Linked Life Data

10913287

Source:http://linkedlifedata.com/resource/pubmed/id/10913287

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
30
pubmed:dateCreated
2000-8-24
pubmed:abstractText
Expression of heterologous SERCA1a ATPase in Cos-1 cells was optimized to yield levels that account for 10-15% of the microsomal protein, as revealed by protein staining on electrophoretic gels. This high level of expression significantly improved our characterization of mutants, including direct measurements of Ca(2+) binding by the ATPase in the absence of ATP, and measurements of various enzyme functions in the presence of ATP or P(i). Mutational analysis distinguished two groups of amino acids within the transmembrane domain: The first group includes Glu771 (M5), Thr799 (M6), Asp800 (M6), and Glu908 (M8), whose individual mutations totally inhibit binding of the two Ca(2+) required for activation of one ATPase molecule. The second group includes Glu309 (M4) and Asn796 (M6), whose individual or combined mutations inhibit binding of only one and the same Ca(2+). The effects of mutations of these amino acids were interpreted in the light of recent information on the ATPase high-resolution structure, explaining the mechanism of Ca(2+) binding and catalytic activation in terms of two cooperative sites. The Glu771, Thr799, and Asp800 side chains contribute prominently to site 1, together with less prominent contributions by Asn768 and Glu908. The Glu309, Asn796, and Asp800 side chains, as well as the Ala305 (and possibly Val304 and Ile307) carbonyl oxygen, contribute to site 2. Sequential binding begins with Ca(2+) occupancy of site 1, followed by transition to a conformation (E') sensitive to Ca(2+) inhibition of enzyme phosphorylation by P(i), but still unable to utilize ATP. The E' conformation accepts the second Ca(2+) on site 2, producing then a conformation (E' ') which is able to utilize ATP. Mutations of residues (Asp813 and Asp818) in the M6/M7 loop reduce Ca(2+) affinity and catalytic turnover, suggesting a strong influence of this loop on the correct positioning of the M6 helix. Mutation of Asp351 (at the catalytic site within the cytosolic domain) produces total inhibition of ATP utilization and enzyme phosphorylation by P(i), without a significant effect on Ca(2+) binding.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
1
pubmed:volume
39
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
8758-67
pubmed:dateRevised
2010-11-18
pubmed:meshHeading
pubmed-meshheading:10913287-Adenosine Triphosphate, pubmed-meshheading:10913287-Animals, pubmed-meshheading:10913287-COS Cells, pubmed-meshheading:10913287-Calcium, pubmed-meshheading:10913287-Calcium-Transporting ATPases, pubmed-meshheading:10913287-Catalysis, pubmed-meshheading:10913287-Chickens, pubmed-meshheading:10913287-DNA, Complementary, pubmed-meshheading:10913287-Enzyme Activation, pubmed-meshheading:10913287-Humans, pubmed-meshheading:10913287-Muscle Fibers, Fast-Twitch, pubmed-meshheading:10913287-Mutagenesis, Site-Directed, pubmed-meshheading:10913287-Phosphates, pubmed-meshheading:10913287-Phosphorylation, pubmed-meshheading:10913287-Rabbits, pubmed-meshheading:10913287-Sarcoplasmic Reticulum Calcium-Transporting ATPases, pubmed-meshheading:10913287-Structure-Activity Relationship, pubmed-meshheading:10913287-Transfection
pubmed:year
2000
pubmed:articleTitle
Detailed characterization of the cooperative mechanism of Ca(2+) binding and catalytic activation in the Ca(2+) transport (SERCA) ATPase.
pubmed:affiliation
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't