title: Functional consequences of alterations to Gly310, Gly770, and Gly801 located in the transmembrane domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.
abstract: Site-specific mutagenesis was used to replace Gly310, Gly770, and Gly801, located in the transmembrane domain of the sarcoplasmic reticulum Ca(2+)-ATPase, with either alanine or valine. In addition, Gly310 was substituted with proline. In the Gly310----Ala mutant, the Vmax for Ca2+ transport and ATPase activity was reduced to about 40% of the wild type activity, but the apparent Ca2+ affinity was close to normal. The Gly310----Val and Gly310----Pro mutants were devoid of Ca2+ transport or ATPase activity and displayed more than a 20-fold reduction in the apparent Ca2+ affinities measured in the phosphorylation assays with either ATP or Pi. In these mutants, the rate of phosphoenzyme hydrolysis was reduced, and the ADP-insensitive phosphoenzyme intermediate accumulated. The apparent affinity for Pi was increased in the absence, but not in the presence, of dimethyl sulfoxide. The properties of this new class of Ca(2+)-ATPase mutants ("E2/E2P" type) are consistent with a conformational state in which the protein-phosphate interaction is stabilized and the Ca(2+)-protein interaction is destabilized. The Gly770----Ala mutant transported Ca2+ with a Vmax close to that of the wild type, but displayed more than a 20-fold reduction of apparent Ca2+ affinity. The Gly770----Val mutant was not phosphorylated from either ATP or Pi. The Gly801----Ala mutant transported Ca2+ with a Vmax of 126% that of the wild type, hydrolyzed ATP at the same Vmax as the wild type in the presence of calcium ionophore, and displayed a 3-fold reduction in apparent Ca2+ affinity. The Gly801----Val mutant was unable to transport Ca2+ and to be phosphorylated from ATP, even at a Ca2+ concentration of 1 mM, but Ca2+ in the micromolar range inhibited phosphorylation from Pi. The ability to bind ATP with normal affinity was retained. The properties of this mutant are consistent with a disruption of one of the two Ca2+ binding sites required for phosphorylation with ATP.
authors.affiliation: Danish Biomembrane Research Centre, University of Aarhus.
authors.forename: J P
authors.initials: JP
authors.lastname: Andersen
authors.forename: B
authors.initials: B
authors.lastname: Vilsen
authors.forename: D H
authors.initials: DH
authors.lastname: MacLennan
mesh_terms: D001667:Binding, Competitive; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D005998:Glycine / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
chemical_list: D000252:Calcium-Transporting ATPases; D002118:Calcium; D005998:Glycine