Partial Purification and Properties of an Acyl Coenzyme A:sn-Glycerol 3-Phosphate Acyltransferase from Rat Liver Mitochondria

Monroy, Gladys; Kelker, H C; Pullman, Maynard E.

doi:10.1016/s0021-9258(19)44084-2

Cited by 96 publications

(11 citation statements)

References 48 publications

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“…The substrate specificity determined with our preparation is similar to that reported with crude rat liver mitochondrial extracts. Overall, mitochondrial GPAT prefers saturated fatty acyl-CoA as a substrate (Monroy et al, 1973;Kelker & Pullman, 1979;Monroy et al, 1972). The preference for saturated fatty acyl donors was also demonstrated in E. coli GPAT, consistent with the known positional distribution of saturated fatty acids (Green et al, 1981).…”

Section: Purification and Reconstitution Of Gpat Expressed In Sf9supporting

confidence: 73%

“…This may be due to the inhibitory effect of detergent on GPAT activity. A decrease in GPAT activity by various detergents such as Triton X-100, Lubrol, and Brij 35 and an increase in activity by subsequent removal of detergent were previously reported (Monroy et al, 1973;Green et al, 1981). It is also possible that addition of exogenous phospholipids does not fully restore GPAT activity.…”

Section: Purification and Reconstitution Of Gpat Expressed In Sf9mentioning

confidence: 83%

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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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Section: Purification and Reconstitution Of Gpat Expressed In Sf9supporting

confidence: 73%

Section: Purification and Reconstitution Of Gpat Expressed In Sf9mentioning

confidence: 83%

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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“…There are four known isoforms of GPAT, two microsomal isoforms located in the endoplasmic reticulum (GPAT3 and GPAT4) and two located in mitochondria (mtGPAT1 and mtGPAT2). , mtGPAT1 displays a strong preference for incorporating palmitoyl-CoA (16:0), thereby primarily producing saturated phospholipids, whereas the other three enzymes are not selective. , For recombinant mouse mtGPAT1, activity is 2-fold greater with palmitoyl-CoA than with various unsaturated chains 18 and 20 carbons in length, and in rat liver, kidney, and heart, mtGPAT1 incorporates palmitoyl CoA 3−10 times more effectively than other long-chain saturated or unsaturated acyl-CoAs. ,− Of the four isoforms, only mtGPAT1 is affected by changes in diet or exercise. When excess calories are available from a high-carbohydrate diet, mtGPAT1 mRNA expression increases, resulting in greater mtGPAT1 activity. − Mice that remain stationary for 10 hours following a prolonged exercise regimen experience an increase in mtGPAT1 activity compared to mice that did not exercise at all, resulting in a significant overshoot of triacylglycerol (TAG) synthesis …”

Section: Introductionmentioning

confidence: 99%

Design and Synthesis of Small Molecule Glycerol 3-Phosphate Acyltransferase Inhibitors

Wydysh

Medghalchi²,

Vadlamudi³

et al. 2009

J. Med. Chem.

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The incidence of obesity and other diseases associated with an increased triacylglycerol mass is growing rapidly, particularly in the United States. Glycerol 3-phosphate acyltransferase (GPAT) catalyzes the rate-limiting step of glycerolipid biosynthesis, the acylation of glycerol 3-phosphate with saturated long chain acyl-CoAs. In an effort to produce small molecule inhibitors of this enzyme, a series of benzoic and phosphonic acids was designed and synthesized. In vitro testing of this series has led to the identification of several compounds, in particular 2-(nonylsulfonamido)benzoic acid (15g), possessing moderate GPAT inhibitory activity in an intact mitochondrial assay.

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“…There are four mammalian GPATs documented to-date. In the 1970s, using density-gradient centrifugation and radioactive tracers, enzymatic activities of GPATs were mapped predominantly to mitochondria and microsomes (Yamashita and Numa, 1972;Monroy et al, 1973). However, it was not until 2006 and 2008 that the microsomal GPAT3 and GPAT4 were firstly identified, respectively (Cao et al, 2006;Chen et al, 2008;Nagle et al, 2008).…”

Section: Glycerol-3-phosphate Acyltransferasementioning

confidence: 99%

Married at Birth: Regulation of Cellular Fat Metabolism by ER–Lipid Droplet Crosstalk

Cao

Mak

2020

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The endoplasmic reticulum (ER) is a hub that coordinates neutral lipid synthesis, storage, and export. To fulfill this role, the ER maintains close contact with lipid droplets (LDs), which are evolutionarily conserved organelles for the storage of neutral lipids. Decades of biochemical evidence points to fatty acid modification and neutral lipid synthesis in the ER. Conceptually, lipid export into extracellular space or lipid retention intracellularly require the subsequent remodeling of an ER membrane leaflet that faces the lumen or cytoplasm, respectively. This is because LDs and very-low-density lipoprotein particles are all structures surrounded by a phospholipid monolayer. While the export of neutral lipids via very-low-density lipoprotein production is well characterized, there has been increasing interest in the mechanisms that underlie neutral lipid retention in LDs. Structural determination, in vitro reconstitution, and localization of key proteins by advanced microscopy techniques collectively enrich models of ER-LD engagement. In this review, we consider current concepts on how LDs emerge from the ER in a directional manner and how sustained ER-LD contacts support LD expansion.

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Partial Purification and Properties of an Acyl Coenzyme A:sn-Glycerol 3-Phosphate Acyltransferase from Rat Liver Mitochondria

Cited by 96 publications

References 48 publications

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

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

Design and Synthesis of Small Molecule Glycerol 3-Phosphate Acyltransferase Inhibitors

Married at Birth: Regulation of Cellular Fat Metabolism by ER–Lipid Droplet Crosstalk

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