[Study of Ca-dependent ATPase of sarcoplasmic reticulum by the use of selectively bound spin labels]

The dependence of the ESR spectral parameters of spin labelled sarcoplasmic reticulum membranes on the amount of bound maleimide spin label is found which is shown to result from non-equivalence of the different SH groups rather than from enzyme activity changes on modification. Some structural characteristics (local polarity, the accessibility to water soluble reagents, the distance from ATP binding site) of the fast and slowly reacting SH groups were studied using selective labelling technique. The main contribution to the ESR absorption for both types of thiol groups gives the spin labelled Ca-ATPase. The slow rotational mobility of both types of strongly immobilized spin labels is measured using microwave saturation technique. This mobility is shown to be due essentially to intramolecular motion in different regions of the Ca-ATPase. The frequency of this motion correlates with the enzyme activity and it is sensitive also to the interaction of the enzyme with the substrates (ATP, acetylphosphate) and to the changes of lipid-protein interaction by temperature variation.