Abstract
In order to investigate the roles of the physical states of phospholipid and protein in the enzymatic behavior of the Ca2+ -ATPase from sarcoplasmic reticulum, we have modified the lipid phase of the enzyme, observed the effects on the enzymatic activity at low temperatures, and correlated these effects with spectroscopic measurements of the rotational motions of both the lipid and protein components. Replacement of the native lipids with dipalmitoyl phosphatidylcholine inhibits ATPase activity and decreases both lipid fluidity, as monitored by EPR spectroscopy on a stearic acid spin label, and protein rotational mobility, as monitored by saturation transfer EPR spectroscopy on the covalently spin-labeled enzyme. Solubilization of the lipid-replaced enzyme with Triton X-100 reverses all three of these effects. Ten millimolar CaCl2 added either to the enzyme associated with the endogenous lipids or to the Triton X-100 soulbilized enzyme inhibits both ATPase activity and protein rotational mobility but has no detectable effect on the lipid mobility. These results are consistent with the proposal that both lipid fluidity and protein rotational mobility are essential for enzymatic activity.
Highlights
From the Department of Muscle Research, Boston Biomedical Research Institute, Department of Neurology, Harvard Medical School, Boston, Massachusetts
In order to investigate the roles of the physical states of phospholipid and protein in the enzymatic behavior of the Ca’+-ATPase from sarcoplasmic reticulum, we have modified the lipid phase of the enzyme, observed the effects on the enzymatic activity at low temperatures, and correlated these effects with spectroscopic measurements of the rotational motions of both the lipid and protein components
The protein was incubated with Triton X-100 for 2 min at 0°C before starting the reaction; addition to the reaction solution resulted in a IO-fold dilution of the Triton/protein mixtures
Summary
Replacement of the native lipids with dipalmitoyl phosphatidylcholine inhibits ATPase activity and decreases both lipid fluidity, as monitored by EPR spectroscopy on a stearic acid spin label, and protein rotational mobility, as monitored by saturation transfer EPR spectroscopy on the covalently spin-labeled enzyme. The inhibition takes place at temperatures at which the mobility of the lipid hydrocarbon chains is strongly restricted (8), indicating that the physical state of the phospholipids associated with the Ca2’-ATPase enzyme is an important factor in determining the rate of ATP hydrolysis in SR. Parallel studies in SR of the effect of temperature on enzymatic activity, lipid fluidity, and mobility of protein-bound spin labels have indicated a correlation among these variables. Protein as a whole or of large The results indicate that, at low temperatures, replacement of the endogenous
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