Veronica Fitzgerald

Novel quantitative method for determination of molecular species of phospholipids and diglycerides

A novel method is described for the quantitative analysis of subclasses (alk-1-enylacyl, alkylacyl, and diacyl types) and molecular species within each subclass of glycerophosphatides. Diradylglycerols from phospholipase C hydrolysis of the phospholipids are converted to benzoate derivatives, the benzoates are separated into their respective subclasses by thin-layer chromatography, and quantitated by measuring absorbance at 230 nm. Molecular species within individual subclasses are separated using a combination of argentation thin-layer chromatography and reversed-phase high-performance liquid chromatography with direct, on-line quantitation at 230 nm. We applied the method to the analysis of ethanolamine phosphatides from beef brain and were able to quantitate the three diradylglycerol subclasses (alk-1-enylacyl, alkylacyl, and diacyl types) as well as ca. 29 molecular species within each of these subclasses. This new quantitative approach for the analysis of specific molecular species of glycerolipids should be applicable to studies involving a variety of biologically important lipids, such as phosphatidylcholine, phosphatidylinositol, platelet activating factor, plasmalogens, and neutral type glycerolipids including diacylglycerols.

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.

Substrate specificity in the biocleavage of the O-alkyl bond: 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a hypotensive and platelet-activating lipid) and its metabolites☆

Abstract 1-Alkyl-2-acetyl- sn -glycero-3-phosphocholine possesses both hypotensive and platelet-activating properties. Our data show that after removal of the acetyl group at the sn -2 position by an acetyl hydrolase, the product, 1-alkyl-2-lyso- sn -glycero-3-phosphocholine, can be cleaved by a microsomal tetrahydropteridine-dependent alkyl monooxygenase in the liver and spleen of rats. Results obtained for the tetrahydropteridine requirement, tissue distribution, responses to thermal inactivation and catalase, and substrate inhibition of the enzyme indicate that the ether linkage of 1-alkyl-2-lyso- sn -glycero-3-phosphocholine is hydrolyzed by a monooxygenase that appears to be identical to the one responsible for cleavage of the O -alkyl moiety of alkylglycerols.

Phase transitions of alkyl ether analogs of phosphatidylcholine.

The phase-transition temperatures of aqueous dispersions of diester, monoether and diether analogs of phosphatidylcholine were determinmed using transparinaric acid as a fluorescent probe. The diether analog of phosphatidylcholine has a higher phase-transition temperature, whilst the monoether analog has a lower phase-transition temperature than their diester counterpart.

BIOSYNTHESIS OF PLATELET-ACTIVATING FACTOR AND ENZYME INHIBITORS

Platelet-activating factor (PAF) is known to be synthesized by either a remodeling or de novo pathway. The enzymes responsible have been extensively studied by a number of laboratories. All evidence indicates the remodeling route is activated during inflammation and other hypersensitivity responses, whereas the de novo pathway is thought to be the source of PAF required for physiological functions. This article provides an update of what is currently known about the enzymatic systems that generate PAF as well as some preliminary findings we have obtained using potential inhibitors of the specific enzymes involved. Recent progress from our laboratory toward understanding the role of the CoA-independent and Co-A dependent transacylases in the formation of lyso-PAF and PAF is summarized.

Molecular Species of Sphingomyelin in Sphingomyelinase-Sensitive and Sphingomyelinase-Resistant Pools of HL-60 Cells

This study of sphingomyelin molecular species in undifferentiated and differentiated (granulocytic form) HL-60 cells demonstrated only minor differences in the distribution of species between the sphingomyelinase-sensitive and sphingomyelinase-resistant pools of sphingomyelin in these cells. The two most prominent species of sphingosine present in both the undifferentiated and differentiated cells were those containing 16∶0 (slightly higher in the sphingomyelinase-resistant membranes) and 24∶1N-acyl moieties. Cell differentiation exerted little effect on the distribution of molecular species of sphingomyelin between the sphingomyelinase-sensitive and sphingomyelinase-resistant pools in HL-60 cells, although the levels ofN-palmitoyl sphinganine were significantly lower and theN-nervonoyl sphingosine higher in both pools from the differentiated cells. Our results indicate the same species of sphingomyelin, available at both the outer layer of the plasma membrane and inner layer of the plasma membrane (plus intracellular membranes) of HL-60 cells, serve as precursors for generation of the ceramides that participate in signal transduction processes initiated by cell activation.

Activation of alkyldihydroxyacetone phosphate synthase by detergents

Abstract The microsomal enzyme alkyldihydroxyacetone phosphate synthase, which synthesizes alkyldihydroxyacetone phosphate from fatty alcohols and acyldihydroxyacetone phosphate, was examined in the harderian gland and Ehrlich ascites carcinoma. Although the enzyme possessed a higher specific activity in the harderian gland than in the Ehrlich ascites carcinoma, similar results were obtained using the enzyme from both sources. Alkyldihydroxyacetone phosphate synthase was stimulated approximately 50% by the addition of deoxycholate or Triton X-100 to the assay system. The concentration of detergent needed to elicit this response was below the critical micellar concentration of these detergents. The reaction rate as a function of pH was essentially constant in the absence of detergent, but, when Triton X-100 was added to the system, the synthase showed a distinct pH optimum of 8.0–8.5. Unlike cytochrome b 5 , a marker for the surface of the microsomal vesicles, the alkyldihydroxyacetone phosphate synthase activity was not removed from microsomal preparations treated with trypsin. Also, alkyldihydroxyacetone phosphate synthase was not solubilized by treatment of the microsomes with deoxycholate concentrations below the critical micellar concentration of this detergent. However, enzymatic activity was efficiently removed from microsomes treated in combination with deoxycholate and trypsin. We suggest from these data that the catalytic portion of alkyldihydroxyacetone phosphate synthase is exposed to the luminal surface of the microsomal vesicles. Since both dihydroxyacetone phosphate and glyceraldehyde 3-phosphate were inactive in the assay system in the presence and absence of detergent, the possibility that acyldihydroxyacetone phosphate functions in the system as a lipophilic molecule that facilitates the transport of a triose phosphate into the microsomal lumen is unlikely.

Coupling of the biosynthesis of fatty acids and fatty alcohols

Abstract A cell-free system for the biosynthesis of fatty alcohols in the pink portion of the rabbit harderian gland is described. The radiolabeled substrates for the fatty acid reductase were generated using soluble fatty acid synthase from the gland in the presence of acetyl-CoA, malonyl-CoA, and NADPH. Harderian gland microsomes, ATP, and Mg2+ were absolute requirements for the synthesis of fatty alcohols and if any of these components were deleted from the assay mixture, no alcohols were detected. We were also unable to detect formation of fatty alcohols if acyl-CoAs were substituted for fatty acid synthase with either NADPH or NADH as reducing agents. The reductase was localized in the microsomal fraction and appears to be on the cytosol-membrane interface of the vesicles, as indicated in experiments using detergents and trypsin. The fatty alcohols formed by the system had the same chain length distribution as the fatty acids made by the fatty acid synthase. The alkyl moieties of the ether lipids in the harderian gland are exclusively saturated and the properties of the alcohol-synthesizing system described in this report can account for the observed exclusion of unsaturated alkyl moieties from the ether lipids of this gland.

Conversion of 1-alkyl-2-acetyl-sn-glycerols to platelet activating factor and related phospholipids by rabbit platelets

The metabolic pathway for 1-alkyl-2-acetyl-sn-glycerols, a recently discovered biologically active neutral lipid class, was elucidated in experiments conducted with rabbit platelets. The total lipid extract obtained from platelets incubated with 1-[1,2-(3)H]alkyl-2-acetyl-sn-glycerols or 1-alkyl-2-[3H]acetyl-sn-glycerols contained at least six metabolic products. The six metabolites, identified on the basis of chemical and enzymatic reactions combined with thin-layer or high-performance liquid chromatographic analyses, corresponded to 1-alkyl-sn-glycerols, 1-alkyl-2-acetyl-sn-glycero-3-phosphates, 1-alkyl-2-acyl (long-chain)-sn-glycero-3-phosphoethanolamines, 1-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamines, 1-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamines, 1-alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholines, and 1-alkyl-2-acetyl-sn-glycero-3-phosphocholines (platelet activating factor). These results indicate that the metabolic pathway for enzymatic activities: choline- and ethanolamine- phosphotransferases, acetyl-hydrolase, an acyltransferase, and a phosphotransferase. The step responsible for the biosynthesis of platelet activating factor would appear to be the most important reaction in this pathway and this product could explain the hypotensive activities previously described for alkylacetyl-(or propionyl)-glycerols. Of particular interest was the preference exhibited for the utilization of the 1-hexadecyl-2-acetyl-sn-glycerol species in the formation of platelet activating factor.

Stimulation of the microsomal alkylglycerol monooxygenase by catalase

Liver cytosol contains a heat-sensitive nondialyzable soluble factor necessary for maximal activity of the alkylglycerol monooxygenase present in rat liver microsomes. In this report, we demonstrate that the stimulatory component in rat liver supernatant is catalase. Catalase functions to protect the enzyme from inactivation by H2O2 in addition to its documented ability to retard the noenzymatic oxidation of the pterin cofactor. The protective effect of catalase on alkylglycerol monooxygenase and on the aromatic amino acid hydroxylase systems indicates that H2O2 sensitivity is a general feature of pterin-dependent hydroxylases.