Very-long-chain fatty acid-containing phospholipids accumulate in fatty acid synthase temperature-sensitive mutant strains of the fission yeast Schizosaccharomyces pombe fas2/lsd1.

Fission yeast lsd1 strains show aberrant mitosis with a lsd phenotype, large and small daughter nuclei, and a very thick septum, the phenotypic expression being temperature-sensitive. The lsd1(+) gene is the homologue of the budding yeast FAS2 gene encoding the fatty acid synthase alpha-subunit as reported previously (S. Saitoh, K. Takahashi, K. Nabeshima, Y. Yamashita, Y. Nakaseko, A. Hirata, M. Yanagida, J. Cell Biol. 134 (1996) 949--961). In this paper, lsd1 is considered to represent fas2. Here, three fas2 strains were investigated and found to have missense point mutations at different sites in the gene encoding the alpha-subunit of fatty acid synthase. The mutation affected only slightly the enzymatic activities monitored in vitro. Unexpectedly, abnormal phospholipids, phosphatidylcholine and phosphatidylethanolamine, both of which contain a very-long-chain fatty acyl residue (1-melissoyl-2-oleolyl-sn-glycero-3-phosphocholine and 1-melissoyl-2-oleolyl-sn-glycero-3-phosphoethanolamine), accumulated in fas2 strains in a temperature-sensitive manner. Rescue of the fas2 strains by addition of palmitate to the medium at restrictive temperature was accompanied by disappearance of these abnormal phospholipids. Accumulation of these lipids in membranes may cause alteration of various cellular functions.

Transfection of the plasma-type platelet-activating factor acetylhydrolase gene attenuates glutamate-induced apoptosis in cultured rat cortical neurons.

Using an adenoviral vector, we induced overexpression of the plasma type of platelet-activating factor acetylhydrolase in cultured rat neurons. Neurons overexpressing this enzyme showed a decrease in glutamate-induced injury, mainly, apparent as decreased apoptosis. Reduction of lipid peroxidation by this enzyme and protection of mitochondrial function were demonstrated, and these may be the basis of the resistance to glutamate-induced neuronal injury that we observed.

Simultaneous separation of lysophospholipids from the total lipid fraction of crude biological samples using two-dimensional thin-layer chromatography.

A novel solvent system for two-dimensional thin-layer chromatography was shown to simultaneously separate lysophospholipid standards, including lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, lysophosphatidylglycerol, lysophosphatidic acid, lysosphingomyelin (sphingosylphosphorylcholine), and sphingosine-1-phosphate from diradylphospholipids, glycosphingolipids, and neutral lipids. Lysophospholipids contained in the total lipid fraction of activated platelets were also well separated by the same system. The present system is a useful tool for the metabolic and structural analysis of lysophospholipids in biological samples.

The Escherichia coli pldC gene encoding lysophospholipase L(1) is identical to the apeA and tesA genes encoding protease I and thioesterase I, respectively.

We deduced the amino acid sequence of Escherichia coli lysophospholipase L(1) by determining the nucleotide sequence of the pldC gene encoding this enzyme. The translated protein was found to contain 208 amino acid residues with a hydrophobic leader sequence of 26 amino acid residues. The molecular weight of the purified enzyme (20,500) was in good agreement with the predicted size (20,399) of the processed protein. A search involving a data bank showed that the nucleotide sequence of the pldC gene was identical to those of the apeA and tesA genes encoding protease I and thioesterase I, respectively. Consistent with the identity of the pldC gene with these two genes, the enzyme purified from E. coli overexpressing the pldC gene showed both protease I and thioesterase I activities.

Deficient platelet-activating factor and related enzymes in hemimegalencephaly.

Platelet-activating factor (PAF) may influence neuronal migration, and gyral anomaly in hemimegalencephaly is believed to result from a neuronal disorder. A 7-month-old girl with hemimegalencephaly presented with intractable seizures, for which graded hemispherectomy was performed. In the resected specimen, we could not detect PAF, and related enzyme activities were low. These results suggest a role of PAF in neuronal migration.

Changes in composition of newly synthesized sphingolipids of HeLa cells during the cell cycle -- suppression of sphingomyelin and higher-glycosphingolipid synthesis and accumulation of ceramide and glucosylceramide in mitotic cells.

Sphingolipid biosynthesis in synchronized HeLa cells was studied by pulse labeling with [14C]Ser or [14C]Gal and a simple TLC method. The major HeLa cell sphingolipids are ceramide (Cer), sphingomyelin, glucosylceramide (GlcCer), lactosylceramide (LacCer), globotriaosylceramide (Gb3Cer), N-acetylneuraminosylgangl iotriaosylceramide (GM2) and sialylparagloboside (G[M1-GlcNAc]). The sphingolipid biosynthetic profiles of HeLa cells in the G1, G1/S boundary, S and G2 phases were similar, but significant changes occurred during M phase, when incorporation of radioactivity into sphingomyelin, Gb3Cer and a mixture of GM2 and G(M1-GlcNAc) decreased, and those of Cer and GlcCer increased. These data indicate that transfer of phosphocholine and galactose to Cer and GlcCer, respectively, decreased in mitotic cells, resulting in accumulation of Cer and GlcCer. Analysis of LacCer synthase activity revealed that GlcCer accumulation was not due to reduced activity of this enzyme. The results suggest that Cer and GlcCer accumulation in mitotic cells resulted from suppression of sphingomyelin and LacCer synthesis, probably caused by vesiculation of membranous organelles, such as the endoplasmic reticulum and Golgi apparatus.

Activated mast cells release extracellular type platelet-activating factor acetylhydrolase that contributes to autocrine inactivation of platelet-activating factor.

IgE-dependent and -independent activation of mouse bone marrow-derived mast cells (BMMC) elicited rapid and transient production of platelet-activating factor (PAF), which reached a maximal level by 2-5 min and was then degraded rapidly, returning to base-line levels by 10-20 min. Inactivation of PAF was preceded by the release of PAF acetylhydrolase (PAF-AH) activity, which reached a plateau by 3-5 min and paralleled the release of beta-hexosaminidase, a marker of mast cell exocytosis. Immunochemical and molecular biological studies revealed that the PAF-AH released from activated mast cells was identical to the plasma-type isoform. In support of the autocrine action of exocytosed PAF-AH, adding exogenous recombinant plasma-type PAF-AH markedly reduced PAF accumulation in activated BMMC. Furthermore, culture of BMMC with a combination of c-kit ligand, interleukin-1beta and interleukin-10 for > 24 h led to an increase in plasma-type PAF-AH expression, accompanied by a reduction in stimulus-initiated PAF production. Collectively, these results suggest that plasma-type PAF-AH released from activated mast cells sequesters proinflammatory PAF produced by these cells, thereby revealing an intriguing anti-inflammatory aspect of mast cells.

Cloning, expression and characterization of plasma platelet-activating factor-acetylhydrolase from guinea pig.

In a previous study, we purified PAF-acetylhydrolase, which converts PAF to an inactive metabolite, lysoPAF, from peritoneal fluid of guinea pigs subjected to experimental endotoxin shock and found that this purified enzyme had similar biochemical properties to the plasma enzyme [Karasawa, K., Yato, M., Setaka, M., and Nojima, S. (1994) J. Biochem. 116, 374-379]. In this study, we isolated a homogeneous enzyme preparation from guinea pig plasma using a similar procedure. The molecular mass of this purified enzyme, as determined by SDS-PAGE was 58-63 kDa, larger than that (43 kDa) of the human enzyme. To elucidate the molecular structure of this enzyme and clarify its relationships with PAF-acetylhydrolases of other species, we isolated and sequenced a cDNA encoding this enzyme. Its cDNA contains an open reading frame encoding 436 amino acids and its predicted molecular mass (49 kDa) is lower than that of the native enzyme, suggesting that guinea pig plasma PAF-acetylhydrolase, unlike the human enzyme, is modified post-translationally, perhaps by glycosylation.

A distribution study of 11C platelet-activating factor (PAF) analogs in normal and tumor-bearing mice.

As a preliminary study to image platelet-activating factor (PAF) receptors in vivo, comparative study of biodistribution between 1-O-hexadecy1-2-O-N, N-dimethylcarbamoyl-sn-glycero-3-phosphocholine [choline-methyl-11C](L-[11C]dimethylcarbamoyl-PAF) and nonspecific PAF analog, 3-O-hexadecyl-2-O-N,N-dimethylcarbamoyl-sn-glycero-1-phosphocholine [choline-methyl-11C](D-[11C]-dimethylcarbamoyl-PAF) was carried out in both normal and tumor-bearing mice. Higher accumulation of L-[11C]dimethylcarbamoyl-PAF than D-[11C]dimethylcarbamoyl-PAF was observed in normal mice spleen. The co-administration of PAF antagonists dose-dependently reduced the radioactivity level of the L-isomer only in the spleen. In mice bearing Ehrlich tumors and Sarcoma 180, more L-than the D-[11C]-isomer was accumulated in the tumor and spleen. We found that specific accumulation sites for L-[11C]dimethylcarbamoyl-PAF exist in the spleen and tumors than in other tissues. Moreover, the comparison of accumulation between L- and D-[11C] dimethylcarbamoyl-PAF would be a useful procedure for estimation of PAF receptors in vivo.

Clear differences in ceramide metabolism between glycosphingolipids and sphingomyelin in a human promyelocytic leukemia cell line HL-60 stimulated by a differentiation inducer.

Although the ceramide components of both glycosphingolipids (GSLs) and sphingomyelin (SM) in HL-60 cells were identical, the molecular species of the ceramides preferentially used in biosynthesis were quite different in GSLs and SM. When HL-60 cells were stimulated to differentiate into macrophage-like cells by phorbol ester after their sphingolipids had been metabolically labeled with L-[3-14C]serine to saturation point, marked changes in the radioactivities of the ceramide residues were observed in GSLs, showing the activation of a biosynthetic pathway of ganglioside GM3. No significant changes were, however, observed in the ceramide residues of SM. These results indicate that it is necessary to consider the overall metabolism of ceramides, including their origin, when investigating the functions of ceramides in signal transduction systems.

Differences related to the production of arachidonic acid between collagen- and thrombin-stimulated human platelets.

The effects of collagen and thrombin on the liberation of free arachidonic acid were investigated in human platelets by fluorometric high-performance liquid chromatography. Collagen induced a concentration-dependent increase in the extent of platelet aggregation, as well as an accumulation of arachidonic acid in human platelets. By contrast, thrombin effectively provoked a potent aggregation at relatively low concentration without any accumulation of free arachidonic acid, although the accumulation of arachidonic acid was detected at a high concentration of thrombin (> 0.1 U/ml) that induced full aggregation. The selective liberation of arachidonic acid was found in thrombin-stimulated platelets. Non-selective liberation of fatty acids occurred in platelets that had been stimulated with a high concentration of collagen (10 micrograms/ml), as well as in platelets stimulated with A23187. The net amount of free arachidonic acid in collagen-stimulated platelets was estimated by use of eicosatetraenoic acid (ETYA), which is an inhibitor of both cyclooxygenase and lipoxygenase. ETYA markedly potentiated the accumulation of free arachidonic acid in collagen-stimulated platelets without changing the amounts of other fatty acids in the cell.

Accumulation of platelet-activating factor acetylhydrolase in the peritoneal cavity of guinea pig after endotoxin shock.

We examined the production of PAF, a mediator of shock, and LysoPAF, an inactive metabolite of PAF, in the guinea pig peritoneal cavity after i.p. administration of Escherichia coli LPS. Within 1 h of LPS administration, the level of PAF in the peritoneal fluid increased from 4.9 to 37.2 pmol/animal and decreased to the control value thereafter. In contrast, the level of lysoPAF gradually rose from 63.5 to 268 pmol/animal for up to 6 h. The activity of acetylhydrolase, which converts PAF to lysoPAF, in the peritoneal cavity increased in parallel with the increase in the lysoPAF level. The enzyme was distinguishable from phospholipase A2, because p-bromophenacylbromide (p-BPB), Ca2+, and ethylenediaminetetraacetic acid (EDTA) did not affect its enzymatic activity. In addition, this acetylhydrolase revealed similar biochemical properties to that detected in plasma. Both acetylhydrolases were resistant to trypsin treatment and had the same apparent molecular weight, as shown by gel-filtration column chromatography. These results suggest that the acetylhydrolase, which accumulates in the peritoneal cavity, infiltrates from the plasma in response to LPS, and then participates in the exclusion of PAF during endotoxin shock.

Purification and characterization of platelet-activating factor acetylhydrolase from peritoneal fluid obtained from guinea pigs after endotoxin shock.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) acetyl-hydrolase is an enzyme that hydrolyzes the acetyl ester of PAF. We purified this enzyme, which accumulated in the peritoneal cavity during endotoxin shock, by ammonium sulfate precipitation, and sequential use of butyl-Toyopearl, heparin-Sepharose, Con A-Sepharose, chelating-Sepharose, and MonoQ column chromatographies. We identified a monomeric polypeptide with a molecular weight of approximately 63 kDa on SDS-PAGE. This molecular weight differs from those of previously described acetylhydrolases. The purified enzyme did not degrade phospholipids with a long chain fatty acyl group at the sn-2 position. In addition, the enzyme activity was not inhibited by either pBPB or quinacrine. Accordingly, this enzyme is distinct from phospholipase A2. In addition, this enzyme also hydrolyzed some oxidatively fragmented phospholipids with PAF-like biological activities such as 1-O-hexadecyl-2-glutaroyl-sn-glycero-3-phosphocholine and 1-O-hexadecyl-2-succinoyl-sn-glycero-3-phosphocholine.

Possible influence of lysophospholipase on the production of 1-acyl-2-acetylglycerophosphocholine in macrophages.

The rate of production of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (acylPAF) was measured in macrophages following the incorporation of [3H]acetate. Upon activation by A23187, guinea pig alveolar macrophages incorporated [3H]acetate into PAF, but a little radioactivity was found in acylPAF. However, labeling of acylPAF and PAF with [3H]acetate was greatly enhanced in A23187-stimulated alveolar macrophages that had been pretreated with phenylmethanesulphonyl fluoride (PMSF). [3H]PAF was predominantly converted to 1-[3H]alkyl-2-acyl glycerophosphocholine, but [14C]acylPAF rapidly hydrolyzed to 14C-labeled free fatty acid by the incubation with lysates prepared from macrophages. The deacetylation of [14C]acylPAF and [3H]PAF by acetylhydrolase and also the hydrolysis of [14C]lysoPC by lysophospholipase were strongly inhibited in macrophages that had been pretreated with PMSF, while PMSF failed to inhibit the activities of acetyltransferase and acyltransferase. The relative proportions of PAF and acylPAF were quite different in different types of cells. In contrast to alveolar macrophages, peritoneal macrophages, neutrophils and spleen cells from guinea pigs incorporated 2-4 times more [3H]acetate into acylPAF than into PAF. The presence of high levels of acylPAF in peritoneal macrophages was confirmed by GLC-MS analysis. The activities of lysophospholipase, acetylhydrolase and acetyltransferase were measured in alveolar and peritoneal macrophages to determine whether the preferential formation of acylPAF as compared to PAF in peritoneal macrophages was due to differences in these activities between alveolar and peritoneal macrophages. The activity of acetylhydrolase of peritoneal macrophages was almost the same as that in alveolar macrophages. The activity of acetyltransferase in peritoneal macrophages was about half of that in alveolar macrophages. However, the activity of lysophospholipase in peritoneal macrophages was one-sixth of that in alveolar macrophages. These results suggest that lysophospholipase is one of the primary factors involved in the control of the production of acylPAF in activated cells, and that it acts by modulating the availability of lysoPC for the synthesis of acylPAF. Furthermore, high levels of activity of lysophospholipase allow the preferential formation of PAF, via the rapid hydrolysis of lysoPC which would act as a competitive inhibitor of the incorporation of acetate into lysoPAF.

Radioimmunoassay for platelet-activating factor.

A radioimmunoassay (RIA) for measurement of platelet-activating factor (PAF) was developed. At a final antiserum dilution of 1:640, the lowest detection limit of PAF was 0.1 pmol (50 pg). The standard curve obtained was suitable for measurement of PAF in amounts ranging from 0.1 pmol to 30 pmol. The antiserum showed high specificity. Cross-reaction for lysoPAF, lysophosphatidylcholine and long-chain phosphatidylcholines was very low (less than 0.025%). 1-Palmitoyl-2-acetyl-sn-glycero-3-phosphocholine cross-reacted slightly (6.25%). PAF exogenously added to macrophage suspensions was quantitatively determined by RIA after solvent extraction and high-performance liquid chromatographic separation. RIA was also used to estimate PAF formation after stimulation of rabbit alveolar macrophages in suspension with calcium ionophore A23187.

Specific binding of antibodies to platelet-activating factor (PAF) as demonstrated by thin-layer chromatography/immunostaining.

The specificity of rabbit antibodies produced by injection of 1-O-(15'-carboxypentadecyl)-2-N,N-dimethylcarbamoyl-sn-glycero-3- phosphocholine bovine serum albumin (BSA) conjugates was examined by a thin-layer chromatography (TLC/immunostaining method. Phosphatidylcholine (PC), lysophosphatidylcholine (lysoPC), lyso platelet-activating factor (lysoPAF), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), sphingomyelin (SM), phosphatidylinositol (PI), phosphatidic acid (PA) and cardiolipin (CL) were not immunostained. Among several synthetic PAF-related compounds, the antibodies only bound to PAF agonists which have the activity to induce washed rabbit platelet aggregation. The results suggest that the binding sites of the antibodies on the PAF molecule are the acetyl group at the sn-2 position and the choline moiety at the sn-3 position of glycerol, both of which are essential for exerting the biological function of PAF and for binding to the PAF receptors located on cellular membranes.

Antibodies to synthetic platelet-activating factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) analogues with substituents at the sn-2 position.

We obtained rabbit antibodies by injecting immunogenic conjugates which were prepared by combining covalently 1-O-(15'-carboxypentadecyl)-2-O-acetyl-sn-glycero-3- phosphocholine(acetyl-CPGPC), 1-O-(15'-carboxypentadecyl)-2-O-N,N-dimethylcarbamoyl-sn-glycero-3 - phosphocholine (dimethylcarbamoyl-CPGPC), or 1-O-(15'-carboxypentadecyl)-2-O-N-butyl-carbamoyl-sn-glycero-3-pho sphocholine (butylcarbamoyl-CPGPC) with protein (BSA or KLH), respectively, and examined the specificity of the resulting antibodies by comparison with inhibition of the binding of iodolabeled CPGPC derivatives to the antibodies by corresponding or related phospholipids. Acetyl-CPGPC and dimethylcarbamoyl-CPGPC possessed haptenic activity causing production of antibodies reactive with PAF. Changes of the substituents at sn-2 in the antigens affected the specificity of the resulting antibodies. The affinity of the substituents to the antibodies decreased in the following order: acetyl much greater than dimethylcarbamoyl and butylcarbamoyl for antibodies to acetyl-CPGPC-KLH; dimethylcarbamoyl greater than acetyl much greater than butylcarbamoyl for antibodies to dimethylcarbamoyl-CPGPC-BSA; and butylcarbamoyl greater than dimethylcarbamoyl greater than acetyl for antibodies to butylcarbamoyl-CPGPC-BSA. Naturally occurring phospholipids, including lysoPAF, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, revealed no cross-reactivities with these antibodies. Anti-dimethylcarbamoyl-CPGPC-BSA IgG and anti-acetyl-CPGPC-KLH IgG inhibited a PAF-induced aggregation of washed rabbit platelets in a dose-dependent manner. In contrast, anti-butylcarbamoyl-CPGPC-BSA IgG did not affect a PAF-induced platelet aggregation, nor did preimmune IgG.

Purification and characterization of (H+ + K+)-ATPase from hog gastric mucosa.

A (H+ + K+)-ATPase-enriched membrane fraction derived from the fundic portion of hog gastric mucosa was obtained by a combination of differential and repeated 7% Ficoll gradient centrifugation. The microsomal membrane fraction isolated by repeated 7% Ficoll gradient centrifugation was free of ouabain-sensitive (Na+ + K+)-ATPase, 5'-nucleotidase and succinate dehydrogenase; and it was highly enriched in (H+ + K+)-ATPase and K(+)-stimulated p-nitrophenylphosphatase (p-NPPase). The (H+ + K+)-ATPase had a pH optimum of 7.4 and was stimulated by Tl+, K+, Rb+ and NH4+ with Ka values of 0.0667, 0.526, 0.667 and 3.03 mM, respectively, at this pH. On the other hand, monovalent cations such as Na+, Li+ and (CH3)4N+ as well as divalent cations such as Cu2+, Ca2+, Ba2+, Sr2+ and Cd2+ inhibited this enzyme activity concentration-dependently. Ouabain and oligomycin had no effect, whereas omeprazole, a specific (H+ + K+)-ATPase inhibitor, inhibited this enzyme activity in a pH-dependent manner. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed a major band (greater than or equal to 90% of protein) at 97,400 daltons, which was phosphorylated in the presence of Mg2+ and [gamma-32P]-ATP and dephosphorylated in the presence of K+. The present method was very simple, and the (H+ + K+)-ATPase activity of the microsomal fraction obtained by this method was much higher compared with those obtained by other methods such as free-flow electrophoresis.

Antibody to platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF).

Specific antibodies to platelet activating factor (PAF) were prepared by immunizing rabbits with a hapten-bovine serum albumin (BSA) conjugate. As the hapten we used the synthetic PAF derivative which is resistant against enzymatic inactivation by plasma or tissues and which can bind to BSA through covalent bonding. Antibody activity was determined by an enzyme-linked immunosorbent assay (ELISA). Anti-PAF IgG reacted strongly with PAF. By means of the ELISA inhibition assay, we found that the antibody did not cross-react with phosphocholine, glycerophosphocholine, dilaurylglycerophosphocholine or PAF analogues which have ethanolamine-type polar head groups instead of choline group.