Abstract
The sn-glycerol-3-phosphate (glycerol-P) acyltransferase of Escherichia coli cytoplasmic membrane was purified in Triton X-100 (Green, P. R., Merrill, A. H., Jr., and Bell, R. M. (1981) J. Biol. Chem. 256, 11151-11159) and incorporated into mixed micelles containing Triton X-100, phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and beta-octyl glucoside. Enzyme activity was quantitatively reconstituted from the mixed micelle into single-walled phospholipid vesicles by chromatography over Sephadex G-50. Activity coeluted with vesicles of 90-nm average diameter on columns of Sepharose CL-4B and Sephacryl S-1000. These vesicles contained less than 2 Triton X-100 and 5 beta-octyl glucoside molecules/100 phospholipid molecules. Calculations suggested that up to eight 91,260-dalton glycerol-P acyltransferase polypeptides were incorporated per 90-nm vesicle. The pH dependence and apparent Km values for glycerol-P and palmitoyl-CoA of the glycerol-P acyltransferase reconstituted into vesicles were similar to those observed upon reconstitution by mixing of the enzyme in Triton X-100 with a 20-fold molar excess of sonicated phosphatidylethanolamine:phosphatidylglycerol:cardiolipin, 6:1:1. The integrity of vesicles containing glycerol-P acyltransferase was established by trapping 5,5'-dithiobis-(2-nitrobenzoic acid). Chymotrypsin inactivated greater than 95% of the glycerol-P acyltransferase in intact vesicles and cleaved the 91,260-dalton polypeptide into several vesicle-bound and several released peptides, indicating that critical domains of the enzyme are accessible in intact vesicles. Trinitrobenzene sulfonate and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene caused greater than 90% loss of glycerol-P acyltransferase in vesicles. Disruption of vesicles with Triton X-100 did not reveal significant latent activity. These data strongly suggest that the glycerol-P acyltransferase was reconstituted asymmetrically into the vesicles with its active site facing outward.
Highlights
The sn-glycerol-3-phosphate acyltrans- committed step of membrane phospholipid biosynthesis (1, ferase of Escherichia coli cytoplasmic membrane was 2)
The amount of phospholipid synthesized in Escherichia purified in TritonX-100
Calculations suggested that up to eight 91,260- fied the plsB gene product as an83,000 M, polypeptide’ [24], dalton glycerol-P acyltransferase polypeptides were purified the enzyme to nearhomogeneity [25], sequenced the incorporated per 90-nm vesicle
Summary
The sn-glycerol-3-phosphate (glycerol-P) acyltrans- committed step of membrane phospholipid biosynthesis (1, ferase of Escherichia coli cytoplasmic membrane was 2). The cytoplasmic membrane bound acyl-CoA:sn-glycerol-3phosphate 0-acyltransferase(EC2.3.1.15) catalyzes the initial these goals and establish an asymmetric orientation of the enzyme in the transverse plane of the reconstituted vesicle bilayer. Sulting vesicles containing glycerol-P acyltransferase had a maximum of 2 Triton X-100 and 5 P-octyl glucoside molecules/100 phospholipid molecules (Fig. 1).This is an upper limit.
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