Dipak Haldar

Phosphatidic Acid Synthesis in Mitochondria TOPOGRAPHY OF FORMATION AND TRANSMEMBRANE MIGRATION

The topography of formation and migration of phosphatidic acid (PA) in the transverse plane of rat liver mitochondrial outer membrane (MOM) were investigated. Isolated mitochondria and microsomes, incubated with sn-glycerol 3-phosphate and an immobilized substrate palmitoyl-CoA-agarose, synthesized both lyso-PA and PA. The mitochondrial and microsomal acylation of glycerophosphate with palmitoyl-CoA-agarose was 80–100% of the values obtained in the presence of free palmitoyl-CoA. In another series of experiments, both free polymyxin B and polymyxin B-agarose stimulated mitochondrial glycerophosphate acyltransferase activity approximately 2-fold. When PA loaded mitochondria were treated with liver fatty acid binding protein, a fifth of the phospholipid left the mitochondria. The amount of exportable PA reduced with the increase in the time of incubation. In another approach, PA-loaded mitochondria were treated with phospholipase A2. The amount of phospholipase A2-sensitive PA reduced when the incubation time was increased. Taken together, the results suggest that lysophosphatidic acid (LPA) and PA are synthesized on the outer surface of the MOM and that PA moves to the inner membrane presumably for cardiolipin formation.

Glycerophosphate acyltransferase from liver

Publisher Summary In mammalian organs, glycerophosphate acyltranferase is present in both the endoplasmic reticulum (microsomes) and mitochondrial outer membrane. In liver, the glycerophosphate acyltransferase activity is nearly equal in these two organelles, whereas in other organs the microsomal acyltransferase is approximately 10 times more active than the mitochondrial enzyme. The enzyme activity can be measured by following the conversion of sn -[2- 3 H]glycerol 3-phosphate to l-butanol-extractable lipid. The liver microsomal glycerophosphate acyltransferase is inhibited by all proteases. The mitochondrial enzyme, on the other hand, is not inhibited by trypsin and chymotrypsin. Non-site-specific proteases, such as proteinase K and subtilisin, inhibit the mitochondrial enzyme. The degree of inhibition increases if the ionic strength of the incubation medium is lowered. Both microsomal and mitochondrial glycerophosphate acyltransferase have been only partially purified. In contrast, the Escherichia coli glycerophosphate acyltranferase has been completely purified, its properties studied, and the gene cloned and sequenced. The successful purification of the prokaryotic enzyme has been facilitated by massive overproduction of the enzyme owing to molecular cloning.

Further distinguishing properties of mitochondrial and microsomal glycerophosphate acyltransferase and the transmembrane location of the mitochondrial enzyme

Abstract The acylation of sn -glycerol 3-phosphate with palmityl-CoA was compared in mitochondria and microsomes isolated from rat liver. Polymyxin B, an antibiotic known to alter bacterial membrane structure, stimulated the mitochondrial glycerophosphate acyltransferase but inhibited the microsomal enzyme. When mitochondrial and microsomal fractions were incubated at 4–6 °C for up to 4 h, the mitochondrial enzyme remained virtually unchanged while the microsomal enzyme lost about one-half of its activity. Incubations at higher temperatures also revealed that the mitochondrial enzyme was comparatively more stable under the conditions employed. The mitochondrial acyltransferase showed no sensitivity to bromelain, papain, Pronase, and trypsin, all of which strongly inhibited the microsomal enzyme. The differential sensitivity to trypsin was observed in mitochondria and microsomes isolated from other rat organs. However, the liver mitochondrial glycerophosphate acyltransferase was inhibited by trypsin in the presence of either 0.05% deoxycholate or 0.1% Triton X-100. The trypsin sensitivity of the mitochondrial glycerophosphate acyltransferase in the presence of detergent was not due to the presence, in the mitochondrial fraction, of a trypsin inhibitor which became inactivated by Triton X-100 or deoxycholate. The results suggest that the catalytic site of mitochondrial glycerophosphate acyltransferase is not exposed to the cytosolic side and it is located in the inner aspect of the outer membrane.

Effect of disulfiram (DS) on mitochondria from rat hippocampus: Metabolic compartmentation of DS neurotoxicity

This experiment was designed to study the acute effects of disulfiram on mitochondrial enzymes in nonsynaptic and synaptic mitochondria from rat hippocampus. Cytochromec oxidase, monoamine oxidase-B, glycerolphosphate acyltransferase and betahydroxybutyrate dehydrogenase were studied. Differences in enzyme activity were seen in controls. Cytochromec oxidase activity was higher in synaptic mitochondria whereas glycerolphosphate acyltransferase activity was higher in nonsynaptic mitochondria. Mitochondria from disulfiram treated rats, particularly synaptic mitochondria, exhibited lower specific activities of cytochromec oxidase and monoamine oxidase-B. These alterations were not limited to either the inner or outer mitochondrial membrane. Transmission electron microscopy revealed that mitochondria from disulfiram treated rats were severely altered in isolated preparations as well as in those from whole tissue. This study shows that disulfiram exerts a differential effect on mitochondrial subpopulations.

A lysophosphatidic acid-binding cytosolic protein stimulates mitochondrial glycerophosphate acyltransferase.

Rat liver cytosolic fraction caused up to five fold stimulation of mitochondrial glycerophosphate acyltransferase apparently by removing the lysophosphatidic acid formed by the acyltransferase. When mitochondria were incubated with palmityl-CoA, [2-3H]-sn-glycerol 3-phosphate and the cytosolic fraction and the supernatant fluid of the incubated mixture was passed through a Sephadex G-100 column, labeled lysophosphatidic acid eluted in three peaks with Mrs (i) 60-70 kDa, (ii) 10-20 kDa, and (iii) less than 5 kDa. Proteins, responsible for binding of lysophosphatidic acid in peaks (i) and (ii), were purified to near homogeneity as judged by electrophoretic analysis. The lysophosphatidic acid binding protein in peak (i) appears to be serum albumin and peak (iii) represents largely unbound lysophosphatidic acid. The 15 kDa protein, purified from peak (ii), bound lysophosphatidic acid, stimulated the acyltransferase and export of lysophosphatidic acid from mitochondria.

Acyl-Coa: sn-glycerol 3-phosphate acyltransferase in mitochondria and microsomes of adult and fetal guinea pig lung.

Summary Guinea pig lung mitochondrial and microsomal glycerophosphate acyltransferase (GAT) was measured using palmityl- or oleyl-CoA as acyl donor. The activities of the two fractions differed in substrate specificity and sensitivity to N-ethylmaleimide, trypsin, and acetone. In comparison to adult lung, the fetal lung had markedly higher subcellular GAT activity. The results demonstrate a bimodal distribution of GAT in lung and suggest that this enzyme may plan an important role in lung development.

Kinetic analysis of cardiolipin synthase: a membrane enzyme with two glycerophospholipid substrates.

Mitochondrial cardiolipin synthase catalyzes the transfer of a phosphatidyl moiety from phosphatidyl-CMP PtdCMP) to phosphatidylglycerol (PtdGro) in the presence of specific divalent cations. The synthase was solubilized fromSaccharomyces cerevisiae mitochondria and purified about 300-fold. The partially purified enzyme was part of a medium-size, mixed micelle which had to bind to a foreign substrate/detergent micelle before catalysis could occur. The kinetics of cardiolipin synthase were studied by changing the molar fraction of substrate in the micelles. The enzyme obeyed Michaelis-Menten kinetics in relation to PtdCMP with aKm of 0.03 mol%. PtdGro caused sigmoidal kinetics with a low apparent affinity. it is speculated that it was involved in docking the enzyme to the substrate/detergent micelle. Cardiolipin synthase did not catalyze isotope exchange between [14C]CMP and PtdCMP, virtually excluding a ping-pong catalytic mechanism. Mg2+stimulated the activity by increasing the turnover number rather than the substrate affinity, a mechanism which was also found for the Co2+-activation of rat liver cardiolipin synthase. It is concluded that a direct association of the metal ion and the enzyme forms the active cardiolipin synthase which has a very high affinity for PtdCMP and a lower affinity for PtdGro.

Two-dimensional gel electrophoretic resolution of the polypeptides of rat liver mitochondria and the outer membrane

The proteins of highly purified rat liver mitochondria were resolved by two-dimensional polyacrylamide gel electrophoresis, and detected by staining with either Coomassie blue or silver. Approximately 250 polypeptides were detected with silver staining which is 2- to 3-times that observed with Coomassie blue. Silver staining was especially more effective than Coomassie blue for detecting polypeptides of less than 50 000 daltons. A two-dimensional gel pattern of rat liver microsomes was distinct from that of the mitochondria. The mitochondrial outer membrane was prepared from purified mitochondria either with digitonin or by swelling in a hypotonic medium. As assessed by marker enzymes, the latter method yielded a considerably purer outer membrane preparation (20-fold purification) than the former (2.6-fold purification). Approximately 50 polypeptides were observed in a two-dimensional gel (pH 3-10) of the highly purified outer membrane fraction. Three isoelectric forms of the pore (VDAC) protein were observed with pI values of 8.2, 7.8 and 7.1. Monoamine oxidase was identified as a polypeptide of Mr 60 000. About 50 polypeptides were also resolved in a reverse polarity non-equilibrium pH gradient electrophoresis gel of the outer membrane, pH 3-10, with at least six isoelectric forms of the VDAC protein observed under these conditions. The six isoforms of the VDAC protein were also observed in a non-equilibrium gel with 2 micrograms of the purified protein.

Acyl‐CoA: sn‐Glycerol‐3‐Phosphate O‐Acyltransferase in Rat Brain Mitochondria and Microsomes

Abstract: The subcellular distribution of acyl‐CoA: sn‐glycerol‐3‐phosphate O‐acyltransferase between brain mitochondria and microsomes was investigated. The activities associated with purified rat brain mitochondrial and microsomal preparations could be distinguished by differences in their acyl‐CoA specificity, products of acylation, and sensitivity to N‐ethylmaleimide, trypsin, acetone, and polymyxin B. It was concluded that both brain mitochondria and microsomes possess the acyltransferase.

Transcriptional regulation of mitochondrial glycerophosphate acyltransferase is mediated by distal promoter via ChREBP and SREBP-1

We have recently identified two promoters, distal and proximal for rat mitochondrial glycerophosphate acyltransferase (mtGPAT). Here we are reporting further characterization of the promoters. Insulin and epidermal growth factor (EGF) stimulated while leptin and glucagon inhibited the luciferase activity of the distal promoter and the amounts of the distal transcript. Conversely, luciferase activity of the proximal promoter and proximal transcript remained unchanged due to these treatments. Only the distal promoter has binding sites for carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1 (SREBP-1). Electromobility shift assays and chromatin immunoprecipitation assays demonstrated that ChREBP and SREBP-1 bind to the mtGPAT distal promoter. Insulin and EGF increased while glucagon and leptin decreased the binding of SREBP-1 and ChREBP to the distal promoter. Thus, the distal promoter is the regulatory promoter while the proximal promoter acts constitutively for rat mtGPAT gene under the influence of hormones and growth factor.