Open AccessThe Effects of ADP, Phosphate and Arsenate on Ca Efflux from Sarcoplasmic Reticulum Vesicles
Author(s) -
PICK Uri,
BASSILIAN Sara
Publication year - 1983
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1983.tb07276.x
Subject(s) - arsenate , chemistry , efflux , endoplasmic reticulum , atpase , phosphate , vesicle , dissociation constant , biochemistry , biophysics , membrane , arsenic , enzyme , biology , receptor , organic chemistry
1 The effects of ADP, phosphate and arsenate on Ca efflux from Ca‐loaded sarcoplasmic reticulum vesicles have been investigated by utilizing a separation step of the Ca‐loaded vesicles from the loading medium on Sephadex columns. 2 ADP and phosphate [Barlogie, B., et al. (1971) FEBS Lett. 12 , 267–268], or arsenate in the absence of ADP [Hasselbach, W., et al. (1972) FEBS Lett. 20 , 311–315] stimulate Ca efflux. The arsenate‐stimulated Ca efflux is optimally activated at pH 6 and the apparent arsenate dissociation constant drops from 0.6 mM at pH 6 to 5 mM at pH 8. 3 ADP has a dual effect on Ca efflux: μM ADP concentrations stimulate Ca efflux in the presence of inorganic phosphate [ K d (ADP) = 3–5 μM], whereas high ADP concentrations inhibit both arsenate and phosphate stimulated Ca‐efflux. The inhibition is non‐competitive with respect to either phosphate or arsenate and the calculated ADP dissociation constants are 180 μM and 200 μM respectively. 4 The phosphorylation of the sarcoplasmic reticulum Ca‐ATPase by inorganic phosphate is competitively inhibited by arsenate [ K d (As i ) = 1.8 mM] and is non‐competitively inhibited by ADP [ K d (ADP) = 120 μM]. 5 Modification of the ATP binding site of the sarcoplasmic reticulum Ca‐ATPase by fluorescein isothiocyanate [Pick, U. (1981) Eur. J. Biochem. 121 , 187–195] prevents both the stimulation of Ca efflux by low ADP concentration and the inhibition of arsenate‐stimulated Ca efflux by high ADP concentrations. 6 The molecular mechanism of action of arsenate and ADP are discussed. It is proposed that the mechanism of arsenate‐stimulated Ca efflux involves the hydrolysis of an arsenylated intermediate containing occluded Ca ions. It is also suggested that ADP inhibits Ca efflux by binding to the E 2 conformational state of the enzyme (E 2 –the low‐affinity Ca‐binding state of the Ca‐ATPase). The results are consistent with a single ATP binding site existing in two interconvertible configurations in the sarcoplasmic reticulum Ca‐ATPase [Pick, U. (1981) Eur. J. Biochem. 121 , 187–195].