Robert L. Wykle

The retention of arachidonic acid in ethanolamine plasmalogens of rat testes during essential fatty acid deficiency

To assess the possible role of plasmalogens in essential fatty acid deficiency, the metabolism of [6,8,9,11,12,14,15-3H7]arachidonic acid was examined in testicular tissue of rats maintained on essential fatty acid-deficient and control diets. The findings demonstrated that this essential fatty acid is metabolized more rapidly in the diacyl phospholipids and is retained to a greater extent in the plasmalogens of testicular lipids of the animals fed the essential fatty acid-deficient diet. It would appear that the build-up of labeled arachidonic acid in the plasmalogens from rats fed the fat-free diet is explained by an acyl exchange reaction, since the quantity of ethanolamine plasmalogens in the testes remained constant.

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).

Studies of lysophospholipase D of rat liver and other tissues

Abstract Lysophospholipase D (EC 3.1.4.-) activity was demonstrated in rat kidneys, intestines, lungs, testes, and liver. The liver enzyme was studied in greatest detail and its labeled products were identified by chemical and Chromatographic techniques. This enzyme hydrolyzes 1-[1- 14 C]hexadecyl- sn -glycero-3-phosphoethanolamine and 1-[1- 14 C]hexadecyl- sn -glycero-3-phosphocholine to yield 1-[1- 14 C]hexadecyl- sn -glycero-3-phosphate; the initial product is subsequently dephosphorylated by a phosphohydrolase in microsomes to form 1-[1- 14 C]hexadecyl- sn -glycerol. The possibility that phospholipase C and a phosphotransferase were responsible for the formation of 1-[1- 14 C]hexadecyl- sn -glycero-3-phosphate was ruled out. Neither 1-[1- 14 C]hexadecyl-2-acyl- sn -glycero-3-phosphoethanolamine nor 1-[1- 14 C]hexadecyl-2-acyl- sn -glycero-3-phosphocholine was hydrolyzed. The enzyme requires Mg 2+ , is inhibited by Ca 2+ , and is stimulated by high salt concentrations; it is localized in the microsomal fraction and has a pH optimum between 7.0 and 7.6. Inhibition by sulfhydryl reagents and protection by glutathione and dithiothreitol suggest that a sulfhydryl group is required for activity. The enzyme is inhibited by detergents and by organic solvent extraction. It appears to be tightly bound to the microsomes, since repeated freeze-thawing or sonication did not release the activity, and trypsin digestion (either in the presence or in the absence of 0.04% deoxycholate) did not destroy the activity. Lysophospholipase D was previously known to occur only in brain (R. L. Wykle and J. M. Schremmer, 1974, J. Biol. Chem. , 249 , 1742–1746) .

The glycerol source for the biosynthesis of alkyl glycerol ethers

Abstract We have used a specific inhibitor to distinguish between glyceraldehyde-3-P and dihydroxyacetone-P as the glycerol source of alkyl glyceryl ethers. Only dihydroxyacetone-P reacted with fatty alcohols in the presence of the inhibitor and microsomes from preputial gland tumors to produce the O -alkyl linkage (CoA, ATP, and Mg ++ are essential cofactors). The products formed have chromatographic properties consistent with the reaction sequence proposed by us in an earlier paper.

The biosynthesis of plasmalogens from labeled O-alkylglycerols in Ehrlich ascites cells.

Abstract The role of O-alkyl lipids as precursors for plasmalogens has been investigated in vivo. We have demonstrated that rac-O-[ i -14C]hexadecyl[2-3H]glycerol (chimyl alcohol) is incorporated into O-alkyl lipids by intact Ehrlich ascites cells and is subsequently converted to plasmalogens having essentially the same 3H/14C ratio as the chimyi alcohol administered. A labeled O-alkylglycerol intermediate with a polar group in the O-alkyl moiety was found in these studies and it had the same 3H/14C ratio as the O-alkyl and O- alk - i - enyl lipids. The unidentified O-alkyl lipid has a number of Chromatographie properties that are similar to those of synthetic i -O-(β-hydroxy) hexadecyl glycerol. Our data indicate that O-alkyl lipids are converted to O- alk- i -enyl lipids via a substitution reaction on the O-alkyl moiety.

The enzymic incorporation of arachidonic acid into ether-containing choline and ethanolam1ne phosphoglycerides by deacylation-acylat1on reactions

Abstract Microsomal preparations and mitochondrial supernatant fractions from rat testes incorporated [ 3 H]arachidonic acid into the 2-position of [ 14 C]alkylacylglycero-phosphorylcholine and endogenous alk-1-enyl-acylglycerophosphorylethanolamine. The reaction required ATP and CoA and was stimulated by Mg 2+ when microsomes washed with EDTA were used. Under the same conditions, only insignificant quantities of [ 3 H]palmitic acid were incorporated into [ 14 C]alkylacylglycerophosphorylcholine or the alk-I-enylacylglycerophosphorylethanolamine. However, both the [ 3 H]palmitic and the [ 3 H]arachidonic acids were incorporated into diacylglycero-phosphorylcholine and diacylglycerophosphorylethanolamine; the palmitate was found predominately in the 1-position and the arachidonate in the 2-position. When α-glycerophosphate was added to the system, incorporation of the 3 H-labeled acids into phosphatidic acid showed little positional selectivity. These findings indicated that the labeled acids were incorporated into the choline- and ethanolamine-containing phospholipids by deacylation-acylation reactions.

The biosynthesis of plasmalogens in a cell-free system

Until now, the biosynthesis of plasmalogens has not been demonstrated in a cell-free system. In our earlier studies on the enzymic synsthesis of Oalkyl bonds in glycerolipids [ 1-5] , we suggested that the formation of the O-alkyl linkage from fatty alcohols and dihydroxyacetone-P might be the initial step in plasmalogen biosynthesis. In v ivo data obtained with O-alkylglycerols [6, 7] and fatty alcohols [5, 8, 9] labeled with ~4C, 3H, and 180 indicate that the O-alkyl glycerolipids are precursors of O-alk-l-enyl glycerolipids. This report describes the biosynthesis of plasmalogens (O-alk-l-enyl glycerolipids) in homogenates and mitochondrial supernatants prepared from Ehdich ascites ceils. Substrates and cofactors (dihydroxyacetone-P, fatty alcohols, CoA, ATP, and Mg 2÷) required for the biosynthesis of O-alkyl lipids [ 1-5 ], plus NADP ÷, were used for optimal incorporation of radioactivity from 1-~4C-hexadecanol into the O-alk1-enyl moiety. Almost all the radioactivity in the plasmalogens of the phospholipids was present in the ethanolamine-containing fraction.

The biosynthesis of ethanolamine plasmalogens by a postmitochondrial fraction from rat brain

Abstract Ethanolamine plasmalogens were synthesized from 1-14C-alkyl-sn-glycero-3-phosphorylethanolamine by mitochondrial supernatants of rat brain. The data indicate that the desaturation of the O-alkyl moiety to the O-alk-l-enyl moiety occurs on intact 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamine.

Microsomal Enzymes Involved in the Metabolism of Ether-Linked Glycerolipids and Their Precursors in Mammals

The pathway by which ether-linked glycerolipids are synthesized in animals remained obscure until 1968. Metabolic in vivo studies and structural analysis of alkyl-linked glycerolipids indicated that the alkyl chains were derived from fatty alcohols (Keenan et al., 1961; Friedberg and Greene, 1967; Ellingboe and Karnovsky, 1967; Snyder and Blank, 1969; Schmid and Takahashi, 1970) and that alk-1-enyl-linked glycerolipids (plasmalogens) were formed by desaturation of alkyl glycerolipids (Horrocks and Ansell, 1967; Bickerstaffe and Mead, 1968; Malins, 1968; Thompson, 1968; Wood and Snyder, 1969; Wood and Healy, 1970; Blank et al., 1970; Wood et al., 1970; Bell et al., 1971; Debuch et al., 1970, 1971; Paltauf, 1971a,b; Stoffel and LeKim, 1971). Thompson and Hanahan (1963) found that radioactivity from [6–14C]glucose was incorporated into ether lipids by bone marrow and that the label was almost exclusively in the sn-3 carbon of the glycerol moiety. Later Friedberg and Greene (1968) concluded from in vivo experiments with Tetrahymena and 14C., 3H-labeled glycerol that the glycerol portion of ether lipids is derived from a triose phosphate or some other carbohydrate instead of α-glycerophosphate.

Dietary control of stearyl CoA and alkylacylglycerophosphorylethanolamine desaturases in tumor

Abstract The responses of stearyl CoA desaturase and alkylacylglycerophosphorylethanolamine desaturase were compared in Fischer R-3259 tumors of rats maintained on normal and fat-free diets. The stearyl CoA desaturase activity significantly increased in control livers, host livers, and tumors in rats maintained on the fat-free diet over that in the same tissues from animals maintained on laboratory chow; however, no increase of alkylacylglycerophosphorylethanolamine desaturase was observed in tumors of rats maintained on the fat-free diet. These findings suggest that the two desaturase systems are not identical and that they are controlled by different components of the microsomal electron transport system. Stearyl CoA desaturase activity was lower in the livers of tumor-bearing animals than in those of control animals. This finding supports the concept of others that indicates host livers tend to acquire the enzymic composition of immature livers.