Hepatic lipid metabolism: effect of spermine, albumin, and Z protein on microsomal lipid formation.

Effects of spermine, bovine serum albumin, and Z protein on microsomal lipid formation from sn-glycerol 3-phosphate and [¹⁴C]palmitoyl CoA were investigated. In the presence of these agents, microsomal lipid formation was stimulated. This was attributed to the activation of sn-glycerol 3-phosphate acyltransferase and to the inhibition of palmitoyl CoA hydrolase. In addition to palmitoyl CoA, spermine also reacted with microsomal membranes in causing their aggregation, and ATP reversed the effect of spermine. Further studies indicated that the interaction of spermine with palmitoyl CoA, rather than with microsomal membranes, was responsible for the activation of glycerolipid formation or to the inhibition of palmitoyl CoA reductase. Examination of the intravesicular distribution of sn-glycerol 3-phosphate acyltransferase and palmitoyl CoA hydrolase and the effects of structural integrity of microsomal vesicles on these two membrane-bound enzymes indicated that the activation of glycerolipid formation and the inhibition of palmitoyl CoA hydrolase by spermine, bovine serum albumin, or Z protein may be closely linked with the structural integrity of microsomal vesicles.     

Sites of phospholipid biosynthesis during induction of intracytoplasmic membrane formation in Rhodopseudomonas sphaeroides

A rapid, gratuitous and cell-division uncoupled induction of intracytoplasmic photosynthetic membrane formation was demonstrated in low-aeration suspensions of chemotrophically grown Rhodopseudomonas sphaeroides. Despite a nearly 2-fold increase in phospholipid levels, no significant increases were detected in the specific activities of CDP-1,2-diacyl-sn-glycerol:sn-glycerol-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase, EC 2.7.8.5) and CDP-1,2-diacyl-sn-glycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase, EC 2.7.8.8), the first committed enzymes of anionic and zwitterionic phospholipid biosyntheses, respectively. The distribution of phosphatidylglycerophosphate and phosphatidylserine synthase activities after rate-zone sedimentation of cell-free extracts indicated that intracytoplasmic membrane phospholipids were synthesized mainly within distinct domains of the conserved cytoplasmic membrane. Labeling studies with ³²P_i and L-[³H]phenylalanine suggested that preexisting phospholipid was utilized initially as the matrix for insertion of intracytoplasmic membrane protein that was synthesized and assembled de novo during induction.Abbreviations BChl bacteriochlorophyll a - B800-850, B875 peripheral and core light-harvesting BChl-protein complexes, respectively, identified by near-IR absorption maxima This paper is dedicated to Professor Gerhart Drews on the occasion of his sixtieth birthday     

Digestion and absorption of a sulphoxide analogue of triacylglycerol in the rat

The stereochemistry of fat digestion and absorption was studied by feeding a triacylglycerol analogue to rats with a thoracic duct cannula. The analogue, rac-1,2-dioleoyl-3-S-tetradecyl-3-thioglycerol-S-oxide was chosen since its enantiomers exhibited high rotation in optical rotatory dispersion (ORD) and circular dichroism (CD). In the chyle, triacylglycerol was the major lipid but X-1,2-diacyl-3-S-tetradecyl-3-thioglycerol-S-oxide constituted 8% of lipid weight. It was resolved by thin-layer chromatography (TLC) into two diastereomers. Each of the diastereomers were analyzed for the proportions of 1-thio-sn-glycerol/3-thio-sn-glycerol isomers by ORD and CD. The 1-thio-sn-glycerol isomers dominated for both compounds indicating that they were enriched during the absorption processes, since a racemic compound was fed. The stereospecificities are probably exerted by acyltransferase(s) during chyle lipid synthesis. The methods used will be valuable tools in studies on the metabolism of enantiomeric glycerides, and also for characterization of naturally occurring sulphur-containing lipids.     

Metabolism of hepatic haem and `green pigments'' in rats given 2-allyl-2-isopropylacetamide and ferric citrate. A new model for hepatic haem turnover

The acyl specificities of several acyltransferases located in the microsomal fraction of lactating rat mammary gland have been investigated using palmitate and oleate as substrates along with CoA, ATP and Mg²⁺, bovine serum albumin and NaF. With either sn-glycerol 3-phosphate or dihydroxyacetone phosphate (plus NADPH) as acyl acceptor, phosphatidic acid containing palmitate preferentially esterified at position-2 and oleate at position-1 was the major product. Dihydroxyacetone phosphate and sn-glycerol 3-phosphate competitively inhibited each other's acylations, suggesting that a single enzyme might be responsible for both esterifications and oleate was the preferred substrate for the formation of acyldihydroxyacetone phosphate. The specificities of the acyl-CoA–1-monoacyl-sn-glycerol 3-phosphate and the acyl-CoA–2-monoacyl-sn-glycerol 3-phosphate acyltransferases were also studied. The specificities observed combined with the relative velocities of these reactions suggest that phosphatidic acid is formed in the mammary gland with the first acylation occurring at position-1 favouring oleate followed by the second acylation at position-2 favouring palmitate. This is consistent with the unusual structure found in the triacylglycerols of rat milk. When a mouse liver microsomal fraction was used the opposite specificities were observed consistent with the structure of the triacylglycerols of mouse liver. The microsomal acylation of the monoacyl-sn-glycerol 3-phosphocholines was also investigated. Although no marked acyl specificity could be detected when the 2-monoacyl-sn-glycerol 3-phosphocholine was used as the acyl acceptor, both oleate and linoleate were esterified in preference to palmitate to the 1-monoacyl-sn-glycerol 3-phosphocholine.     

Enzymatic aspects of the reduction of microsomal glycerolipid biosynthesis after perfusion of the liver with dibutyryl adenosine-3',5'-monophosphate.

Livers from fed male rats were perfused in a nonrecycling system for 60 min with a medium containing 100 mg/dl glucose, 3 g/dl bovine serum albumin, and ~0.5 mm oleic acid, with or without 20 μm dibutyryl cyclic adenosine-3′,5′-monophosphate (Bt₂cAMP). At the termination of the experiment, microsomes were isolated from these livers. In agreement with data reported previously, Bt₂cAMP decreased output of triacylglycerol, but stimulated ketogenesis and output of glucose; uptake of free fatty acid was unaffected by the nucleotide. Perfusion with Bt₂AMP decreased the biosynthesis of triacylglycerol, diacylglycerol, and phosphatidate from sn-[U-¹⁴C]glycerol-3-phosphate by microsomes isolated from these livers. Perfusion with Bt₂cAMP also decreased incorporation of sn-glycerol-3-phosphate into phosphatidate by microsomes isolated from the livers, when the microsomes were incubated with NaF to inhibit phosphatidate phosphohydrolase, and when fatty acid, coenzyme A and ATP were replaced by the acyl coenzyme A derivative; the formation of phosphatidate under these conditions was used as an estimate of the activity of sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15). However, the activities of microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) and diacylglycerol acyltransferase (EC 2.3.1.20), measured with microsomal bound substrate, were increased by Bt₂cAMP. These data have been interpreted to mean that Bt₂cAMP inhibits hepatic microsomal synthesis of triacylglycerol at a step prior to the formation of phosphatidate, presumably at the glycerophosphate acyltransferase (EC 2.3.1.15) step(s).     

Metabolic interrelations of hydroxy-substituted ether-linked glycerolipids in the pink portion of the rabbit harderian gland.

The pink portion of the rabbit harderian gland is known to contain a preponderance of ether-linked glycerolipids consisting primarily of 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols and smaller amounts of 1-alkyl-2,3-diacyl-sn-glycerols. In the present study, we have used a combination of chemical, enzymatic, and chromatographic techniques to identify two minor lipid components in the gland as 1-hydroxyalkyl-2-acyl-sn-glycerols and 1-hydroxyalkyl-2,3-diacyl-sn-glycerols. The long-chain acyl groups occurring in the 1-hydroxyalkyl-2-acyl-sn-glycerols and 1-hydroxyalkyl-2,3-diacyl-sn-glycerols are almost exclusively hexadecanoic acid, whereas the 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols have a ratio of hexadecanoic acid to octadecanoic acid of $\text{2}\text{1}$. The 1-(O-acyl) hydroxyalkyl-2,3-diacyl-sn-glycerols and the 1-hydroxyalkyl-2,3-diacyl-sn-glycerols also contain a short-chain acyl moiety identified as 3-methylbutanoic acid (isovaleric acid). This acid was found to occupy the 3-position of the glycerol backbone in these lipid classes.Metabolic experiments demonstrate that 3-methylbutanoic acid in the lipids of the gland is derived from the catabolism of l-leucine. Pulse-chase data show a precursor-product relation between the 1-hydroxyalkyl-2,3-diacyl-sn-glycerols and 1-(O-acyl-hydroxyalkyl-2,3-diacyl-sn-glycerols and rule out direct hydroxylation of 1-alkyl-2,3-diacyl-sn-glycerols as a possible biosynthetic route to the 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols.Characterization of the alkyl and acyl groups and the positional distributions of the acyl moieties in combination with the metabolic information indicated the acylation sequence involved in the formation of 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerol is 1-hydroxyalkyl-2-acyl-sn-glycerols → 1-hydroxyalkyl-2,3-diacyl-sn-glycerols → 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols. The data also suggest that hydroxylation of the alkyl side-chain occurs before or at the alkylacylglycerol stage.     

Synthesis of monoacid 2,3-diacyl-sn-glycerols via 1,6-ditrityl-d-mannitol

Stereochemically pure 2,3-dipalmitoyl-sn-glycerol and 2,3-dioleoyl-sn-glycerol were prepared in an overall yield of 20% by a new and facile method starting from D-mannitol. The synthetic intermediates were 1,6-ditrityl-D-mannitol (1), 1-trityl-sn-glycerol (2), and 1-trityl-2,3-diacyl-sn-glycerol (3). The key reaction was the oxidation of 1 with lead tetraacetate followed by reduction with sodium borohydride. The product (2) was readily separated from the only byproduct, tritylethyleneglycol.     

The relationship between palmitoyl-coenzyme A synthetase activity and esterification of sn-glycerol 3-phosphate in rat liver mitochondria

1. The specific activities for palmitoyl-CoA synthetase and for sn-glycerol 3-phosphate esterification, with palmitoyl-CoA generated either by the endogenous synthetase or from palmitoyl-(−)-carnitine, CoA and excess of carnitine palmitoyltransferase, were measured with rat liver mitochondria. 2. The mean specific activity of palmitoyl-CoA synthetase was approximately five- and seven-fold the rates of sn-glycerol 3-phosphate esterification from palmitate and palmitoyl-(−)-carnitine respectively. No significant correlation was found in different rats between the activities of palmitoyl-CoA synthetase and sn-glycerol 3-phosphate esterification from either acyl precursor. However, there was a significant correlation (r=0.83, P<0.001) between the rates of glycerolipid synthesis from palmitate and palmitoyl-(−)-carnitine. 3. The mean molar composition of the glycerolipid synthesized from palmitate was 58% lysophosphatidate, 31% phosphatidate and 11% neutral lipid. With palmitoyl-(−)-carnitine the equivalent values were 70, 23 and 7%, which were significantly different. 4. When palmitoyl-CoA synthetase had been inactivated by 60–70% after preincubation of mitochondria at 37°C, it became rate-limiting in glycerolipid biosynthesis. Additions of 1–5mm-ATP prevented inactivation of palmitoyl-CoA synthetase. 5. Preincubation also inhibited the oxidation of palmitate, palmitoyl-CoA, palmitoyl-(−)-carnitine and malate plus glutamate. These inhibitions could not be prevented by addition of ATP. 6. Diversion of palmitoyl-CoA to form palmitoyl-(−)-carnitine did not inhibit sn-glycerol 3-phosphate esterification. 7. The palmitoyl-CoA pool synthesized by the palmitoyl-CoA synthetase was augmented by adding partially purified synthetase or carnitine palmitoyltransferase and palmitoyl-(−)-carnitine. No stimulation of palmitate incorporation into glycerolipids occurred. 8. At low concentrations of Mg²⁺, palmitate, ATP and CoA the velocity with palmitoyl-CoA synthetase decreased more than that of glycerolipid synthesis from palmitate. 9. It is concluded that in the presence of optimum substrate concentrations the activity of sn-glycerol 3-phosphate acyltransferase and not of palmitoyl-CoA synthetase is rate-limiting in the synthesis of phosphatidate and lysophosphatidate in isolated rat liver mitochondria.     

Lysolecithin as regulator of de novo lecithin synthesis in rat liver microsomes.

1-Acyl-sn-glycero-3-phosphocholine (lysolecithin) was found to affect 1,2-diacyl-sn-glycerol:CDPcholine cholinephosphotransferase (CPT; EC 2.7.8.2) activity of rat liver microsomes in a concentration dependent, characteristic manner. Cholinephosphate transfer was activated at lysolecithin concentrations below 0.5 mM with a maximum stimulation occurring at 75–100 μM lysolecithin levels. At concentrations above 0.5 mM, CPT activity was inhibited by lysolecithin. It was shown that CPT inhibition by lysolecithin is competitive (Ki ≈ 0.6 mM) with respect to CDPcholine. The possible role of lysolecithin as regulator of de novo lecithin synthesis in vivo is outlined.     

Role of spermidine in the activity of sn-glycerol-3-phosphate acyltransferase from Escherichia coli

The integral membrane protein, sn-glycerol-3-phosphate acyltransferase, catalyzes the first committed step in phospholipid synthesis, and both acyl-CoA and acyl-acyl carrier protein can be used as acyl donors in this reaction. We found that spermidine increased the specific activity of the acyltransferase when either substrate was used as the acyl donor. Magnesium, as well as other cations, also increased acyltransferase activity but were not nearly as effective as spermidine. Two roles for spermidine in this reaction were deduced from our data. First, spermidine dramatically lowered the K_m for glycerol 3-phosphate resulting in an overall rate enhancement when either substrate was used as the acyl donor. This effect was attributed to the modification of the acyl-transferase environment due to the binding of spermidine to membrane phospholipids. A second effect of spermidine was evident only when acyl-acyl carrier protein was used as substrate. Using this acyl donor, a pH optimum of 7.5 was found in the absence of spermidine, but in its presence, the pH optimum was shifted to 8.5. Between pH 7.5 and 8.5, palmitoyl-acyl carrier protein undergoes a conformational change to a more expanded, denatured state and its activity in the acyltransferase assay decreases dramatically. Spermidine restored the native conformation of palmitoyl-acyl carrier protein at pH 8.5, thus accounting for the majority of rate enhancement observed at elevated pH.     

Chemical and physicochemical studies of lysylphosphatidylglycerol derivatives. Occurrence of a2' yields 3' lysyl migration

Syntheses of 1,2-didodecanoyl-sn-glycero-3-phosphoryl-1′-(3′-O-L-lysyl)-sn-glycerol (IV) and 1,2-didodecanoyl-sn-glycero-3-phosphoryl-1′-(2′-O-L-lysyl)-sn-glycerol (VIII) as well as 1,2-didodecanoyl-sn-glycerol-3-phosphoryl-1′-sn-glycerol (XII) are described. 2′- and 3′-lysylphosphatidylglycerol are obtained as pure isomers and can be distinguished spectroscopically (infrared, 100 and 300 MHZ NMR). By these criteria a migration of the lysyl group from the 2′ to the 3′ position of the glycerol occurs in the presence of a strong acid catalyst such as HCl. On the other hand, a weak acid such as acetic acid appears ineffective in inducing lysyl migration, even at very high concentrations.Spectroscopic analysis furthermore demonstrated that lysylphosphatidylglycerol extracted from the Staphylococcus aureus membrane, is a 3′-isomer.     

Formation of phospholipids from sn-glycerol-3-phosphate and free fatty acids or their derivatives by homogenates of soybean cotyledons

Cell-free homogenates of soybean cotyledons contain a sn-glycerol-3-phosphate acyltransferase system which incorporated [U-¹⁴C]-sn-glycerol-3-phosphate into 5 labelled lipids when incubated with palmitic acid in the presence of ATP and CoA. In decreasing order of incorporation of label, the lipids were: lysophosphatidic acid, monoacylglycerol, phosphatidic acid, diacylglycerol and triacylglycerol. The substrate specificity of the acyltransferase system was investigated with the fatty acids shown in order of decreasing rates of reaction; palmitate > stearate > oleate > linoleate > linolenate > laurate. Making these acids more soluble as triethanolamine salts or as polyoxyethylene sorbitan esters did not greatly enhance these rates of reaction. Activity was found in a 10000 g pellet containing plastids, mitochondria and glyoxysomes and also in the lipid layer; the activity in these particulate fractions was enhanced by the addition of cytosol which itself had little activity when gentle methods of cell disruption were used. During cotyledon development the total acyltransferase activity increased, although its specific activity slowly declined due to more rapid synthesis of other proteins. During germination total activity decreased but there was a transient increase in specific activity due to more rapid degradation of other proteins.     

An improved method for the preparation of 1,2-isopropylidene-SN-glycerol

A simple and fast route for the preparation of 1,2-isopropylidene-sn-glycerol from D-mannitol in 45% yield is described. The value of optical rotation, [α]_D²⁰ + 15.2°, is higher than usual indicating considerable racemization for other procedures. Since 1,2-isopropylidene-sn-glycerol serves as general intermediate for the synthesis of glycerides and of phosphoglycerides these lipids contain substantial amounts of the isomer, for instance 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine may consist of up to 15% of 2,3-dipalmitoyl-sn-glycerol-1-phosphocholine in earlier preparations.     

Transient inhibition by ribose 5-phosphate of photosynthetic O2 evolution in a reconstituted chloroplast system

Photosynthetic oxygen evolution by a reconstituted chloroplast system utilising sn-phospho-3-glycerol (3-phosphoglycerate) ceases upon the addition of ribose 5-phosphate even though the presence of this metabolite permits a rapid and immediate CO₂ fixation. The period of cessation is appreciable at 0.1 mM ribose 5-phosphate. It is lengthened as the amount of added ribose 5-phosphate is increased and by the addition of dithiothreitol, a known activator of ribulose-5-phosphate kinase. Ribulose 1,5-bisphosphate is without effect. A similar interruption of O₂ evolution may also be brought about by the addition of ADP or by ADP-generating systems such as glucose plus hexokinase. Spectrophotometric experiments indicate that the reoxidation of NADPH in the presence of sn-phospho-3-glycerol is similarly affected.The transient inhibition by ribose 5-phosphate is not observed in the presence of an active ATP-generating system or in the presence of sufficient dl-glyceraldehyde to inhibit ribulose-5-phosphate kinase activity.It is concluded that ribose 5-phosphate inhibits photosynthetic O₂ evolution by adversely affecting the steady-state ATP/ADP ratio and consequently the reduction of sn-phospho-3-glycerol to glyceraldehyde 3-phosphate. The results are discussed in their relation to ADP regulation of photosynthetic carbon assimilation and metabolite transport.     

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252–6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 μmol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.     

SUITABILITY OF DIFFERENT MOLECULAR SPECIES OF 1,2-DIACYLGLYCEROLS AS SUBSTRATES FOR DIACYLGLYCEROL KINASE IN RAT BRAIN MICROSOMES

The relative suitability of different molecular species of 1,2-diacyl-sn-glycerols as substrates for the diacylglycerol kinase (ATP: 1,2-diacyl-sn-glycerol phosphotransferase) in rat brain microsomes was investigated. The diacylglycerols tested were a mixture of the 1-[³H]palmitoyl and 1-[¹⁴C]stearoyl homologues of either the 2-oleoyl (monoenoic), 2-linoleoyl (dienoic), 2-arachidonoyl (tetraenoic), or 2-docosahexaenoyl (hexaenoic) diacylglycerols with an isotope ratio (³H/¹⁴C) approximately equal to 1.00. At substrate concentrations of 0.125 mM and 0.60 mM, only a modest preference of the kinase for total (1-palmitoyl plus 1-stearoyl homologues) monoenoic over total hexaenoic species was indicated. The tetraenoic diacylglycerols gave reaction rates which were not significantly different from the monoenes, dienes, or hexaenes when the data were analyzed statistically. No significant enzyme selectivity for either the 1-palmitoyl or 1-stearoyl homologues of the various 1-saturated 2-unsaturated diacylglycerols was apparent. The present results, together with data on the composition of free 1,2-diacylglycerols in brain, which reveal a preponderance of tetraenoic molecular species, suggest that the tetraenoic phosphatidic acids (mainly as 1-stearoyl 2-arachidonoyl species) are quite possibly the major products of diacylglycerol kinase activity in rat brain under physiological conditions.     

Coupling of ethanol metabolism to lipid biosynthesis: labelling of the glycerol moieties of sn-glycerol-3-phosphate,a phosphatidic acid and a phosphatidylcholine in liver of rats given [1,1-2H2]ethanol

The mechanism behind ethanol-induced fatty liver was investigated by administration of [1,1-²H₂]ethanol to rats and analysis of intermediates in lipid biosynthesis. Phosphatidic acid and phosphatidylcholine were isolated by chromatography on a lipophilic anion exchanger and molecular species were isolated by high-performance liquid chromatography in a non-aqueous system. The glycerol moieties of palmitoyl-linoleoylphosphatidic acid, the corresponding phosphatidylcholine and free sn-glycerol-3-phosphate were analysed by GC/MS of methyl ester t-butyldimethylsilyl derivatives. The deuterium labelling in the glycerol moiety of the phosphatidic acid was 2–3-times higher than in free sn-glycerol-3-phosphate, indicating that a specific pool of sn-glycerol-3-phosphate was used for the synthesis of phosphatidic acid in liver. The results indicate that NADH formed during ethanol oxidation is used in the formation of a pool of sn-glycerol-3-phosphate that gives rise to triacylglycerol and possibly fatty liver.     

The effect of cadmium ions and cadmium-metallothionein on the activities of phospholipid-synthesizing enzymes of rat liver microsomes in vitro

The effects of cadmium ions or cadmium-metallothionein on the activities of acyl-CoA:1acyl-sn-glycerol 3-phosphoric acid or 1-acyl-sn-glycero 3-phosphocholine acyltransferase of rat liver microsomes have been studied, in vitro. Cadmium ions were found to cause a noncompetitive type inhibition of these two acyltransferases. The K_i values were calculated, and found to be smallest (1.7 × 10^?5m) for palmitoyl-CoA and greatest (1.0 × 10^?4m) for linoleoyl-CoA, among the several fatty acyl-CoA's tested on the 1-acyl-sn-glycerol 3-phosphoric acid acyltransferases. With the 1-acyl-sn-glycero 3-phosphocholine acyltransferase, the K_i values were found to be smallest for the plamitoyl-CoA acyltransferase (3.8 × 10^?5m) and largest for thearachidonoyl-CoA acyltransferase (1.1 × 10^?4m). In contrast, mouse liver cadmium-metallothionein, including 4 mol of cadmium and 2 mol of zinc in one molecule of metallothionein, was not found to be inhibitory or rather stimulative on the above two acyltransferases at the same concentration of cadmium tested in the cadmium ion inhibitor experiments. The above results demonstrate that there is a strong and irreversible inhibition by cadmium ions on acyl-CoA acyltransferases, but that when cadmium acts on the enzyme in the form of a cadmium-metallothionein complex, the inhibition effect does not occur. These findings may reflect differing degrees of toxicity of these two types of cadmium compounds in mammalian tissues.     

Studies on Phospholipid-synthesizing Enzyme Activities during the Growth of Etiolated Cucumber Cotyledons

The enzymatic incorporation of sn-glycerol 3-phosphate into lipid by extracts of cucumber (Cucumis sativus) cotyledons showed an absolute requirement for ATP (saturation 2 mM). The incorporation was stimulated 4-fold by 0.2 mM oleate. Ethyldiaminetetraacetate stimulated the incorporation at concentrations below 1 mM and inhibited at higher concentrations. Mg²⁺ did not affect the reaction. Triton X-100 and Cutscum inhibited the reaction, while a third detergent, Span 80, was stimulatory. p-Mercuribenzoate was inhibitory. The enzymatic reaction has a pH optimum in the range of 8.8 to 9.6. The Michaelis constant was 112 μM for sn-glycerol 3-phosphate. The major amount of product was phosphatidic acid, the remainder was diacylglycerol, monoacylglycerol, and an unknown phospholipid.     

Solubilization of microsomal-associated phosphatidylinositol synthase from germinating soybeans

CDP-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):myo-inositol phosphatidyltransferase (EC 2.7.8.11, phosphatidylinositol synthase) catalyzes the final step in the de novo synthesis of phosphatidylinositol in the endoplasmic reticulum fraction of germinating soybeans (Glycine max L. var Cutler 71). A variety of solubilization agents were examined for their ability to release phosphatidylinositol synthase activity from the microsome fraction. The most effective agent to solubilize the enzyme was the nonionic detergent Brij W-1. A 2.1-fold increase in specific activity was achieved using 1% Brij W-1 with 69% activity solubilized.