A specific acetylhydrolase that inactivates platelet activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a potent cellular mediator in mammalian cells, by removal of the sn -2 acetyl moiety, has been found in the cytosolic fraction of several postembryonic developmental stages and specific tissues of the corn earworm, Heliothis zea (Boddie). Effects of magnesium, calcium, EGTA, deoxycholate, dithiothreitol, diisopropylfluorophosphate, egg phosphatidylcholine, and an acylacetyl-glycerophosphocholine show that hydrolysis of the acetate moiety is due to a specific acetylhydrolase for PAF. The activity does not appear to be due to a typical cellular phospholipase A 2 that utilizes phospholipid substrates with a long-chain acyl group at position sn -2 of glycerol. Specific activities and properties of the acetylhydrolase from this insect match closely with those described from tissues of vertebrate animals.

1-Alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase catalyzes the conversion of biologically inactive lysophospholipid to bioactive platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) by an acetylation reaction. The activity of this enzyme in eosinophils isolated from patients with eosinophilia is stimulated (up to 4-fold) in a dose-, time-, and Ca2+/Mg2+-dependent manner after exposure to the eosinophil chemotactic factor of anaphylaxis (ECF-A), C5a, formyl-methionylleucylphenylalanine (fMLP), or ionophore A23187. The three naturally occurring chemotactic factors (ECF-A, C5a, and fMLP) cause a rapid and transient increase of enzyme activity, with a maximum at 1 or 3 min, whereas ionophore A23187 maintains an elevated level for up to 15 min. The activity of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase, an enzyme that catalyzes the breakdown of PAF to lyso-PAF, is not affected by C5a, fMLP, or ionophore A23187. The presence of PAF in eosinophils was established by demonstrating the lipid nature of the compound, the RF value being identical with that of synthetic 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine on thin layer chromatograms, and by its ability to induce serotonin release from rabbit platelets. Furthermore, ECF-A, C5a, fMLP, and ionophore A23187 all induce the secretion of PAF from eosinophils. These findings suggest that the generation and release of PAF could be a consequence of eosinophil chemotactic activation and may thus function in inflammatory and allergic reactions in which eosinophils participate.

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.

Long-chain alcohols are synthesized in the mouse preputial gland tumor (ESR-586) by NADPH:acyl-CoA oxidoreductase. In this study, a series of labeled acids was tested as substrates for the oxidoreductase in a cell-free system from the tumor, and the distribution of label into alcohols, waxes, and other products was determined. The system contained the labeled acid, an acyl-CoA-generating system, an NADPH-generating system, and tumor homogenate. The highest rates of alcohol synthesis were obtained with palmitic (16:0), heptadecanoic (17:0), stearic (18:0), myristic (14:0), elaidic (18:1 trans), and linoleic (18:2) acids, which yielded, respectively, 151, 124, 102, 76, 65, and 35 pmol alcohol/min per mg protein. Decanoic (10:0), lauric (12:0), oleic (18:1 cis), linolenic (18:3), arachidonic (20:4), and behenic (22:0) acids all gave lower activities. Acyl-CoA formation did not appear to be rate limiting with any of the substrates tested except behenic acid. In addition to the fatty alcohol product, a small amount of fatty aldehyde was formed in the system. Incorporation of the labeled fatty acids into wax esters was examined and the distribution of label between the alcohol and acid components of the waxes was determined. Incubation of [1-(14)C]palmitic acid yielded 3.4% free alcohol, 8.3% alcohol esterified in waxes, and 7.7% palmitoyl groups esterified into waxes, whereas, at the other extreme, [1-(14)C]linolenic acid yielded 0.8%, 0.6%, and 38%, respectively, into the homologous components.-Wykle, R. L., B. Malone, and F. Snyder. Acyl-CoA reductase specificity and synthesis of wax esters in mouse preputial gland tumors.

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.