Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana.

During a search for cDNAs encoding plant sterol acyltransferases, we isolated four full-length cDNAs from Arabidopsis thaliana that encode proteins with substantial identity with animal lecithin : cholesterol acyltransferases (LCATs). The expression of one of these cDNAs, AtLCAT3 (At3g03310), in various yeast strains resulted in the doubling of the triacylglycerol content. Furthermore, a complete lipid analysis of the transformed wild-type yeast showed that its phospholipid content was lower than that of the control (void plasmid-transformed) yeast whereas lysophospholipids and free fatty acids increased. When microsomes from the AtLCAT3-transformed yeast were incubated with di-[1-14C]oleyl phosphatidylcholine, both the lysophospholipid and free fatty acid fractions were highly and similarly labelled, whereas the same incubation with microsomes from the control yeast produced a negligible labelling of these fractions. Moreover when microsomes from AtLCAT3-transformed yeast were incubated with either sn-1- or sn-2-[1-14C]acyl phosphatidylcholine, the distribution of the labelling between the free fatty acid and the lysophosphatidylcholine fractions strongly suggested a phospholipase A1 activity for AtLCAT3. The sn-1 specificity of this phospholipase was confirmed by gas chromatography analysis of the hydrolysis of 1-myristoyl, 2-oleyl phosphatidylcholine. Phosphatidylethanolamine and phosphatidic acid were shown to be also hydrolysed by AtLCAT3, although less efficiently than phosphatidylcholine. Lysophospatidylcholine was a weak substrate whereas tripalmitoylglycerol and cholesteryl oleate were not hydrolysed at all. This novel A. thaliana phospholipase A1 shows optimal activity at pH 6-6.5 and 60-65 degrees C and appears to be unaffected by Ca2+. Its sequence is unrelated to all other known phospholipases. Further studies are in progress to elucidate its physiological role.

Substrate selectivity of plant and microbial lysophosphatidic acid acyltransferases.

Linoleic acid (18:2) is found in a large variety of plant oils but to date there is limited knowledge about the substrate selectivity of acyltransferases required for its incorporation into storage triacylglycerols. We have compared the incorporation of oleoyl (18:1) and linoleoyl (18:2) acyl-CoAs onto lysophosphatidic acid acceptors by sub-cellular fractions prepared from a variety of plant and microbial species. Our assays demonstrated: (1). All lysophosphatidic acid acyltransferase (LPA-AT) enzymes tested incorporated 18:2 acyl groups when presented with an equimolar mix of 18:1 and 18:2 acyl-CoA substrates. The ratio of 18:1 to 18:2 incorporation into phosphatidic acid varied between 0.4 and 1.4, indicating low selectivity between these substrates. (2). The presence of either stearoyl (18:0) or oleoyl (18:1) groups at the sn-1 position of lysophosphatidic acid did not affect the selectivity of incorporation of 18:1 or 18:2 into the sn-2 position of phosphatidic acid. (3). All LPA-AT enzymes tested incorporated the saturated palmitoyl (16:0) acyl group from equimolar mixtures of 16:0- and 18:1-CoA. The ratios of 18:1 to 16:0 incorporation are generally much higher than those of 18:1 to 18:2 incorporation, varying between 2.1 and 8.6. (4). The LPA-AT from oil palm kernel is an exception as 18:1 and 16:0 are utilised at comparable rates. These results show that, in the majority of species examined, there is no correlation between the final sn-2 composition of oil or membrane lipids and the ability of an LPA-AT to use 18:2 as a substrate in in vitro assays.

The treatment of purified maize oil bodies with organic solvents and exogenous diacylglycerol allows the detection and solubilization of diacylglycerol acyltransferase.

In spite of its importance in the biosynthesis of reserve oils in plants, diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) has not been purified to homogeneity, and its study has remained incomplete. We found that the microsomal preparations from developing maize embryos contained substantial amounts of endogenous diacylglycerol (DAG). A solubilization procedure for extracting DAGAT from the microsomes (D. Little, R. Weselake, K. Pomeroy, S.T. Furukawa, J. Bagu, Biochem. J. 304 (1994)) was ineffective in eliminating the endogenous DAG, even after gel filtration. DAG removal through the preparation of acetone powders from the embryos led to the loss of DAGAT activity. Labelled triacylglycerol (TAG) was produced in the standard DAGAT assay when labelled DAG was supplied in benzene solution to the freeze-dried microsomes and the sample was dried and resuspended in an aqueous buffer. In contrast, no labelled TAG was produced when a similar sample supplied with non-labelled DAG was assayed with emulsified labelled DAG and acyl-CoA. Repeated washing of the microsomal freeze-dried fraction with benzene resulted in a complete loss of DAGAT activity in the standard assay, but the activity was restored by the addition of DAG plus phosphatidylcholine or Tween 20 in benzene. Although DAGAT has been reported to be confined mainly to the endoplasmic reticulum, we found that DAGAT activity was high in the purified oil bodies from both developing and mature maize embryos and was not removed by repeated washing with 6 M urea. The DAGAT activity was restored from delipidated oil bodies and from microsomes after the preparations had been resuspended in methanol/acetic acid/water (1:1:1, v/v). Although most of the proteins in the suspension were eluted as a single peak at the void volume after gel filtration chromatography, DAGAT activity was found in later fractions. SDS-PAGE of the peak activity fraction revealed no protein bands after silver staining, and the finding suggest that DAGAT protein is of low abundance and has a high k(cat).

N-(4-hydroxyphenyl)-retinamide increases lecithin:retinol acyltransferase activity in rat liver.

N-(4-Hydroxyphenyl)-retinamide (4-HPR; Fenretinide) is a synthetic retinoid which is undergoing investigation as a cancer chemopreventive agent. However, 4-HPR alters vitamin A kinetics and reduces the concentration of plasma retinol. We have conducted studies to examine the effects of 4-HPR on the activity of the enzyme lecithin:retinol acyltransferase (LRAT). This enzyme is implicated in the absorption and storage of vitamin A and is regulated, in liver, by vitamin A nutritional status. To determine whether 4-HPR, like retinoic acid, is able to induce liver LRAT activity, vitamin A-deficient rats having negligible liver LRAT activity were treated with single doses of 4-HPR (0.02-2.5 mg) and liver homogenates were assayed for LRAT activity using 3H-retinol bound to the cellular-retinol binding protein, CRBP, as substrate. Treatment with 4-HPR resulted in a dose- and time-dependent increase in liver LRAT activity which reached a maximum at 24 h. The activity of LRAT assayed in vitro and of hepatic 3H-retinyl ester content determined after an in vivo pulse of 3H-retinol were highly correlated (r = 0.802, P < 0.0002). When vitamin A-sufficient rats were fed a 4-HPR-supplemented diet for 30 d, LRAT activity differed significantly from control values in the liver (P < 0.0001) but not the small intestines. Changes in hepatic retinol metabolism which favor the esterification of vitamin A may be related to the mechanism by which 4-HPR alters vitamin A kinetics in vivo.

Dietary medium-chain fatty acids raise and (n-3) polyunsaturated fatty acids lower hepatic triacylglycerol synthesis in rats.

The hypothesis tested was that dietary medium-chain or (n-3) polyunsaturated fatty acids, when compared with (n-6) polyunsaturated fatty acids, alter plasma triacylglycerol levels by affecting hepatic triacylglycerol synthesis as reflected by the activities of acetyl-CoA carboxylase, fatty acid synthase and diacylglycerol acyltransferase in liver. In two separate experiments rats were fed purified diets containing (n-6) polyunsaturated fatty acids in the form of corn oil and either (n-3) polyunsaturated fatty acids in the form of fish oil or medium-chain triacylglycerols (MCT). Consumption of MCT significantly raised plasma triacylglycerol concentrations, whereas fish oil feeding had a lowering effect compared with the corn oil-fed group. In individual rats, the hepatic triacylglycerol concentration was directly correlated with the plasma triacylglycerol concentration (r = 0.60, P < 0.001). The MCT oil diet vs. the corn oil diet markedly raised the activities of hepatic acetyl-CoA carboxylase, fatty acid synthase and diacylglycerol acyltransferase. In the rats fed fish oil, the activities of fatty acid synthase and diacylglycerol acyltransferase were significantly reduced, whereas the activity of acetyl-CoA carboxylase was not affected relative to activities in rats fed corn oil. The activities of the three enzymes were directly correlated with plasma triacylglycerol concentrations in individual rats (r = 0.60-0.75, P < 0.001). The type of fat in the diet probably affects the rate of hepatic triacylglycerol synthesis which is an important determinant of plasma triacylglycerol concentrations.

Glutaminyl-cyclase expression in the bovine/porcine hypothalamus and pituitary.

The expression of the glutaminyl cyclase (QC), an enzyme responsible for the post-translational modification of N-terminal pyroglutamyl residues of neuropeptide precursors, was examined in bovine/porcine hypothalamic and pituitary tissue by means of immunocytochemistry and in situ hybridization. In the anterior pituitary a distinct pattern of QC immunoreactivity and mRNA expression was found associated exclusively with somatotrophs. In corticotrophs of the pars intermedia QC expression was undetectable, whereas a small portion of putative pars tuberalis cells in the rostral part of the pars distalis were heavily labelled. The neurointermediate lobe was devoid of signals for QC mRNA, but showed significant QC-immunoreactivity on secretory granules of axonal nerve endings. Also nuclei of the hypothalamus were found to be positive for QC-immunoreactivity. Intense labelling was observed in the nucleus supraopticus and nucleus paraventricularis. Staining of the nucleus periventricularis was found to be moderate, whereas no labelling of perikarya in the nucleus arcuatus, the preoptic area and the nucleus suprachiasmaticus was detectable. Moreover, varicose fibers stained positive for QC-immunoreactivity and could be identified in the main transport route from the hypothalamus to the pars neuronalis (tractus hypothalamo hypophysialis). These results suggest that the enzyme is transported via the same routes as its substrate/product to the median eminence or the neural lobe. Furthermore, the mapping of the cellular QC distribution reveals a strikingly distinctive expression pattern, that should be useful for the identification of yet undiscovered places of peptide synthesis and processing.

Location of glycerol phosphate acyltransferase in the transverse plane of mitochondrial outer membrane of guinea pig lung.

Incubation of guinea pig lung mitochondrial suspension in an isotonic low ionic strength buffer containing various proteolytic enzymes caused significant stimulation of the glycerophosphate acyltransferase activity. The maximal stimulation range between 20 and 105%, and the order was as follows: bromelain greater than chymotrypsin greater than pronase greater than trypsin greater than papain greater than nagarse. Under hypotonic conditions, over 85% of GAT was destroyed by all the proteolytic enzymes. Microsomal enzyme activity was consistently inhibited (greater than 95%) by exposure to any of these proteases even under isotonic conditions. These results suggest that GAT is located on the inner aspect of the mitochondrial outer membrane. Also, it is likely that a portion of this enzyme or that of a modulator is present in the outer side of the outer membrane and proteolysis of this component causes stimulation.

Method for detection of enzymatic activities which use aminoacyl-tRNA as a substrate.

A method for detection of enzymatic activities which use aminoacyl-tRNA is described. It is based on the synthesis of aminoacyl-tRNA in the reaction mixture (in situ) without additional purification. The results are the same as when using purified AA-tRNA.

Effects of morphone on choline acetyltransferase levels in the caudate nucleus of the rat.

1. Choline acetyltransferase (choline-o-acetyltransferase 2.3.1.6.) concentrations were determined in the caudate nucleus, thalamus, and cortex of control and morphine treated rats. The enzyme was assayed using a modified radiochemical method on a number of selected days, one hour after the last injection of 30 mg/kg of morphine and also during the subsequent phase of abstinence from morphine.2. Significant lowering of choline acetyltransferase activity in the caudate nucleus area was found in two cases, one hour after the first dose of morphine and upon subsequent abstinence from morphine.3. The enzyme activity in the two other parts of the brain remained at the normal levels.4. The presence of endogenous inhibitors formed during morphine administration was excluded.5. The relationship of a possible effect of morphine on the tissue binding of the enzyme and the subsequent lowering of its activity was tested by homogenization of the caudate nucleus area in different media. The decrease in enzyme activity occurred in all extraction media one hour after morphine administration.6. Inhibitory effects of in vitro addition of morphine to caudate nucleus homogenate, obtained from normal and morphine treated rats, were found to occur only at very high concentrations of the drug, negating the possibility of direct inhibitory effects of morphine.7. These experiments suggest two possible causes of the observed effects, which can be responsible for the lowering of enzyme activity, and can be operative simultaneously: (1) a negative feedback mechanism of accumulated acetylcholine, occurring after the first dose of morphine, and (2) the possible changes in enzyme configuration produced by morphine treatment.

Acetylcholine and related enzymes in the neural lobe and anterior hypothalamus of the rabbit.

1. Acetylcholine (ACh), cholinesterases and choline acetyltransferase (choline acetylase) were estimated in the neural lobe and hypothalamus of the adult male rabbit. Acetylcholine was also estimated in the neural lobes and hypothalami of some other mammals.2. Acetylcholine-like activity was measured by bio-assay using the leech dorsal muscle preparation.3. Characterization experiments indicated that about 90% of the activity measured was due to acetylcholine.4. Mean acetylcholine content in the neural lobe of the rabbit, after extraction with perchloric acid, was 4.38 +/- 0.98 mug/g fresh tissue, and 4.87 +/- 1.53 mug/g in the hypothalamus.5. Acetylcholine was also found to be present, in comparable concentrations, in the neural lobe of man and in the neural lobes and hypothalami of ox, rat and hedgehog.6. Acetylcholinesterase, present in the neural lobe and hypothalamus of the rabbit, hydrolysed 1.74 +/- 0.11 mu-moles of substrate/min/g and 3.78 +/- 0.60 mu-moles substrate/min/g fresh tissue respectively.7. The concentration of butyrylcholinesterase was about one tenth that of acetylcholinesterase in both tissues.8. Choline acetyltransferase present in the neural lobe and in the hypothalamus synthesized 87 +/- 22 mug ACh/hr/g fresh tissue and 378 +/- 149 mug respectively.

Acylation of sn-glycerol 3-phosphate by cell fractions of rat liver.

The esterification of sn-glycerol 3-(dihydrogen phosphate) with long-chain fatty acids by rat liver microsomal preparations has been studied. A newly modified spectrophotometric assay for glycerolphosphate acyltransferase (GP-acyltransferase) compared favorably with other assay methods, including measurement of the incorporation of sn-glycerol-(14)C 3-(dihydrogen phosphate) into glycerolipids. Cofactor requirements, preliminary kinetic constants, and optimum pH were determined. The product of the reaction was identified as monoacylglycerophosphate by thin-layer chromatography. Albumin activated GP-acyltransferase at low concentrations of acyl CoA but was inhibitory at higher concentrations. Serum -lipoprotein also caused activation of GP-acyltransferase. The effect of albumin could not be attributed to binding of substrate or fatty acids or the provision of metal ions. Diacylglycerophosphate, cytidine triphosphate, sulfhydryl binding agents, and sodium palmitate were identified as inhibitors of microsomal GP-acyltransferase. The physiological significance of these inhibitors remains to be established.