Different sensitivity of Na(+),K(+)-ATPase and Mg(2+)-АТРase to ethanol and arachidonic acid in rat colon smooth muscle under pretreatment of cellular membranes with Ds-Na

Abstract

The methodological procedure provides the detection of the relatively high Na+,K+-ATPase functional activity in the crude cellular membranes of rat colon smooth muscle (CSM) following standard detergent pretreatment (with Ds-Na vs digitonin). It includes the essential discrete steps: detergent membrane permeabilization under optimal detergent/protein ratio and active site protection by ATP (for Ds-Na) prior enzymatic reaction with substantial detergent dilution far below critical micelle concentration in the ATPase medium. The high level of the Na+,K+-ATPase activity, originally detected in CSM, did not differ for two detergents and was comparable with ouabain-resistant Mg2+,АТР-hydrolase activity, The features of ATPase protein-lipid complexes were evaluated by the enzyme sensitivity to the effect of ethanol and arachidonic acid with different membrane disordering effectiveness. The long-chain fatty acid is a more effective inhibitor as compared with aliphatic alcohol for both ATPases. Mg2+,АТР-hydrolase appeared to be much more resistant to inactivation than Na+,K+-ATPase. The data reflect the possible differences in lipid dependence of two enzymatic systems due to the peculiarities of the structural arrangement in membrane and importance of the hydrophobic microenvironment for mechanism of catalysis. Thus, the data represent the approach to the simple and reliable Na+,K+-АТРase activity determination in nonpurified CSM membranes, acceptable for different tissues and appropriate for quantitative comparison in pathophysiological studies and for testing the impact of diverse effectors on Na+,K+-АТРase.