Solubilized monomeric sarcoplasmic reticulum Ca pump protein

Abstract

Phosphorylation (by inorganic phosphate) of sarcoplasmic reticulum Ca pump protein has been studied in a detergent solution in which the protein has been previously shown to exist as a monomer. The course of the reaction is qualitatively similar to that observed for membrane-bound (possibly oligomeric) protein.In particular, the results indicate that alternation between the two principal conformational states of the Ca pump protein persists in the monomeric state, which suggests that the machinery for coupling of ATP hydrolysis to Ca *+ transport is intact. There are quantitative differences between monomeric and membrane-bound protein with respect to phosphorylation, but they are not necessarily related to the state of association.

A model for the phosphorylation of the Ca2+ + Mg2+-activated ATPase by phosphate

“…It was necessary to assume that binding of Mg2+ to these sites was not affected by pH, since otherwise binding constants that give inhibition at pH 6.2 gave too large inhibitions at pH 8.0. Even so, it was impossible to simulate the large decrease in phosphorylation levels reported to occur at the highest concentrations of Mg2+ (Loomis et al, 1982;Martin & Tanford, 1984) without obtaining inhibitions inconsistent with the data of Punzengruber et al Inesi et al (1984). However, since Loomis et al (1982) found that up to 40% of the binding of [32P]phosphate was non-specific at high Mg2+ concentrations, we believe the simulations to be acceptable.…”

“…2 and 3), at high Mg2+ concentrations these levels fall. This has been attributed to the binding of Mg2+ to the Ca2+-binding sites of the El conformation of the ATPase (Loomis et al, 1982;Martin & Tanford, 1984). Fig.…”

“…= 3.3 nmol/mg of protein. The experimental data from Martin & Tanford (1984) show the fractional equilibrium level of phosphorylation 1979) show the equilibrium level of phosphoenzyme (nmol/mg of protein) as a function of free phosphate concentration at pH 7 for Ca2+-loaded vesicles at a free Mg2+ concentration of 10 mm (A) and for non-loaded vesicles at free Mg2+ concentrations of 20 mm (A), 10 mm (O) and 5 mm (*). The curves are simulations with the parameters in Table 1 and [EP]max = 4.0 nmol/mg of protein and, for the Ca2+-loaded vesicles, [Ca2+1int.…”

“…Fig. 4 shows a fit to the experimental data of Martin & Tanford (1984) in terms of such a model, with the parameters listed in Table 2. It was necessary to assume that binding of Mg2+ to these sites was not affected by pH, since otherwise binding constants that give inhibition at pH 6.2 gave too large inhibitions at pH 8.0.…”

Direct demonstration of structural changes in soluble, monomeric Ca2+-ATPase associated with Ca2+ release during the transport cycle.

“…On the other hand, structural studies suggest that the Ca2+-ATPase peptides may form dimeric or higher oligomeric complexes in the membrane, which could be the minimal transport units (5)(6)(7). In favor of the view that the functional unit in Ca2+ transport is a monomeric peptide (8) are findings that show that it is possible to prepare soluble monomeric Ca2+-ATPase in detergent with retention of important functional properties, such as ATP hydrolysis (3) accompanied by release of Ca2+ from the two high-affinity transport sites (9,10) and ability to catalyze resynthesis of ATP from ADP and Pi after a "jump" in Ca2+ concentration (10)(11)(12). However, based on kinetic evidence obtained with soluble preparations of oligomeric and monomeric Ca2+-ATPase, it has been argued that peptide-peptide interactions are required for coupling between the ATPase reaction and Ca2+ translocation (13)(14)(15).…”