Dissociation of calcium from the phosphorylated calcium-transporting adenosine triphosphatase of sarcoplasmic reticulum: Kinetic equivalence of the calcium ions bound to the phosphorylated enzyme

Journal Article · Tue Nov 26 04:00:00 EST 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00111a019· OSTI ID:5488363

Hanel, A M; Jencks, W P ^[1]

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1. Brandeis Univ., Waltham, MA (United States)

The internalization of {sup 45}Ca by the calcium-transporting ATPase into sarcoplasmic reticulum vesicles from rabbit muscle was measured during a single turnover of the enzyme by using a quench of 7 mM ADP and EGTA. Intact vesicles containing either 10-20 {mu}M or 20 mM Ca{sup 2+} were preincubated with {sup 45}Ca for {approximately}20 s and then mixed with 0.20-0.25 mM ATP and excess EGTA to give 70% phosphorylation of E{sub tot} with the rate constant k = 300 s{sup {minus}1}. The two {sup 45}Ca ions bound to the phosphoenzyme (EP) become insensitive to the quench with ADP as they are internalized in a first-order reaction with a rate constant of k = {approximately}30s{sup {minus}1}. The first and second Ca{sup 2+} ions that bind to the free enzyme were selectively labeled by mixing the enzyme and {sup 45}Ca with excess {sup 40}Ca, or by mixing the enzyme with {sup 40}Ca with {sup 45}Ca, for 50 ms prior to the addition of ATP and EGTA. The internalization of each ion into loaded or empty vesicles follows first-order kinetics with k = {approximately}30{sup {minus}1}; there is no indication of biphasic kinetics or an induction period for the internalization of either Ca{sup 2+} ion. A first-order reaction for the internalization of the individual Ca{sup 2+} ions is consistent with a rate-limiting conformational change of the phosphoenzyme with k{sub c} = 30 s{sup {minus}1}, followed by rapid dissociation of the Ca{sup 2+} ions from separate independent binding sites. Finally, the Ca{sup 2+} ions may exchange their positions rapidly on the phosphoenzyme before dissociating.

• OSTI ID: 5488363
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:47; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ALKALINE EARTH ISOTOPES
ANIMALS
ATP-ASE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CALCIUM 40
CALCIUM 45
CALCIUM ISOTOPES
CELL CONSTITUENTS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ENDOPLASMIC RETICULUM
ENZYMES
EVEN-EVEN NUCLEI
EVEN-ODD NUCLEI
HYDROLASES
INTERMEDIATE MASS NUCLEI
ISOTOPES
KINETICS
LIGHT NUCLEI
MAMMALS
MEMBRANE TRANSPORT
MUSCLES
NUCLEI
ORGANIC COMPOUNDS
PHOSPHOHYDROLASES
PHOSPHORYLATION
PROTEINS
RABBITS
RADIOISOTOPES
REACTION KINETICS
SARCOPLASMIC RETICULUM
STABLE ISOTOPES
VERTEBRATES