Abstract

In the proposed study we plan to investigate further the structural and mechanistic features of calcium transport carried out by the ATPase of sarcoplasmic reticulum membranes. The two principal approaches will utilize (1) non-covalent (reversibly bound) probes, such as ATP analogs, and (2) covalent (irreversibly bound) modification, primarily directed towards specific labeling of the nucleotide sites. Determination of the nature and extent of interactions will be done primarily by spectrophotometric, spectrofluorimetric, and enzyme activity measurements. Among the specific parameters to be obtained are stoichiometric, binding strength, and cooperativity constants associated with nucleotide and calcium binding and how these change during the calcium transport cycle; and distances between sites (measured by fluorescence energy transfer). From covalent modification experiments, we expect to identify further the functional groups which participate in substrate binding and catalysis, and to detect protein conformation changes which occur during the calcium transport cycle.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM031083-04A3
Application #
3278995
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1982-09-01
Project End
1993-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
4
Fiscal Year
1988
Total Cost
Indirect Cost

  Institution

Name
University of the Pacific-San Francisco
Department
Type
Schools of Dentistry/Oral Hygn
DUNS #
City
San Francisco
State
CA
Country
United States
Zip Code
94115

  Publications

Murphy, A J; Coll, R J (1993) Formation of a stable inactive complex of the sarcoplasmic reticulum calcium ATPase with magnesium, beryllium, and fluoride. J Biol Chem 268:23307-10
Murphy, A J; Coll, R J (1992) Fluoride binding to the calcium ATPase of sarcoplasmic reticulum converts its transport sites to a low affinity, lumen-facing form. J Biol Chem 267:16990-4
Murphy, A J; Hoover, J C (1992) Inhibition of the Na,K-ATPase by fluoride. Parallels with its inhibition of the sarcoplasmic reticulum CaATPase. J Biol Chem 267:16995-700
Highsmith, S; Murphy, A J (1992) Electrostatic changes at the actomyosin-subfragment 1 interface during force-generating reactions. Biochemistry 31:385-9
Coll, R J; Murphy, A J (1992) Fluoride-inhibited calcium ATPase of sarcoplasmic reticulum. Magnesium and fluoride stoichiometry. J Biol Chem 267:21584-7
Murphy, A J; Coll, R J (1992) Fluoride is a slow, tight-binding inhibitor of the calcium ATPase of sarcoplasmic reticulum. J Biol Chem 267:5229-35
Coll, R J; Murphy, A J (1991) Kinetic evidence for two nucleotide binding sites on the CaATPase of sarcoplasmic reticulum. Biochemistry 30:1456-61
Murphy, A J (1990) Reaction of a carbodiimide adduct of ATP at the active site of sarcoplasmic reticulum calcium ATPase. Biochemistry 29:11236-42
Murphy, A J (1990) Inactivation of Ca2(+)-, Na+K(+)-, and H+K(+)-ATPases with a carbodiimide derivative of ATP. FEBS Lett 263:175-7
Rubtsov, A M; Murphy, A J (1988) Caffeine interaction with the Ca-release channels of heavy sarcoplasmic reticulum. Evidence that 170 kD Ca-binding protein is a caffeine receptor of the Ca-channels. Biochem Biophys Res Commun 154:462-8

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