Characterization of reconstituted partially purified glycerophosphate acyltansferase from Escherichia coli.
A modification of the method of Snider and Kennedy (J. Bacteriol. (1977) 130, 1072-1083) was worked out to solubilize sn-glycero-3-phosphate acyltransferase from whole cells by Triton X-100. The solubilized preparation was used for a systematic study of the reconstitution of enzymatic activity as observed by addition of phospholipid vesicles. Although enzymatic activity was regained by addition of vesicles and not by addition of multilayered liposomes, subsequent Sepharose 4B chromatography revealed the enzyme to be incorporated in large lipid aggregates of undefined structures. Incorporation of glycerophosphate acyltransferase in single bilayer vesicles composed of phosphatidylcholine and phosphatidylglycerol (4:1) was obtained after removal to Triton X-100 from the enzyme solution, co-dispersion of enzyme and phospholipids with cholate and Sephadex G-50 gel filtration of this mixture to remove cholate. The optimal conditions for this reconstitution procedure with respect to phospholipid/protein and phosphatidylcholine/phosphatidylglycerol ratio were established. The active site of glycero-3-phosphate acyltransferase in the reconstituted system was localized for at least 90% at the outside surface of the vesicle, as revealed by proteolysis experiments under conditions of vesicle intactness as shown by C-NMR experiments. The reconstituted systems produced only lysophosphatidate from sn-[14C]glycero-3-phosphate and palmitoyl-CoA and showed identical apparent Km for sn-glycero-3-phosphate and identical pH- and temperature-dependencies as the enzyme in isolated Escherichia coli membranes.
D000217:Acyltransferases / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D012995:Solubility; D013379:Substrate Specificity
D000217:Acyltransferases; D005992:Glycerol-3-Phosphate O-Acyltransferase