Glycerolipid biosynthesis in rat adipose tissue. Effect of polyamines on triglyceride synthesis.

Triacylglycerol formation from sn-glycerol 3-phosphate and 1,2-diacyl-sn-glycerol was markedly elevated in the presence of spermine and spermidine. This was attributed to the activation of microsomal sn-glycerol 3-phosphate acyltransferase and 1,2-diacyl-sn-glycerol acyltransferase and to the inhibition of palmitoyl-CoA hydrolase. Spermine was more effective than spermidine, and putrescine did not stimulate triacylglycerol formation. The stimulatory effect of spermine on triacylglycerol-forming enzymes was observed in the presence of Mg²⁺ and was apparent in the presence or absence of bovine serum albumin. The activation of 1,2-diacyl-sn-glycerol acyltransferase by spermine was specific, and other diacylglycerol-utilizing enzymes were not affected under these conditions. These studies demonstrate that polyamines may be important regulators of triacylglycerol formation in adipose tissue.     

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.     

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.     

The relationship between palmitoyl-coenzyme A synthetase activity and esterification of sn-glycerol 3-phosphate by the microsomal fraction of guinea-pig intestinal mucosa

1. With microsomal fractions of guinea-pig intestinal mucosa the mean specific activity of palmitoyl-CoA synthetase was approx. 1.3-fold the esterification of sn-glycerol 3-phosphate with palmitoyl-CoA generated by the endogenous synthetase. The latter activity was approx. 2.5- and 5-fold that when palmitoyl-CoA was generated from palmitoylcarnitine or when it was added directly to the assay system. 2. There were significant correlations (P<0.001) between the specific activities of palmitoyl-CoA synthetase and glycerolipid synthesis from either palmitate or palmitoylcarnitine. 3. The mean molar composition of glycerolipid synthesized from palmitate or palmitoylcarnitine was approx. 18% lysophosphatidate, 75% phosphatidate and 7% neutral lipid. 4. Glycerolipid synthesis from palmitate was inhibited by 80–90% after preincubation of microsomal fractions at 37°C for 40min and was caused by inactivation of palmitoyl-CoA synthetase. 5. Addition of 100–400mm-KCl inhibited palmitoyl-CoA synthetase activity and glycerolipid synthesis from palmitate but stimulated glycerol phosphate acyltransferase activity. 6. Diversion of palmitoyl-CoA synthesized by the endogenous synthetase to palmitoylcarnitine resulted in an almost stoicheiometric decrease in glycerolipid synthesis. 7. Addition of rac-1-monopalmitin promoted utilization of palmitoyl-CoA by the monoglyceride pathway but did not inhibit phosphatidate biosynthesis. 8. With rate-limiting concentrations of CoA and Mg²⁺ the relative decreases in velocity for palmitoyl-CoA synthetase and glycerolipid synthesis from palmitate were almost identical. However, low concentrations of palmitate and ATP produced greater decreases in synthetase activity than in glycerolipid synthesis. 9. There appears to be a fine balance between the activities of palmitoyl-CoA synthetase and glycerol phosphate acyltransferase, with neither activity being in excess with respect to phosphatidate synthesis.     

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).     

Topography of phosphatidylcholine, phosphatidylethanolamine and triacylgycerol biosynthetic enzymes in rat liver microsomes

The topography of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol biosynthetic enzymes within the transverse plane of rat liver microsomes was investigated using two impermeant inhibitors, mercury-dextran and dextran-maleimide. Between 70 and 98% of the activities of fatty acid : CoA ligase (EC 6.2.1.3), sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were inactivated by mercury-dextran. Dextran-maleimide caused 52% inactivation of the sn-glycerol-3-phosphate acyltransferase. Inactivation of each of these activities except fatty acid : CoA ligase occurred in microsomal vesicles which remained intact as evidenced by the maintenance of highly latent mannose-6-phosphatase activity (EC 3.1.3.9). These glycerolipid biosynthetic activities were not latent, indicating that substrates have free access to the active sites. Moreover, ATP, CDP-choline and CMP appeared unable to penetrate the microsome membrane. These data indicate that the active sites of thease enzymes are located on the external surface of microsomal vesicles. It is concluded that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.     

Enhancement and inhibition of enzymes of heme metabolism by diethyl maleate in the rat kidney

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.     

Evidence for the reversibility of the acyl-CoA:lysophosphatidylcholine acyltransferase in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons and rat liver.

Acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine occurs in the microsomal preparations of developing safflower cotyledons. Evidence is presented to show that the acyl exchange is catalysed by the combined back and forward reactions of an acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23). The back reaction of the enzyme was demonstrated by the stimulation of the acyl exchange with free CoA and by the observation that the added CoA was acylated with acyl groups from position 2 of sn-phosphatidylcholine. Re-acylation of the, endogenously produced, lysophosphatidylcholine with added acyl-CoA occurred with the same specificity as that observed with added palmitoyl lysophosphatidylcholine. A similar acyl exchange, catalysed by an acyl-CoA:lysophosphatidylcholine acyltransferase, occurred in microsomal preparations of rat liver. The enzyme from safflower had a high specificity for oleate and linoleate, whereas arachidonate was the preferred acyl group in the rat liver microsomal preparations. The rate of the back reaction was 3-5% and 0.2-0.4% of the forward reaction in the microsomal preparations of safflower and rat liver respectively. Previous observations, that the acyl exchange in safflower microsomal preparations was stimulated by bovine serum albumin and sn-glycerol 3-phosphate, can now be explained by the lowered acyl-CoA concentrations in the incubation mixture with albumin and in the increase in free CoA in the presence of sn-glycerol 3-phosphate (by rapid acylation of sn-glycerol 3-phosphate with acyl groups from acyl-CoA to yield phosphatidic acid). Bovine serum albumin and sn-glycerol 3-phosphate, therefore, shift the equilibrium in acyl-CoA:lysophosphatidylcholine acyltransferase-catalysed reactions towards the rate-limiting step in the acyl exchange process, namely the removal of acyl groups from phosphatidylcholine. The possible role of the acyl exchange in the transfer of acyl groups between complex lipids is discussed.     

sn-Glycerol-3-phosphate acyltransferases in plants

sn-Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation at sn-1 position of glycerol-3-phosphate to produce lysophosphatidic acid (LPA). LPA is an important intermediate for the formation of different types of acyl-lipids, such as extracellular lipid polyesters, storage and membrane lipids. Three types of GPAT have been found in plants, localizing to the plastid, endoplasmic reticulum, and mitochondria. These GPATs are involved in several lipid biosynthetic pathways and play important biological roles in plant development. In the present review, we will focus on the recent progress in studying the physiological functions of GPATs and their metabolic roles in glycerolipid biosynthesis.     

Inhibition of palmitoyl CoA of EDTA- and Mg2+-ATPase of heavy meromyosin from rabbit skeletal muscle

Palmitoyl CoA inhibited EDTA-ATPase of heavy meromyosin (HMM) prepared from rabbit skeletal muscle. The concentration for half maximum inhibition of EDTA-ATPase was about 18 microM. Myristoyl CoA, the other long chain fatty acyl CoA, also inhibited EDTA-HMM ATPase, but CoA and short chain CoA thioesters, such as butyryl CoA, acetoacetyl CoA and acetyl CoA, at 40 microM hardly inhibited EDTA-ATPase. Less than 20% inhibition of EDTA-HMM ATPase was obtained with Na-palmitate and Na-myristate at 40 microM, whereas about 90% inhibition of the enzyme occurred in the presence of 40 microM palmitoyl CoA and myristoyl CoA. Palmitoyl carnitine, as well as carnitine, failed to inhibit EDTA-HMM-ATPase. The inhibition of palmitoyl CoA of EDTA-ATPase was reversed by bovine serum albumin and spermine. Mg2+-HMM ATPase activity was enhanced by palmitoyl CoA at 2, 5, and 10 microM. About a 25% increase in Mg2+-HMM ATPase activity was obtained at 5 and 10 microM. At higher concentrations than 20 microM, the enzyme was inhibited by palmitoyl CoA and the degree of inhibition was related to the concentration of the CoA thioester. At 80 microM, the activity was about 15% of the maximum value. The efficacy of myristoyl CoA on Mg2+-ATPase was almost the same as that of palmitoyl CoA. Mg2+-ATPase activity was not enhanced by CoA, butyryl CoA, acetoacetyl CoA, Na-myristate, Na-palmitate, palmitoyl carnitine, or carnitine at 10 microM, and was hardly reduced by these substances at 40 microM. Serum albumin and spermine also canceled, to some extent, these effects of palmitoyl CoA on Mg2+-ATPase.     

Acyl coenzyme A: cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at −40°C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

Acyl coenzyme A:cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at -40 degrees C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

Glycerolipid biosynthesis in rat adipose tissue. I. Properties and distribution of glycerophosphate acyltransferase and effect of divalent cations on neutral lipid formation

A sensitive radioactive assay of acyl CoA:sn-glycerol-3-phosphate-O-acyltransferase (EC 2.3.1.15) was developed to study the properties and subcellular distribution of this enzyme in rat epididymal adipose tissue. The esterification of sn-glycerol-3-phosphate was measured in the presence of palmitoyl CoA or palmitate, ATP, CoA, and Mg(2+) at pH 7.5. The presence of glycerophosphate acyltransferase was detected in both mitochondria and microsomes. The product of this reaction was identified as phosphatidate by thin-layer chromatography and dual isotope incorporation studies. Several divalent cations reduced the activity of this enzyme. Although Mg(2+) was not required for the activity of glycerophosphate acyltransferase, its addition to the incubation mixture resulted in an increased formation of neutral lipids at the expense of phosphatidate. This result is explained by an activation of microsomal phosphatidate phosphatase (EC 3.1.3.4). The effect of Mg(2+) was completely abolished by Ni(2+), Co(2+), Mn(2+), and Zn(2+). These studies suggest that the balance between Mg(2+) and several other divalent ions may be important in the regulation of neutral lipid synthesis in adipose tissue.     

Inhibition by ozone of the acylation of glycerol 3-phosphate in mitochondria and microsomes from rat lung

The synthesis of lipids from [U-¹⁴C]glycerol 3-phosphate by mitochondrial or microsomal fractions from rat lung was inhibited by ozone. The susceptible reaction was the first acylation of glycerol 3-phosphate. Enzymes unaffected by the ozone exposure included: acyl-CoA thioesterase, acyl-CoA thiokinase, acyl-CoA:acylglycerol 3-phosphate acyltransferase, acyl-CoA:diacylglycerol acyltransferase, and acyl-CoA:acylglycerophosphocholine acyltransferase. The effect of ozone on lipid synthesis is closely comparable to the inhibition by N-ethylmaleimide suggesting that the effect of ozone is the oxidation of enzyme sulfhydryl groups. There was no indication of lipid oxidation caused by ozone and no indication of the production of a stable toxic compound.     

Topography of phosphatidylcholine,phosphatidylethanolamine and triacylglycerol biosynthetic enzymes in rat liver microsomes

The topography of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol biosynthetic enzymes within the transverse plane of rat liver microsomes was investigated using two impermeant inhibitors, mercury-dextran and dextran-maleimide. Between 70 and 98% of the activities of fatty acid : CoA ligase (EC 6.2.1.3), $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were inactivated by mercury-dextran. Dextran-maleimide caused 52% inactivation of the $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase. Inactivation of each of these activities except fatty acid : CoA ligase occurred in microsomal vesicles which remained intact as evidenced by the maintenance of highly latent mannose-6-phosphatase activity (EC 3.1.3.9). These glycerolipid biosynthetic activities were not latent, indicating that substrates have free access to the active sites. Moreover, ATP, CDP-choline and CMP appeared unable to penetrate the microsome membrane. These data indicate that the active sites of these enzymes are located on the external surface of microsomal vesicles.It is concluded that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.     

Triacylglycerol biosynthesis in developing seeds of Tropaeolum majus L. and Limnanthes douglasii R. Br.

Triacylglycerols of both Tropaeolum majus L. and Limnanthes douglasii R. Br. are predominantly esterified with very long-chain acyl groups at each position of the glycerol backbone. In order to elucidate whether these acyl groups are directly chanelled into the triacylglycerols via the stepwise acylation of glycerol-3-phosphate, seed oil formation has been investigated in developing embryos of both plant species. [1-¹⁴C]Acetate labelling experiments using embryos at different stages of development, as well as the determination of the properties of the microsomal acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) and acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51), revealed differences between the two plant species, especially with respect to the incorporation of very longchain acyl groups into the C2 position of the triacylglycerols. In microsomal fractions of developing embryos of L. douglasii both a glycerol-3-phosphate and a 1-acylglycerol-3-phosphate acyltransferase were detected which utilize very long-chain acyl-CoA thioesters as substrates. Thus, in seeds of L. douglasii very long-chain acyl groups can enter not only the C1, but also the C2 position of the triacylglycerols in the course of de-novo biosynthesis. A comparison of the properties of the acyltransferases of developing embryos with those of the corresponding activities of leaves indicates an embryo specific expression of an erucoyl-CoA-dependent microsomal 1-acylglycerol-3-phosphate acyltransferase in L. douglasii. The microsomal glycerol-3-phosphate acyltransferase of developing embryos of T. majus displayed properties very similar to those of the corresponding activity of L. douglasii. On the other hand, the microsomal 1-acylglycerol-3-phosphate acyltransferases of the two plant species showed strikingly different substrate specificities. Irrespective of the acyl groups of 1-acylglycerol-3-phosphate and regardless of whether acyl-CoA thioesters were offered separately or in mixtures, the enzyme of T. majus, in contrast to that of L. douglasii, was inactive with erucoyl-CoA. These results of the enzyme studies correspond well with those of the [1-¹⁴C]acetate labelling experiments and thus indicate that T. majus has developed mechanisms different from those of L. douglasii for the incorporation of erucic acid into the C2 position of its triacylglycerols.Abbreviations GPAT acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) - LPAT acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51) This work was supported by the Bundesministerium für Forschung und Technologie (Förderkennzeichen 0316600A).     

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.     

Purification of acyl CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase

Acyl coenzyme A:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase (EC 2.3.1.23) is capable of forming lipid bilayer vesicles from its soluble substrates lysophosphatidylcholine (LPC) and oleoyl CoA. This suggested a purification method in which rat liver microsomes are first washed with deoxycholate to increase specific activity of the endogenous acyltransferase approximately fivefold, then solubilized by the detergent effect of excess LPC and oleoyl CoA in 1:1 stoichiometric ratios. As the LPC is converted to phosphatidylcholine by acyl group transfer, the detergent effect is lost and lipid vesicles containing the enzyme activity are produced. Other microsomal proteins are excluded from the vesicles. The vesicles may be separated by density gradient flotation and are found to contain acyltransferase with a specific activity of 9–10 µmol/mg/min. This reflects a purification of approximately 140-fold, about ten times greater than achieved in previous studies.     

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.     

Solubilization and partial purification of dihydroxyacetone-phosphate acyltransferase from guinea pig liver

Dihydroxyacetone-phosphate:acyl coenzyme A acyltransferase (EC 2.3.1.42) was solubilized and partially purified from guinea pig liver crude peroxisomal fraction. The peroxisomal membrane was isolated after osmotic shock treatment and the bound dihydroxyacetone-phosphate acyltransferase was solubilized by treatment with a mixture of KCl-sodium cholate. The solubilized enzyme was partially purified by ammonium sulfate fractionation followed by Sepharose 6B gel filtration. The enzyme was purified 1200-fold relative to the guinea pig liver homogenate and 80- to 100-fold from the crude peroxisomal fraction, with an overall yield of 25–30% from peroxisomes. The partially purified enzyme was stimulated two- to fourfold by Asolectin (a soybean phospholipid preparation), and also by individual classes of phospholipid such as phosphatidylcholine and phosphatidylglycerol. The kinetic properties of the enzyme showed that in the absence of Asolectin there was a discontinuity in the reciprocal plot indicating two different apparent K_m values (0.1 and 0.5 mm) for dihydroxyacetone phosphate. The V_max was 333 nmol/min/mg protein. In the presence of Asolectin the reciprocal plot was linear, with a K_m = 0.1 mm and no change in V_max. The enzyme catalyzed both an exchange of acyl groups between dihydroxyacetone phosphate and palmitoyl dihydroxyacetone phosphate in the presence of CoA and the formation of palmitoyl [³H]coenzyme A from palmitoyl dihydroxyacetone phosphate and [³H]coenzyme A, indicating that the reaction is reversible. The partially purified enzyme preparation had negligible glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity.