca2+) and Vmax, but in the presence of calmodulin the treatment decreased Vmax. only. 6. The results are consistent with the idea that MA reacts readily with the Ca(2+)-ATPase when the enzyme is in an E1 conformation, but not an E2 conformation, and that, reciprocally, treatment of the enzyme with MA shifts the enzyme to E1. 7. Provided that Ca2+ is present, ATP, with low apparent affinity (K0.5 = 195 microM), protected against inactivation by MA. However, MA treatment did not change the Km values of either the high-affinity or the low-affinity site for ATP, suggesting that protection results from a shift to a conformation in which the Lys residues are inaccessible to MA.
Donnet
C
C
Instituto de Química y Fisicoquímica Biólogicas, (UBA-CONICET), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
Caride
A J
AJ
Fernández
H N
HN
Rossi
J P
JP
D000255:Adenosine Triphosphate / Q000378:metabolism; D000328:Adult; D001345:Autoradiography; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007096:Imidoesters / Q000494:pharmacology; D008239:Lysine / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation
D007096:Imidoesters; D000255:Adenosine Triphosphate; C010222:methyl acetimidate; D010744:Phosphoric Monoester Hydrolases; D000252:Calcium-Transporting ATPases; D008239:Lysine