Effect of changes in the phospholipid composition on the enzymatic activity of D-.beta.-hydroxybutyrate dehydrogenase in rat hepatocytes

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Abstract
The phospholipid composition of primary rat hepatocytes was manipulated by supplementing the medium with choline analogs. The unnatural analog l-2-amino-1-butanol was incorporated into membrane phospholipids to the largest extent, whereas the natural choline analogs ethanolamine, N-methylethanolamine, and N,N-dimethylethanolamine were methylated to yield phosphatidylcholine [PC]. When cells were supplemented with [14C]ethanolamine, greater than 25% of the total PC contained radiolabel in the polar head group after 2 days of supplementation. The extent of phospholipid methylation was reduced by depriving the cells of serine and methionine. Under these conditions, N-methylethanolamine and N,N-dimethylethanolamine were incorporated into phospholipids and were not further metabolized to PC. After 3 days of supplementation with N-methylethanolamine, the content of phosphatidylmethylethanolamine went from essentially 0 to 40% of the total phospholipids and surpassed the extent of incorporation of all other analogs. The formation of the new phospholipid species was primarily at the expense of PC and phosphatidylethanolamine. D-.beta.-Hydroxybutyrate dehydrogenase, which requires PC for activity, was assayed in submitochondrial membranes isolated from supplemented cells. For cells supplemented with either l-2-amino-1-butanol or N-methylethanolamine, the Km for NADH increased relative to choline-supplemented cells while the Km for acetoacetate remained the same. For example, after 3 days of supplementation with N-methylethanolamine, the Km for NADH was 3-fold higher than the value for the choline-supplemented control cells. The change in the Km was due to the change in the lipid environment with no alteration in the enzyme itself. The PC molecules necessary to activate the enzyme may exchange with the other phospholipids in the membrane so that the Km of the enzyme reflects the overall content of PC as well as other properties of the membrane phospholipids.