Spin-labelling studies of fragmented sarcoplasmic reticulum

Abstract

Vesicular fragments of sarcoplasmic reticulum (SR) were spin labelled with 2,2,6,6-tetramethyl, 4-isothiocyanate piperidine-1-oxyl (probe “A”) and 2,2,6,6-tetramethyl, 4-amino (N-iodoacetamide) piperidine-1-oxyl (probe “B”). Two to five moles of probe “A” or “B” were covalently bound to 10⁶g of membrane protein, with minimal loss of activity (ATPase, Ca²⁺, uptake). The EPR spectra of labelled SR were then studied in various experimental conditions.

Strongly acid or alkaline pH, protein denaturation with ura, and membrane solubilization with deoxycholate produced marked alterations of the EPR spectra of spin-labelled SR, indicating changes in the local environment surrounding the probes, and the occurrence of conformational changes.

A reversible modification of the EPR spectra of probe “A” and an accelerated efflux of accumulated Ca²⁺ were produced by increasing the temperature of SR suspensions from 30° to 40° C. Such a parallel behavior indicates that reversible structural transitions may control membrane permeability and Ca²⁺ efflux.

ATP modifies the EPR spectra of probe “B”, suggesting that ATP binding to the membrane induces a structural change involving the local environment of certain sulfhydryl groups. The ATP concentration required for this effect is comparable to that requied for activation of ATPase. ADP and ITP are also effective, while pyrophosphate, AMP, and cyclic AMP are not. The effect of ATP is reversible.

In other experiments, 2,2,6,6-tetramethylpiperidine-1-oxyl (probe“C”) was equilibrated with concentrated suspensions of SR. The EPR spectra obtained thereafter indicate that probe “C” binds to the membrane fragments, still maintaining a high degree of motional freedom. These spectra were markedly changed by deoxycholate solubilization of the membrane fragments, while they were little affected by protein denaturation with guanidine. These results confirm the hypothesis that the region of distribution of probe “C” into SR, is prevalently constituted by low-viscosity lipids.