Regulation of the erythrocyte Ca2(+)-ATPase by mutant calmodulins with positively charged amino acid substitutions.
Four mutant calmodulins with site-specific charge alterations have been used to activate the human erythrocyte Ca2(+)-ATPase. These charge alterations were accomplished either by insertion of new Lys residues or by substitution of Lys residues for Glu in two of the seven calmodulin alpha-helices. Two enzyme preparations, purified monomeric Ca2(+)-ATPase and erythrocyte ghost membranes, were used with comparable results. At 100 nM Ca2+, the Ca2(+)-ATPase activity was lowered significantly by charge reversal from negative to positive in both the central alpha-helix and the carboxy-terminal domain. While all mutant calmodulins with charge reversal ultimately stimulated the Ca2(+)-ATPase activity to the same extent, the concentration of mutant calmodulin required for half-maximal activation was from 36-fold (central alpha-helix) to 126-fold higher (alpha-helix in the carboxy-terminal domain) than that of the control calmodulin. There was also a significant difference in the stimulation of Ca2(+)-ATPase activity by the different mutant calmodulins as a function of Ca2+ concentration, being most pronounced at submicromolar Ca2+ concentrations where enzyme activation by calmodulin appears to be a physiologically relevant mechanism. In contrast to the mutant calmodulins with charge reversal, mutant calmodulins in which two positive charges were added in the central alpha-helix activated the Ca2(+)-ATPase in a way undistinguishable from the control calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)
Kosk-Kosicka
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D
Department of Biological Chemistry, University of Maryland, School of Medicine, Baltimore 21201.
Bzdega
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T
D000595:Amino Acid Sequence; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000302:isolation & purification; D002147:Calmodulin / Q000235:genetics / Q000494:pharmacology*; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D014336:Troponin / Q000494:pharmacology; D020107:Troponin T
D002147:Calmodulin; D014336:Troponin; D020107:Troponin T; D000252:Calcium-Transporting ATPases