Phospholipid requirement of acyl coenzyme A:sn-glycerol-3-phosphate acyltransferase from rat liver mitochondria.

Kelker, H C; Pullman, Maynard E.

doi:10.1016/s0021-9258(18)50604-9

Cited by 23 publications

(6 citation statements)

References 55 publications

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“…Previous reports showed preference for saturated fatty acyl CoA as a substrate of mitochondrial GPAT and postulated that, although the principal site for glycerolipid synthesis is the endoplasmic reticulum, this substrate preference of mitochondrial GPAT may explain the asymmetric distribution of saturated fatty acids at the sn-1 position (Kelker & Pullman, 1979;Stern & Pullman, 1978;Haidar et al, 1979). Therefore, to ascertain that the transfected p90 shows the characteristic of the mitochondrial GPAT, we compared palmitoyl CoA to oleoyl CoA as a substrate for mitochondrial GPAT from transfected cells.…”

Section: Resultsmentioning

confidence: 96%

“…It has also been reported that mitochondrial GPAT prefers saturated fatty acyl CoA over unsaturated fatty acids as an acyl donor, whereas the microsomal enzyme does not show this substrate specificity (Kelker & Pullman, 1979; Stem & Pullman, 1978). It has been postulated that the predominance of saturated fatty acids found in the sn-1 position in naturally occurring acylglycerols, in contrast to unsaturated fatty acids at the sn-2 position, is the result of this fatty acyl CoA preference of the mitochondrial GPAT.…”

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confidence: 99%

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Expression and identification of p90 as the murine mitochondrial glycerol-3-phosphate acyltransferase

Yet

Lee²,

Hahm³

et al. 1993

Biochemistry

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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

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Section: Resultsmentioning

confidence: 96%

mentioning

confidence: 99%

Expression and identification of p90 as the murine mitochondrial glycerol-3-phosphate acyltransferase

Yet

Lee²,

Hahm³

et al. 1993

Biochemistry

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“…However, cardiolipin, a major phospholipid in mitochondrial membrane, was not effective in reconstituting GPAT activity to any appreciable level. In fact, it has been previously reported that caridolipin is a potent inhibitor of GPAT activity (Kelker & Pullman, 1979). Although less than 10% as effective compared to crude phosphatidylcholine, phosphatidylethanolamine yielded low GPAT activity when added to the reaction mixture in reconstituting GPAT activity.…”

Section: Purification and Reconstitution Of Gpat Expressed In Sf9mentioning

confidence: 96%

“…Mitochondrial GPAT was shown to prefer saturated to unsaturated fatty acyl-CoA as an acyl donor, whereas the microsomal enzyme showed no substrate specificity (Kelker & Pullman, 1979;Stem & Pullman, 1978). The predominance of saturated fatty acids at the s«-l position in naturally occurring acylglycerols, in contrast to unsaturated fatty acids at the sn-2 position, has been postulated to be the result of the fatty acyl-CoA preference of mitochondrial GPAT (Brindley, 1991).…”

mentioning

confidence: 99%

Purification and Reconstitution of Murine Mitochondrial Glycerol-3-phosphate Acyltransferase. Functional Expression in Baculovirus-Infected Insect Cells

Yet¹,

Moon²,

Sul³

1995

Biochemistry

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Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial step in glycerolipid biosynthesis. We recently cloned a cDNA to a 6.8-kb mRNA, a message that can be induced dramatically by feeding a high-carbohydrate diet [Paulauskis & Sul (1988) J. Biol. Chem. 263, 7049-7054; Shin et al. (1991) J. Biol. Chem. 266, 23834-23839], and identified the open reading frame, p90, as mitochondrial GPAT [Yet et al. (1993) Biochemistry 32, 9486-9491]. To initiate characterization of mitochondrial GPAT, we purified and reconstituted the GPAT activity using phospholipids after expressing functional enzyme in Sf9 insect cells. Infection with recombinant virus containing p90 sequence resulted in high levels of GPAT expression in mitochondria, compared to noninfected cells or cells infected with the reverse orientation insertion baculovirus. There was a dramatic increase in N-ethylmaleimide-resistant mitochondrial GPAT activity. The GPAT protein was not detectable by Western blot in noninfected Sf9 cells or in cells infected with the GPAT sequence in the reverse orientation. However, in cells infected with GPAT in the correct orientation, there was a dramatic increase in the GPAT protein that was readily detectable by Coomassie staining both in total extracts and in the mitochondrial fraction. To ease the purification, we next expressed GPAT as a polyhistidine fusion protein in insect cells. The polyhistidine tag did not interfere with targeting to mitochondria or with the catalytic activity of GPAT.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…The results in Table I show that even the low level of acyltransferase in liver microsomes can acylate concentrations of lysophosphatidylcholine up to three times the initial phosphatidylcholine content. A purified acyltransferase would permit even more efficient reconstitutions, and 466 RAPID COMMUNICATIONS recent successes in isolating acyltransferases and transacylases (2,24,38) suggest that purification of the rat liver enzyme is feasible .…”

Section: Discussionmentioning

confidence: 99%

An enzymatically driven membrane reconstitution from solubilized components.

Deamer¹,

Boatman²

1980

The Journal of Cell Biology

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Acyltransferase activity is present in a variety of membrane species, including liver microsomes. The substrates of this enzyme are lysophosphatides and acyl CoA derivatives. We have found that the detergent effect of these substrates can be used to solubilize rat liver microsomes. If the solubilized fraction is incubated, the acyltransferase acylates the lysophosphatide and thereby degrades the detergent effect so that vesicular membranes re-form . Gel electrophoresis patterns show that the reconstituted membranes contain all of the major protein components of the original microsomes. A marker enzyme for liver microsomes, NADPH-cytochrome c reductase, was present in the reconstituted membranes at 70% of the specific activity in the original microsomes, and freeze-fracture electron microscopy showed intramembrane particles on all fracture faces . This system may provide a useful model for studies of certain membrane biogenesis reactions that utilize acyltransferase in vivo .

show abstract

Phospholipid requirement of acyl coenzyme A:sn-glycerol-3-phosphate acyltransferase from rat liver mitochondria.

Cited by 23 publications

References 55 publications

Expression and identification of p90 as the murine mitochondrial glycerol-3-phosphate acyltransferase

Expression and identification of p90 as the murine mitochondrial glycerol-3-phosphate acyltransferase

Purification and Reconstitution of Murine Mitochondrial Glycerol-3-phosphate Acyltransferase. Functional Expression in Baculovirus-Infected Insect Cells

An enzymatically driven membrane reconstitution from solubilized components.

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