Expression and identification of p90 as the murine mitochondrial glycerol-3-phosphate acyltransferase.
Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and committed step in glycerolipid biosynthesis. Mitochondrial GPAT, unlike the microsomal isozyme, prefers saturated fatty acids as a substrate. We have recently reported cloning of a cDNA to an unidentified 6.8-kb mRNA by a differential hybridization. The mRNA contains an open reading frame of 827 amino acids (p90) with 30% sequence homology in a 300 amino acid stretch to Escherichia coli GPAT. The 6.8-kb mRNA was induced dramatically when fasted mice were refed a high-carbohydrate diet. Here, we have expressed the open reading frame as trpE fusion proteins and used them to generate antibodies. The antibodies recognized a polypeptide of 90 kDa (p90) when the 6.8-kb cDNA sequence was used for in vitro transcription and translation. By Western blot analysis using these antibodies, we detected p90 in mitochondrial fractions of liver, and the p90 level was increased by refeeding. The increase in the p90 level correlated with the increase in mitochondrial GPAT activity. Moreover, p90 was not detectable in 3T3-L1 preadipocytes but markedly increased during adipose conversion. This increase was consistent with the 11-fold increase we observed in N-ethylmaleimide (NEM)-resistant mitochondrial GPAT activity during adipocyte differentiation. In addition, we have expressed p90 in CHO cells by stable transfection. The transfected genes in both correct and reverse orientations produced distinct 3.9-kb transcripts owing to the truncation of a part of the noncoding regions of the endogenous 6.8-kb mRNA before insertion into the pMSXND vector. The transfected CHO cells were treated with 2-aminopurine, an agent that increases expression of exogenous genes.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115.
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D016475:3T3 Cells; D000273:Adipose Tissue / Q000166:cytology / Q000201:enzymology; D000818:Animals; D000906:Antibodies / Q000276:immunology; D016466:CHO Cells; D002454:Cell Differentiation; D006224:Cricetinae; D015870:Gene Expression; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008930:Mitochondria, Liver / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection
D000906:Antibodies; D011993:Recombinant Fusion Proteins; D005992:Glycerol-3-Phosphate O-Acyltransferase