Mechanism for activation of the 4-nitrobenzo-2-oxa-1,3-diazole-labeled sarcoplasmic reticulum ATPase by Ca sup 2+ and its modulation by nucleotides

Journal Article · Tue Aug 07 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00483a022· OSTI ID:6098593

Wakabayashi, Shigeo; Shigekawa, Munekazu ^[1]

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1. National Cardiovascular Center Research Institute, Osaka (Japan)

The mechanism for activation of sarcoplasmic reticulum ATPase by Ca{sup 2+} was investigated in 2 mM MgCl{sub 2} and 0.1 M KCl at pH 6.5 and 11{degree}C by using enzyme preparations in which a specific amino acid residue (Cys-344) was labeled with 4-nitrobenzo-2-oxa-1,3-diazole (NBD). The authors compared the kinetics of binding and dissociation of Ca{sup 2+} from the enzyme with those of the accompanying NBD fluorescence changes. The fluorescence rise following addition of Ca{sup 2+} proceeded monoexponentially. At 2-100 {mu}M Ca{sup 2+} and in the absence of nucleotides, the Ca{sup 2+}-induced fluorescence rise and Ca{sup 2+} binding to the enzyme proceeded at similar rates, which were almost independent of the Ca{sup 2+} concentration. ATP by binding at 1 mol/mol of the phosphorylation site markedly accelerated both the Ca{sup 2+}-induced fluorescence rise and Ca{sup 2+} binding, ADP and AMPPNP but not GTP also being effective. These data are consistent with a reaction model in which binding of Ca{sup 2+} occurs after the conformational transition of the free enzyme from a state (E{sub 2}) having low affinity for Ca{sup 2+} to one (E{sub 1}) having high affinity for Ca{sup 2+} and in which ATP bound at the catalytic site of E{sub 2}, whose affinity for ATP is about 30-fold less than that of E{sub 1}, accelerates this conformational transition.

• OSTI ID: 6098593
• Journal Information: Biochemistry; (USA), Vol. 29:31; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ATP-ASE
ENZYME INDUCTION
CALCIUM 45
CROSS-LINKING
NUCLEOTIDES
BIOLOGICAL EFFECTS
BIOCHEMICAL REACTION KINETICS
CONFORMATIONAL CHANGES
FLUORESCENCE
SARCOPLASMIC RETICULUM
ACID ANHYDRASES
ALKALINE EARTH ISOTOPES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CALCIUM ISOTOPES
CELL CONSTITUENTS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ENDOPLASMIC RETICULUM
ENZYMES
EVEN-ODD NUCLEI
GENE REGULATION
HYDROLASES
INTERMEDIATE MASS NUCLEI
ISOTOPES
KINETICS
LUMINESCENCE
NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
PHOSPHOHYDROLASES
POLYMERIZATION
RADIOISOTOPES
REACTION KINETICS
550201* - Biochemistry- Tracer Techniques