CrATP-induced Ca2+ occlusion in mutants of the Ca(2+)-ATPase of sarcoplasmic reticulum.
Use of the nonphosphorylating beta,gamma-bidentate chromium(III) complex of ATP to induce a stable Ca(2+)-occluded form of the sarcoplasmic reticulum Ca(2+)-ATPase was combined with molecular sieve high performance liquid chromatography of detergent-solubilized protein to examine the ability of the Ca(2+)-ATPase mutants Gly-233-->Glu, Gly-233-->Val, Glu-309-->Gln, Gly-310-->Pro, Pro-312-->Ala, Ile-315-->Arg, Leu-319-->Arg, Asp-703-->Ala, Gly-770-->Ala, Glu-771-->Gln, Asp-800-->Asn, and Gly-801-->Val to occlude Ca2+. This provided a new approach to identification of amino acid residues involved in Ca2+ binding and in the closure of the gates to the Ca2+ binding pocket of the Ca(2+)-ATPase. The "phosphorylation-negative" mutant Asp-703-->Ala and mutants of ADP-sensitive phosphoenzyme intermediate type were fully capable of occluding Ca2+, as was the mutant Gly-770-->Ala. Mutants in which carboxylic acid-containing residues in the putative transmembrane segments had been substituted ("Ca(2+)-site mutants") and mutant Gly-801-->Val were unable to occlude either of the two calcium ions. In addition, the mutant Gly-310-->Pro, previously classified as ADP-insensitive phosphoenzyme intermediate type (Andersen, J.P., Vilsen, B., and MacLennan, D.H. (1992). J. Biol. Chem. 267, 2767-2774), was unable to occlude Ca2+, even though Ca(2+)-activated phosphorylation from MgATP took place in this mutant.
Vilsen
B
B
Danish Biomembrane Research Centre, Institute of Physiology, University of Aarhus.
Andersen
J P
JP
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D009132:Muscles / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014162:Transfection
D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium