Showing papers by "John L. Harwood published in 1992"

Lipid composition of subcellular membranes from larvae and prepupae of Drosophila melanogaster.

Abstract: Subcellular membranes were analyzed for their lipid composition and protein content at two developmental points representing the third instar wandering larvae and prepupal stages of Drosophila. At both stages, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were the major constituents with phosphatidylinositol (PI), phosphatidylserine (PS), diphosphatidylglycerol (DPG) and phosphatidic acid (PA) being relatively minor components. In total homogenates and in the nuclear-enriched fraction there was no significant difference in the phospholipid composition of the wandering larvae and prepupae. In mitochondria only a significant increase in the minor component PS was observed in the prepupae. In lysosomal membranes on the other hand, the relative abundance of the major components PE and PC increased in the prepupae although the molar ratios of the two lipids remained almost constant. The fatty acid composition of the phospholipids remained virtually unchanged in all of the fractions examined, including the lysosomes, and there was no evidence of lipid peroxidation. With regard to cellular degeneration and the involvement of lysosomes, we conclude that mechanisms other than gross modification of the lipid and/or lipid/protein ratio of their membranes are involved in the liberation of the acid phosphatase contents.

Interferon-gamma increases macrophage phospholipid polyunsaturation: a possible mechanism of endotoxin sensitivity.

Abstract: Incubation of murine macrophages or the macrophage-like cell line P388D with interferon-gamma in vitro induced a significant increase in the polyunsaturated fatty acid content of phosphatidylethanolamine. These increases were time and dose-dependent, being maximal at 12 hours and with 5000 U/ml interferon and were inhibited in the presence of anti-interferon-gamma monoclonal antibody. Interferon-gamma induced a significant increase in linoleate in peritoneal macrophages while in the cell line arachidonate was significantly increased. These results are of interest because such increases in the polyunsaturated fatty acid content of phosphatidylethanolamine were previously shown by us to be associated with increased sensitivity to endotoxin in mice in vivo. The implications for interferon-gamma sensitizing to endotoxin are discussed.

Glycerol 3-phosphate acylation by microsomal fractions from avocado mesocarp.

Abstract: Triacylglycerol synthesis in plants occurs via the classical Kennedy pathway [1,2]. The quality and physical properties of the triaqlglycerol products are determined by the substrate specificities of the acyltransferases involved [2]. The initial step is catalysed by glycerol 3-phosphate acyltransferase and involves the acylation of the sn-1 position of lycerol 3-phosphate to form lysophosphatidic acid. This occurs at much slower rates than the second acylation and thus limits formation of phosphatidate, a key intermediate in lipid formation. Little is known about the mechanism of action of the acyltransferases of the Kennedy pathway, as they are membrane bound in the endoplasmic reticulum, and there has been little success in their purification. In this work, a microsome fraction from avocado mesocarp has been used as a source of glycerol 3-phosphate acyltransferase in order to study its mode of action, especially with res ect to substrate specificities. friacylglycerol synthesis by the microsome fraction of avocado mesocarp was stuqsd using radiolabelled substrates. In vitm incubations with [ Clglycerol 3-phosphate showed carbon flux through the intermediates of the Kennedy pathway, includin I sophosphatidic acid, hosphatidic acid and diacylglycerok. b e amounts of labelLd intermediates produced could be varied by altering the incubation conditions. For exam le, inclusion of 10 mM EDTA in the incubations increasei! labellin in lysophos hatidate and phosphatidate by 189% and 2! %, respective&. Incubations with mixed acyl-CoA substates showed that glycerol 3phosphate acyltransferase had higher rates with saturated acyl-CoA species whilst 1-acyl glycerol 3-phosphate acyltransferase utilised monounsaturated acyl-CoA species preferentially. Both the acyltransferases would utilise other substrates thus forming uncharacteristic triacyl lycerol species. Incubations using radiolabelled a 1-CoA su%strates were found to be of limited use because $the high rates of transa lation catalysed by the avocado microsomes. Yn order to determine the site of the glycerol 3phosphate acyltransferase, we carried out limited proteolysis of the microsomal vesicles [3]. In these experiments the total amount of protein di estion was kept to a minimum in order to prevent access of t8e roteinases to the intra-luminal face of the vesicles. Under tgese strictly controlled conditions a neutral proteinase from rat liver [4] and t sin reduced gl cerol 3-phosphate acyltransferase activityyy 95% and l&% res ectivel (Fig.1). This showed that the active site of glycerol fphospiate acyltransferase was accessible to the cfoplasmic side of the endoplasmic reticulum. SDS-PAGE s owed that under the proteolytic conditions used, only a single protein band of approximately 175 KDa was degraded significantly. Solubilisation of the membrane bound glycerol 3phosphate acyltransferase activity has been achieved using detergents and protein denaturants. Treatment with urea results in solubilisation of 30% of the articulate activity. Of a variety of detergents tested CHAPS 6.05% w/v) treatment was most effective and solubilised 15% of particulate activi . This activity was stable at 4OC for 24 hours, but was unstab r e to freezing, even in the presence of glycerol. In contrast, particulate activity fell to 15% of the original after a similar period at 4OC. Application of dye column chromatography methods used in purification of the E.coli glycerol 3-phosphate acyltransferase [5] to the solubilised avocado pre arations was of limited use because of the poor yields opactivity. However, size exclusion chromatography and different affinity columns have yielded preparations with considerably enhanced specific activity. acyation f 0.0 25 50 75 100 125 0