Abstract
Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step of glycerolipid synthesis. Two distinct GPAT isoenzymes had been identified in mammalian tissues, an N-ethylmaleimide (NEM)-sensitive isoform in the endoplasmic reticulum membrane (microsomal GPAT) and an NEM-resistant form in the outer mitochondrial membrane (mtGPAT). Although only mtGPAT has been cloned, the microsomal and mitochondrial GPAT isoforms can be distinguished, because they differ in acyl-CoA substrate preference, sensitivity to inhibition by dihydroxyacetone phosphate and polymixin B, temperature sensitivity, and ability to be activated by acetone. The preponderance of evidence supports a role for mtGPAT in synthesizing the precursors for triacylglycerol synthesis. In mtGPAT(-/-) mice, PCR genotyping and Northern analysis showed successful knockout of mtGPAT; however, we detected a novel NEM-sensitive GPAT activity in mitochondrial fractions and an anti-mtGPAT immunoreactive protein in liver mitochondria, but not in microsomes. Rigorous analysis using two-dimensional gel electrophoresis revealed that the anti-mtGPAT immunoreactive proteins in wild type and mtGPAT(-/-) liver mitochondria have different isoelectric points. These results suggested the presence of a second GPAT in liver mitochondria from mtGPAT(-/-) mice. Characterization of this GPAT activity in liver from mtGPAT null mice showed that, unlike the mtGPAT activity in wild type samples, activity in mtGPAT knockout mitochondria did not prefer palmitoyl-CoA, was sensitive to inactivation by NEM, was inhibited by dihydroxyacetone phosphate and polymixin B, was temperature-sensitive, and was not activated by acetone. We conclude that a novel GPAT (mtGPAT2) with antigenic epitopes similar to those of mtGPAT is detectable in mitochondria from the livers of mtGPAT(-/-) mice.
Highlights
The initial and rate-limiting step of glycerolipid synthesis is the acylation of glycerol 3-phosphate with long-chain fatty acylCoA to form 1-acyl-glycerol 3-phosphate (LPA).1 This reaction is catalyzed by two glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) isoenzymes that are encoded by different genes [1]
Mitochondrial GPATϪ/Ϫ Liver Mitochondria Contain an NEM-sensitive Glycerol-3-phosphate acyltransferase (GPAT) Activity—Mitochondrial GPAT knockout mice were generated by replacing a 0.5-kb genomic sequence encoding part of the active site with neo and confirmed by PCR genotyping [8]
GPAT is a critical enzyme in glycerolipid synthesis because it catalyzes the initial and committed step required for the formation of triacylglycerol and all of the glycerophospholipids
Summary
Chemicals—All chemicals were purchased from Sigma if not otherwise indicated. [2-3H]Glycerol was from PerkinElmer Life Sciences. Purity of the Mitochondrial and Microsomal Fractions—Purity of the liver subcellular fractions was established by measuring the activity of marker enzymes, NADH cytochrome c reductase [17], and cytochrome c oxidase (cytochrome c oxidase kit; Sigma), for endoplasmic reticulum and mitochondria, respectively. Mitochondria (1 mg of protein) were added to 600 l of rehydration buffer containing 7 M urea, 2 M thiourea, 0.5% (v/v) Biolytes 3–10 (Bio-Rad), 50 mM Tris-HCl, 1% ASB-14 (Calbiochem) and 1% Triton X-100, and 2 mM tributyl phosphine. Immobilized pH gradient gel strips (Bio-Rad) were rehydrated overnight (12–16 h) at 50 V with rehydration buffer containing the protein sample. The immobilized pH gradient gel strips were incubated for 10 min with shaking in equilibration buffer
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
