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

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.