Boyd Malone

Activities of enzymes that metabolize platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in neutrophils and eosinophils from humans and the effect of a calcium ionophore

Abstract Enzymatic systems in human blood cells are described for the activation and inactivation of a biologically active phospholipid (1-alkyl-2-acetyl- sn -glycero-3-phosphocholine) with hypotensive, platelet-aggregating, and inflammatory properties. The results document the presence of alkyldihydroxyacetone-phosphate synthase (forms the O -alkyl linkage in lipids), 1-alkyl-2-lyso- sn -glycero-3-phosphocholine:acetyl-CoA acetyltransferase (produces the biologically active molecule), and 1-alkyl-2-acetyl- sn -glycero-3-phosphocholine: acetylhydrolase (destroys the biological activity) in human neutrophils and eosinophils. Both the acetyltransferase and acetylhydrolase activities are increased severalfold after treatment of normal neutrophils with ionophore A23187; however, alkyldihydroxyacetone-phosphate synthase activity is not influenced by the ionophore. Eosinophils isolated from patients with eosinophilia have significantly greater activities of all the enzymes studied than the eosinophils isolated from normal individuals. Our results indicate the acetyltransferase responsible for 1-alkyl-2-acetyl- sn -glycero-3-phosphocholine synthesis may serve an important role in human blood cells that release this biologically active phospholipid. Moreover, the acetyltransferase activity was found to be dramatically influenced by calcium flux.

Enzymic interconversion of fatty alcohols and fatty acids

The enzymic reduction of l-14C-palmitic acid to hexadecanol and the enzymic oxidation of l-14C-hexadecanol to palmitic acid have been demonstrated in unwashed microsomes from mouse preputial gland tumors. NAD is required for oxidation and NADPH is required for reduction. ATP, CoA, and Mg++ are also required for the reduction of fatty acids (presumably for activation) but they are not necessary for the oxidation of fatty alcohols.

1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) stimulates calcium influx in rabbit platelets

Summary 1-Alkyl-2-acetyl- sn -glycero-3-phosphocholine (platelet-activating factor) induces an influx of Ca 2+ in rabbit platelets that is time-, temperature-, and concentration-dependent. The ionophoretic activity of 1-alkyl-2-acetyl- sn -glycero-3-phosphocholine is much higher (>2-fold at 10 −10 M) than 3-hexadecyl-2-acetyl- sn -glycero-l-phosphocholine, 1-hexadecyl-2-lyso- sn -glycero-3-phosphocholine, 1-acyl-2-acetyl- sn -glycero-3-phosphocholine, or 1-hexadecanoyl-2-lyso- sn -glycero-3-phosphocholine.

The biosynthesis of alkyl ether bonds in lipids by a cell-free system

Abstract Investigators have found the biological significance and the biosynthetic pathways of ether-linked lipids containing glycerol difficult to determine (1). Despite available knowledge concerning enzymes for the biocleavage (2,3), deacylation (4), acylation (5,6), and phosphorylation (7) of glyceryl ethers, the biosynthesis of the ether bond in these compounds has not previously been demonstrated in a cell-free system. This note describes an active enzyme complex capable of converting intact l- 14 C-labeled fatty alcohols into alkyl glyceryl ethers. The reaction has been demonstrated in whole homogenates and in microsomal-plus-supernatant fractions of transplantable preputial tumors in mice. These tumors are typical of neoplasms containing high quantities of alkyl ether linkages in both neutral- and phospho-glycerides (8).

Stimulation of calcium uptake by 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) in rabbit platelets: possible involvement of the lipoxygenase pathway.

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) induces an increase of Ca2+ uptake in rabbit platelets. This process depends upon the extracellular concentration of Ca2+ with the maximum stimulation occurring at 1-3 mM; uptake under these conditions is blocked by verapamil, a calcium-entry blocker. Increase of calcium uptake by the bioactive phospholipid was independent of ADP-induced platelet responses and of metabolites of arachidonic acid metabolism formed through the cyclooxygenase pathway. However, mepacrine, p-bromophenacyl bromide, eicosatetraynoic acid, and nordihydroguaiaretic acid significantly or totally inhibited the stimulation of Ca2+ uptake by 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine. When arachidonic acid was given sufficient time to be metabolized to other products by the platelets, stimulation of Ca2+ uptake also occurred. Arachidonic acid and platelet-activating factor did not produce an additive or synergistic effect. Our data suggest that a metabolite(s) generated from arachidonic acid through the lipoxygenase pathway may be the mediator(s) responsible for the action of platelet-activating factor in the induction of increased Ca2+ uptake in rabbit platelets.

Acyltransferases and the biosynthesis of pulmonary surfactant lipid in adenoma alveolar type II cells.

Abstract Acyltransferases are present in microsomes from alveolar type II cell adenomas (produced by urethan injections) that transfer palmitic acid in the presence of CoA, ATP, and Mg++ to sn -glycerol-3-P to form phosphatidic acid, to dihydroxyacetone-P to form acyldihydroxyacetone-P, and to 1-acyl- sn -glycero-3-phosphocholine to form 3- sn -phosphatidylcholine. The data clearly demonstrate that the microsomal preparations can catalyze significant incorporation of palmitic acid into the 2-position of the disaturated species of 3-sn-phosphatidylcholine independently of phosphatidic acid formation as evidenced by the fact that sn -glycerol-3-P and calcium ions (which inhibit choline phosphotransferase) did not influence the incorporation of palmitic acid into the main surfactant lipid. Thus, a deacylation-acylation reaction involving 2-lysophosphatidylcholine appears to be an important pathway for the synthesis of surfactant lipid in alveolar type II cells; the control of acyl specificity at the 2-position is determined by the relative concentrations of the coparticipating substrates, l-palmitoyl- sn -glycero-3-phosphocholine and palmitoyl-CoA.

The biosynthesis of alkyl glyceryl ethers by microsomal enzymes of digestive glands and gonads of the starfish, Asterias forbesi

Abstract 1. 1. A microsomal enzyme system that can synthesize alkyl ether linkages in glycerolipids has been isolated from the digestive glands and gonads of the starfish, Asterias forbesi . The total lipids of the microsomes from the digestive glands contain approx. 4.5% alkyl glyceryl ethers and 5.5% alk-1-enyl glyceryl ethers. The chain lengths of alkyl moieties consist of 16:0 (31%), 18:0 (39%) and 20:1 (13%), whereas the chain lengths of the alk-1-enyl moieties consist mainly of 18:0 (79%). 2. 2. The biosynthetic system for alkyl ethers utilizes long-chain fatty alcohols and glyceraldehyde 3-phosphate as substrates and ATP, CoA, and Mg 2+ as cofactors. The time-course for the reaction is linear for at least 90 min, and the products produced have chromatographic properties identical to those which we have found in microsomes from neoplastic cells. No alk-1-enyl glyceryl ethers are formed in this system. Gas-liquid chromatography of the 14 C-labeled O -alkylglycerol isopropylidene derivatives (prepared after LiA1H 4 reduction of total lipids) revealed that approx. 90% of the 14 C was associated with the C 16:0 glyceryl ether fraction after incubation of 14 C labeled C 16:0 fatty alcohol.

Enzymic synthesis of O-alkyl glycerolipids in brain and liver of rats during fetal and postnatal development

Abstract O -Alkyl glycerolipid synthesizing enzymes were measured in brains and livers of fetal and postnatal rats. Highest activities occurred during the early stages of postnatal growth; at this time, enzymes that cleave the O -alkyl moiety are present in liver but not in brain. Our data are compatible with the possible precursor role of the O -alkyl lipids in the formation of plasmalogens during myelination.

Biosynthesis of pulmonary surfactant: Comparison of 1-palmitoyl-sn-glycero-3-phosphocholine and palmitate as precursors of dipalmitoyl-sn-glycero-3-phosphocholine in adenoma alveolar type II cells☆

Abstract Urethan-induced pulmonary adenomas of mice are composed of cells that appear to be morphologically identical to alveolar type II cells and synthesize disaturated diacyl- sn -glycero-3-phosphocholine, the major component of pulmonary surfactant. 1-[1- 14 C]Palmitoyl- sn -glycero-3-phosphocholine and [1- 14 C]palmitic acid were compared as precursors of disaturated diacyl- sn -glycero-3-phosphocholine in the adenoma type II cells by incubating both substrates with whole adenomas. When the precursors were compared at equal concentrations (100 μ m ) in the presence of albumin (1 mg/ml), the rates of incorporation of 1-[1- 14 C]palmitoyl- sn -glycero-3-phosphocholine and [1- 14 C]palmitic acid into diacyl- sn -glycero-3-phosphocholine were 5.2 and 2.9 nmol/min · g tissue, respectively. The concentration of monoacyl- sn -glycero-3-phosphocholine (lysolecithin) in the blood plasma of BALB/c mice was 150 μ m . In short-term labeling experiments, the label in disaturated diacyl- sn -glycero-3-phosphocholine was equally distributed between the sn -1 and sn -2 positions when 1-[1- 14 C]palmitoyl- sn -glycero-3-phosphocholine was the precursor, whereas 75 to 80% was in the sn -2 position when [1- 14 C]palmitic acid was the precursor. The ratios are consistent with incorporation of 1-palmitoyl- sn -glycero-3-phosphocholine via the lysolecithin:lysolecithin transacylase reaction and incorporation of palmitate via acylation of 1-palmitoyl- sn -glycero-3-phosphocholine by acyl-CoA:lysolecithin acyltransferase. 1-[1- 14 C]Palmitoyl- sn -glycero-3-phospho-[ 3 H- methyl ]choline was incorporated into total cellular diacyl- sn -glycero-3-phosphocholine with an isotope ratio similar to that of the precursor; the disaturated species was more enriched in 14 C. These findings indicate the cells take up intact monoacyl- sn -glycero-3-phosphocholine and incorporate it into diacyl- sn -glycero-3-phosphocholine. The ability of the cells to utilize intact lysophosphoglycerides for synthesis of cellular lipids was further demonstrated by showing that ether analogs, 1-alkyl- sn -glycero-3-phosphocholine and 1-alkyl- sn -glycero-3-phosphoethanolamine, are taken up and acylated by the cells. Activities of lysolecithin:lysolecithin transacylase and acyl-CoA:lysolecithin acyltransferase were measured in subcellular fractions of the adenoma type II cells; the specific activities of the enzymes were 2.1 nmol/min · mg soluble protein and 21 nmol/min · mg microsomal protein, respectively. The total activity of the acyltransferase in the cell fractions was about four-fold higher than the activity of the transacylase. Characteristics of the two enzymes were studied and are discussed. The findings indicate that exogenous 1-palmitoyl- sn -glycero-3-phosphocholine and palmitic acid both serve as efficient precursors of disaturated diacyl- sn -glycero-3-phosphocholine in the adenoma alveolar type II cells.

The participation of 1- and 2-isomers of O-alkylglycerols as acyl acceptors in cell-free systems

Abstract Cell-free homogenates from several neoplastic tissues and normal rat liver contain enzymes that acylate O -alkylglycerols. Acylation of rac-1- O -alkylglycerols produced only 1-alkyl-3-acylglycerols, whereas the acylation of 2-alkylglycerols produced 1,3-diacyl-2-alkylglycerols; neither of the isomeric forms of the alkylglycerols was phosphorylated. ATP, CoA, and Mg 2+ were required as cofactors for acylation.