Abstract
Abstract Long chain fatty acids inhibit the Mg2+-dependent Na+ + K+-stimulated ATPase of rat brain microsomal membrane preparations without any significant effect on the basic Mg2+-stimulated component. The magnitude of inhibition increases with the chain length, reaching a maximum with myristate followed by a decline with higher fatty acids. Myristate (5 x 10-5 m) produces about 45% inhibition of the Na+ + K+-stimulated ATPase. Unsaturated fatty acids are more inhibitory than their corresponding saturated congeners; e.g. at 5 x 10-5 m, oleate produces 66% inhibition compared with 15% by stearate. The inhibitory effects are not due to the binding of Mg2+ by fatty acids as the effects are not reversed by increasing the Mg2+ concentration in the reaction. The inhibition is freely reversible and does not appear to be time dependent. Kinetic analysis of the action of myristate on the Na+ + K+-stimulated ATPase indicates that the inhibition is uncompetitive with respect to ATP and competitive with respect to K+. The primary site of action of myristate appears to be an inhibition of K+ activation of the enzyme. The Na+ activation of the enzyme is also inhibited, but only when the concentration of Na+ is very low; in the presence of higher concentrations of Na+, the action of myristate can be interpreted as a second (in addition to Na+) competitive inhibitor of K+. K+-stimulated p-nitrophenyl phosphatase is also inhibited in a competitive fashion by myristate. The myristate inhibition of the Na+ + K+-stimulated ATPase is as well elicited in the presence of ouabain or oligomycin. The effects of these mixed inhibitors are of a cumulative nature suggesting separate binding sites for each of the inhibitors.
Highlights
Unsaturated fatty acids are more inhibitory than their corresponding saturated congeners; e.g. at 5 X low6 M, oleate produces 66% inhibition compared with 15% by stearate
The inhibitory effects are not due to the binding of Mg2f by fatty acids as the effects are not reversed by increasing the Mgz+ concentration in the reaction
The foregoing results point to a site of action of free fatty acids on the cellular metabolism, i.e. inhibition of Na+ + K+stimulated ATPase which is believed to be involved in membrane transport
Summary
MaterialsThe detailsconcerningthe materialsusedand preparation of solutions etc. have been published previously [15, 16]. Free fatty acids were obtained from Applied ScienceLaboratories, CollegePark, Pennsylvania. They were converted to Tris or sodiumsalts prior to use. Free fatty acidshave beenshownto affect severalmetabolic activities in tissues. Na+ + K+-stimuIated ATPase activity wasisolatedexactIy as describedby Ahmed and Judah [16]. Fatty Acids and Membrane ATPase Vol 246, Eo. 1. M&f Tris-HCl and 1 mu EDTA, pH 7.4, and was stored frozen, in small aliquots. Assay of Enzyme Activities-The reaction mixture for the determination of Mg2+ + Na+ + K+-stimulated ATPase activity consisted of 3 mM MgCl2, 3 mM ATP (Tris salt), 115 InM
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