Effect of lipids on the reconstitution of D-lactate oxidase in Escherichia coli membrane vesicles.
An unsaturated fatty acid auxotroph lacking D-lactate dehydrogenase activity has been isolated from Escherichia coli ML 308-225 dld-3. While NADH oxidase activity in membrane vesicles prepared from the mutant cells grown in a variety of unsaturated fatty acids is comparable to that of previously isolated fatty acid auxotrophs, D-lactate oxidase activity is absent. However, D-lactate oxidase ativity can be restored when vesicles are incubated with a purified preparation of D-lactate dehydrogenase obtained from wild type cells. The effect of altering the fatty acid composition of the membrane on the reconstitution of D-lactate oxidase activity was examined. Binding of purified D-lactate dehydrogenase was not affected by either the lipid composition of the membrane vesicles or the temperature during reconstitution. However, the reconstitution of D-lactate oxidase activity was strongly influenced by the fatty acid composition of the membrane lipids. The temperature dependence of the reconstituted activity was analyzed. Temperature transitions were not observed with membrane vesicles supplemented with oleic or linolenic acid but palmitelaidic acid-enriched vesicles exhibited a transition temperature at 30 degrees. Attempts to reconstitute the elaidic-supplemented vesicles at temperatures below 30 degrees failed to yield active D-lactate oxidase and the vesicles aggregated. At 42 degrees, aggregation did not occur and D-lactate oxidase activity was obtained to a level of 10% of that found with membranes from the parent strain (ML 308-225). These results suggest that although binding of D-lactate dehydrogenase is independent of the physical state of the membrane, reconstitution of the D-lactate oxidase activity in membrane vesicles is dependent upon the fatty acid composition of the phospholipid, hence the physical state of the membrane.
D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000172:deficiency / Q000378:metabolism; D007773:Lactates / Q000378:metabolism; D008563:Membrane Lipids / Q000502:physiology; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D013696:Temperature
D005231:Fatty Acids, Unsaturated; D007773:Lactates; D008563:Membrane Lipids; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D007770:L-Lactate Dehydrogenase; C021497:lactate 2-monooxygenase; D009247:NADH, NADPH Oxidoreductases