Pressure effects on the binding of vanadate to the sarcoplasmic reticulum calcium‐transport enzyme

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Abstract
The effect which hydrostatic pressure exerts on the binding of vanadate to the calcium-transport enzyme was determined. The recent unavailability of radioactive vanadate prevented direct measurements of vanadate binding. The vanadate-free enzyme fraction was instead monitored by phosphorylating it with ATP according to Medda and Hasselbach [Medda, P. & Hasselbach, W. (1983) Eur. J. Biochem. 137, 7–14]. Vanadate binding is reduced with rising pressure at first markedly and subsequently, above 30 MPa, relatively little. The biphasic pressure-binding relationship was analysed by applying a biexponential fitting procedure to the experimental data. The biphasicity of the pressure-binding relationship indicates that the description of vanadate binding requires at least a two-step reaction sequence. The volume increments which predominate at lower pressure values, range from 200–400 ml · mol−1 depending on the composition of the reaction medium containig 5 μM and 20 μM vanadate and no or 15% (by vol.) Me2SO. The binding volumes deduced for the higher pressure range amount to 20–40 ml · mol−1. Vanadate binding is reduced in the presence of 30 μM calcium, and simultaneously both binding volumes are diminished by 100 ml · mol−1 and 20 ml · mol−1 for the low and high pressure values, respectively, as one can expect for mutual interactions between the two ligands of the transport enzyme

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