The two calcium ions initially bound to nonphosphorylated sarcoplasmic reticulum Ca sup 2+ -ATPase can no longer be kinetically distinguished when they dissociate from phosphorylated ATPase toward the lumen

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

DOI:https://doi.org/10.1021/bi00111a020· OSTI ID:5488348

Orlowski, S; Champeil, P ^[1]

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1. URA Centre National de la Recherche Scientifique, Gif-sur-Yvette (France)

Using rapid filtration, the authors investigated the kinetics of release toward the lumen of sarcoplasmic reticulum vesicles of the two Ca{sup 2+} ions transported by the Ca{sup 2+}-dependent ATPase of these vesicles. Release rates at 20C were measured by three methods, with vesicles previously made leaky with an ionophore. First, they measured the rate at which {sup 45}Ca{sup 2+} bound to ATPase approached its steady-state level after addition of ATP to the {sup 45}Ca{sup 2+}-equilibrated ATPase. At pH 6 in the absence of potassium, the observed kinetics did not reveal any very fast phase of {sup 45}Ca{sup 2+} dissociation from phosphorylated ATPase. Second, they measured the kinetics of {sup 45}Ca{sup 2+} dissociation from phosphorylated ATPase in a 'chase' experiment, by isotopic dilution of calcium under turnover conditions in the presence of potassium. They found that these kinetics were essentially monophasic. The third method was based on the fact that phosphorylation-induced {sup 45}Ca{sup 2+} occlusion occurred faster than {sup 45}Ca{sup 2+} dissociation from nonphosphorylated ATPase: here, they measured the rate of {sup 45}Ca{sup 2+} internalization on addition to {sup 45}Ca{sup 2+}-saturated ATPase of an unlabeled ATP-containing medium. This method allowed separate observation of the dissociation kinetics of each of the two {sup 45}Ca{sup 2+} ions bound to phosphorylated ATPase, after either one or the other had been labeled by a preliminary partial isotopic exchange in the nonphosphorylated state of the ATPase. The results suggest that, after sequential binding to the nonphosphorylated ATPase, the two Ca{sup 2+} ions transported by the phosphorylated ATPase become kinetically indistinguishable when they are released toward the lumenal side of the SR vesicles.

• OSTI ID: 5488348
• 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
ATP-ASE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOCHEMISTRY
CALCIUM 45
CALCIUM ISOTOPES
CELL CONSTITUENTS
CHEMICAL REACTIONS
CHEMISTRY
DAYS LIVING RADIOISOTOPES
DISSOCIATION
ENDOPLASMIC RETICULUM
ENZYMES
EVEN-ODD NUCLEI
FILTRATION
HYDROLASES
INTERMEDIATE MASS NUCLEI
ISOTOPES
KINETICS
MEMBRANE TRANSPORT
NUCLEI
ORGANIC COMPOUNDS
PH VALUE
PHOSPHOHYDROLASES
PHOSPHORYLATION
PROTEINS
RADIOISOTOPES
REACTION KINETICS
SARCOPLASMIC RETICULUM
SEPARATION PROCESSES