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

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 -141. Vanadate binding is reduced with rising pressure at first markedly and subsequently, above 30 MPa, relatively little. The biphasic pressurebinding 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 . rno1-l depending on the composition of the reaction medium containing 5 pM and 20 pM vanadate and no or 15% (by vol.) MezSO. The binding volumes deduced for the higher pressure range amount to 20-40 ml . mol-l. Vanadate binding is reduced in the presence of 30 pM calcium, and simultaneously both binding volumes are diminished by 100 ml . mol-' and 20 ml . mol-' for the low and high pressure values, respectively, as one can expect for mutual interactions between the two ligands of the transport enzyme.In recent studies we tried to monitor structural changes of the sarcoplasmic reticulum calcium pump connected with its transport cycle [l -31. We determined reaction volume changes by measuring the effect of pressure on the calciumdependent hyrolysis of two ATP substrate analogues, 4-nitrophenyl phosphate and dinitrophenyl phosphate. The intention of these studies was to find out if the transition between the well-characterized reaction intermediates could be related to the kinetically derived volume changes. Two characteristic reaction volumes were obtained. We assigned a small volume change of approximately + 20 ml . mol-' to the slow transition between the phosphate-free intermediates Ez and El and a large one of 80-100 ml . rno1-l to the transition of EIPCa to E2P which is connected to ion translocation.Since the resulting volume changes were obtained from the effect of pressure on the reaction rates of the forward running pump, only activation volumes could be derived. Their values cannot be directly related to net volume changes connected with the transition between corresponding reaction intermediates.To determine real volume changes unambiguosly it is necessary to measure the effect of pressure on equilibria between the intermediates under consideration. As a reaction equilibrium which can most simply be established we considered to study phosphorylation of the calcium-free protein Ez which results in the reaction intermediate EzP having a low calcium affinity. In the past, formation and decay of this intermediate could quite easily be studied due to its acid Correspondence to W. Hasselbach, Max-Planck-lnstitut fur Medizinische Fo...

Cardioprotective Effect of Vanadyl Sulfate on Ischemia/Reperfusion‐Induced Injury in Rat Heart In vivo Is Mediated by Activation of Protein Kinase B and Induction of FLICE‐Inhibitory Protein

“…Several studies have showed that prolonged treatment with vanadyl sulfate has no toxic effects (Cam et al 1993; Mongold et al 1990), whereas orthovanadate was toxic because of inhibition of growth‐factor‐induced protein synthesis (VinA als et al, 2001). Vanadate is a known inhibitor of a number of phosphatases (Nechay et al 1986) including Ca 2+ ‐ATPases (Ronzani et al 1991), CF1‐ATPase (Carmeli et al 1992), Na + /K + ‐ATPase (Cantley et al 1977), and H + /K + ‐ATPase (Dafnis et al 1992) and oral administration has a large toxic effect on the rat myocardium (Pytkowski and Jagodzinska‐Hamann 1996). The vanadyl ion is less toxic than the vanadate ion, as judged by the LD 50 values in several animals (Llobet and Domingo 1984; Waters 1977), and the vanadyl state is likely the therapeutically active form of vanadium in cells (Nakai et al 1995; Sakurai et al 1990).…”

Pressure-induced dissociation of fluorescein from the anti-fluorescein single-chain antibody 4-4-20

“…0006-2960/93/0432-10929S04.00/0 © 1993 American Chemical Society Despite broad interest in the use of SCA, little has been done on characterization of the molecular basis of antigen binding by SCA molecules as well as on definition of thermodynamic parameters involved in this interaction. Hydrostatic pressure is now established as a valuable tool in the study of interactions between protein subunits (Paladini & Weber, 1981;Thompson & Lakowicz, 1984; Silva et al, 1986;Coelho-Sampaio et al, 1991) and between proteins and ligands (Li et al, 1976a; Masson & Balny, 1990; Ronzani et al, 1991). Using hydrostatic pressure, it is possible to derive information regarding the thermodynamics of biological processes under isothermal conditions (Weber & Drickamer, 1983;Weber, 1987Weber, , 1992.…”