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Stimulation of Ca2+ efflux from sarcoplasmic reticulum by preincubation with ATP and inorganic phosphate.

ABSTRACT: Preincubation of sarcoplasmic reticulum with 1 mM-ATP completely inhibits Ca2+ accumulation and stimulates ATPase activity by over 2-fold. This effect of ATP is obtained only when the preincubation is carried out in the presence of Pi, but not with arsenate, chloride or sulphate. The inhibition by ATP of Ca2+ accumulation is pH-dependent, increasing as the pH is increased above 7.5. Inhibition of Ca2+ accumulation is observed on preincubation with ATP, but not with CTP, UTP, GTP, ADP, adenosine 5'-[beta gamma-methylene]triphosphate or adenosine 5'-[beta gamma-imido]triphosphate. The presence of Ca2+, but not Mg2+, during the preincubation, prevents the effect of ATP + Pi on Ca2+ accumulation. The ATP + Pi inhibition of Ca2+ accumulation is not due to modification of the ATPase catalytic cycle, but rather to stimulation of a rapid Ca2+ efflux from actively or passively loaded vesicles. This Ca2+ efflux is inhibited by dicyclohexylcarbodi-imide. Photoaffinity labelling of sarcoplasmic-reticulum membranes with 8-azido-[alpha-32P]ATP resulted in specific labelling of two proteins, of approx. 160 and 44 kDa. These proteins were labelled in the presence of Pi, but not other anions.

SUBMITTER: Shoshan-Barmatz V

PROVIDER: S-EPMC1148441 | biostudies-other | 1987 Nov

REPOSITORIES: biostudies-other

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Interactions of phosphate groups of ATP and Aspartyl phosphate with the sarcoplasmic reticulum Ca2+-ATPase: an FTIR study.

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Correlation between Ca2+ uptake, Ca2+ efflux and phosphoenzyme level in sarcoplasmic-reticulum vesicles.

Project description:Previously [Inesi & de Meis (1989) J. Biol. Chem. 264, 5929-5936] it was shown that dimethyl sulphoxide (Me2SO) increases the amount of Ca2+ accumulated by sarcoplasmic-reticulum vesicles. This effect was related to a decrease in the enzyme affinity for ADP from less than 20 microM to 1 mM. In the present work the apparent affinity of the ADP-sensitive phosphoenzyme for ADP was determined by measuring the rate of ATP synthesis in vesicles previously loaded with Ca2+, at different pH values and in the presence and absence of Me2SO (20%, v/v) and KCl. In all conditions tested, addition of Me2SO never promoted an increase of the apparent Km for ADP to a value higher than 25 microM. ADP inhibits the phosphorylation of the enzyme by Pi. Two components, with Ki values of 80 microM and 8 mM, were detected when vesicles previously loaded with Ca2+ were used. The high-affinity component (Ki 80 microM) was abolished after addition of Me2SO to the medium. Empty vesicles, on the other hand, exhibited only the low-affinity component (Ki 8 mM). During ATP synthesis in a totally aqueous medium, there was a decrease in the phosphoenzyme formed by Pi, after addition of 80-100 microM-ADP to the medium. In the presence of Me2SO this decrease was smaller. The sum of the fractions of phosphoenzyme formed by ATP and by Pi during Ca2+ uptake was higher in the presence of Me2SO than in a totally aqueous medium. Me2SO decreased the passive efflux of Ca2+ measured in the presence of 0.1 mM-Pi and 0.1 mM-MgCl2. In a totally aqueous medium the same decrease was observed when Pi and MgCl2 concentrations were raised to 4 mM. These data suggest that ADP binds to two different enzyme conformations. The binding to one of these conformations (*E) inhibits the phosphorylation of the enzyme by Pi, increases the efflux of Ca2+ and decreases the amount of Ca2+ accumulated by the vesicles.

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