ROTATIONAL MOBILITY OF CA2-ATPASE OF SARCOPLASMIC-RETICULUM IN VISCOUS MEDIA()

The rotational diffusion of Ca2+-ATPase [Ca2+,Mg2+-activated ATP phosp hohydrolase E.C. 3.6.1.38] was studied in native sarcoplasmic reticulu m membrane by saturation transfer ESR spectroscopy after covalent labe lling of intramembranous sulfhydryl groups with nitroxyl derivative of maleimide (5-MSL) as a function of sucrose and glycerol in the suspen ding medium. The relative enzymatic activity of sarcoplasmic reticulum was followed by increasing the viscosity of the aqueous phase. The AT P hydrolysing activity of the enzyme decreased differently on adding s ucrose and glycerol. In the case of sucrose the reciprocal of power de pendence of viscosity was observed, whereas for glycerol an exponentia l decay law was obtained, indicating solvent-protein interaction. On i ncreasing the viscosity of the aqueous phase by either sucrose or glyc erol, no changes were observed in the intramembranous viscosity as mea sured using intercalated spin-labelled stearic acid (16-SASL). The eff ective rotational correlation time of the protein was measured, as a m obility parameter, using saturation transfer ESR spectroscopy and foun d to be increased linearly with the viscosity of the sucrose containin g medium and for the extramembranous size a height of 6.8 nm was obtai ned, indicating that approx. 82% of the volume of Ca2+-ATPase protein is external to the sarcoplasmic reticulum. The addition of glycerol pr obably promoted protein-protein interaction, as indicated by the large r changes in rotational diffusion and non-linear viscosity dependence.