Two distinct classes of nucleotide binding sites in sarcoplasmic reticulum Ca-ATPase revealed by 2‘,3‘-O-(2,4,6-trinitrocyclohexadienylidene)-ATP.

Y Dupont,R Pougeois,M Ronjat,S Verjovsky-Almeida

doi:10.1016/s0021-9258(17)39599-6

Abstract

It was previously reported that 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) (TNP)-nucleotides bind with high affinity to the sarcoplasmic reticulum Ca-ATPase (Dupont, Y., Chapron, Y., and Pougeois, R. (1982) Biochem. Biophys. Res. Commun. 106, 1272-1279 and Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). Here we report a study of the Ca-ATPase nucleotide binding sites using TNP-nucleotides. Competition at equilibrium between TNP-nucleotides and ATP was measured in the absence of calcium; it was found that TNP-nucleotides and ATP competitively bind to two classes of sites of equal concentration (3.5 nmol/mg). The ATP dissociation constants for the two classes of sites were found to be sensitive to H+ and Mg2+ concentrations. In the absence of Mg2+ (independently of pH) or at acid pH (independently of Mg2+ concentration), the nucleotide sites behave like one single family of sites of intermediate affinity (Kd = 20 microM). They split into two classes of sites of high (Kd = 2-4 microM) and low (Kd greater than 1 mM) affinity at pH values higher than neutral and in the presence of Mg2+. The calcium-activated ATP hydrolysis is accelerated by TNP-ATP (or TNP-AMP-PNP) binding on the phosphorylated enzyme. It is concluded 1) that the Ca-ATPase enzyme possesses two classes of ATP binding sites, 2) that the affinity of these two sites and the nature of their interaction is modulated by the H+ and Mg2+ concentrations, and 3) that the hydrolytic activity of the high affinity ATP binding site is activated by ATP or TNP-AMP-PNP (or TNP-ATP) binding in a low affinity ATP binding site.

Highlights

The affinity for TNP-AMP-PNP was not evaluated with accuracy in this series of experiments, but it was around 0.2 p ~a v, alue not very different from that found in our previous studies with TNP-ATP or TNP-AD(PK d = 50-150 nM).This is in contrast with Watanabe andInesi (17) who reported two families of binding sites of much higher dissociation constants: 2 and 160 pM, respectively
It is concluded 1) that the CaATPase enzyme possesses two classes of ATP binding sites, 2) that the affinity of these two sites and the nature of their interactionis modulated by the H+and M&+ concentrations, and 3)that the hydrolytic activity of the high affinity ATP binding site is activated by ATP or TNP-AMP-PNP bindingin a low affinity ATP bindinsgite
Dupont et al (16) and Watanabe and Inesi (17) reported that the Ca-ATPase vesicles present a slow TNP-ATP magnesium-dependent hydrolytic activity and that thiasctivity is calcium-independent (17)or only slightly activated by calcium (16)

Summary

Introduction

The affinity for TNP-AMP-PNP was not evaluated with accuracy in this series of experiments, but it was around 0.2 p ~a v, alue not very different from that found in our previous studies with TNP-ATP or TNP-AD(PK d = 50-150 nM).This is in contrast with Watanabe andInesi (17) who reported two families of binding sites of much higher dissociation constants: 2 and 160 pM, respectively.

Results

Conclusion