Transfer Of Phosphocholine Research Articles

33] 1-Alkyl-2-acetyl-sn-glycerol cholinephosphotransferase

Publisher Summary A dithiothreitol (DTT)-insensitive cholinephosphotransferase catalyzes the transfer of phosphocholine from CDPcholine to l-alkyl-2-acetyl- sn -glycerol in the final step of the biosynthesis of platelet-activating factor (PAF) in the de novo pathway. In contrast, DTT strongly inhibits the cholinephosphotransferase activity that catalyzes the formation of phosphatidylcholine from diacylglycerols and CDPcholine. Therefore, the terms DTT-insensitive and DTT-sensitive are useful in distinguishing these two types of cholinephosphotransferases. Because the two types of cholinephosphotransferase activities differ in a number of other properties, different proteins might be responsible for synthesizing PAF and phosphatidylcholine. The importance of the DTT-insensitive cholinephosphotransferase in maintaining PAF at physiological is significant because the cosubstrate (CDPcholine) formed by cytidylyltransferase can be rate limiting in the synthesis of PAF by the de novo route. Yet, an additional function of the DTT-insensitive cholinephosphotransferase could be its regulation of the cellular levels of alkylacetylglycerols, because the latter can also induce biological responses by influencing protein kinase C activity and by inducing cell differentiation (for example, converting HL-60 cells to macrophage-like cells7). The DTT-insensitive cholinephosphotransferase occupies a central position in the PAF de novo pathway because it can utilize one type of mediator (alkylacetylglycerols) as a substrate to produce another (PAF) with completely different activities.

Changes in phospholipid composition and calcium flux in LLC-PK cells cultured at low magnesium concentrations

Monolayers of porcine kidney cells (LLC-PK) were grown in a series of Nu-Serum-supplemented media containing different Mg 2+ concentrations (480, 250, 25, 6.3 or 2.6 μM) to study the effect of Mg 2+ depletion on cellular phospholipid changes and the consequent effect on the membrane permeability to Ca 2+. Cells grown on 6.3 or 2.6 μM Mg 2+ showed a decrease in PE, PS, Sph, PI and an increase of PC. These changes were attributed mainly to the decreased rate of Sph synthesis through the transfer of phosphocholine from PC to ceramide, or due to the increase of PE N-methylation as found in Mg 2+-deficient cells. The 45Ca uptake was increased in cells grown on 25.0 μM Mg 2+, while it was decreased in cells grown on 6.3 or 2.6 μM Mg 2+. These changes in Ca 2+ uptake were related to changes of cellular phospholipids and fatty acids which affect adenylate cyclase activity in the membrane, as well as the membrane fluidity.

Evidence for a high activity of sphingomyelin biosynthesis by phosphocholine transfer from phosphatidylcholine to ceramides in lung lamellar bodies

Biosynthesis of sphingomyelin from ceramides was investigated in lung subcellular fractions by incubating a lyophilized mixture of albumin and subcellular fraction (0.1–0.2 mg of protein) coated with [acyl- 14 C]-ceramide and phosphatidyl[methyl- 3 H]choline in Iris-buffer. The lamellar-body-rich fraction exhibited the highest specific activity for sphingomyelin biosynthesis measured by 14C incorporation into sphingomyelins or by [ 3H]phosphocholine transfer from phosphatidylcholines. Plasma membranes formed the next most active fraction, followed by the ‘smooth’ and, then, the ‘rough’ endoplasmic reticulum. Sphingomyelin biosynthesis by lamellar bodies was optimum at pH 7.4 and was inhibited by sphingomyelins formed. Slight inhibitory effects were also observed with Mn 2+, Ca 2+ and lysophosphatidylcholine. Phosphocholine transfer from CDPcholine was not observed under the reaction conditions employed. Ceramide conversion and phosphocholine transfer increased with ceramide concentration to reach a maximum at about 0.06 mM. The highest conversion rate was observed when 18:1 ceramide was used as an acceptor. When 1-palmitoyl-2-oleoylphosphatidylcholine was the phosphocholine donor, the overall biosynthesis of sphingomyelin was much higher than when using dipalmitoylphosphatidylcholine. These results suggest the possible involvement of the studied reaction in the control of the degree of saturation of the surfactant phosphatidylcholine.

A new de novo pathway for the formation of 1-alkyl-2-acetyl-sn-glycerols, precursors of platelet activating factor. Biochemical characterization of 1-alkyl-2-lyso-sn-glycero-3-P:acetyl-CoA acetyltransferase in rat spleen.

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC, platelet activating factor (PAF] can be biosynthesized either by acetylation of alkyllyso-GPC through a remodeling pathway or by the transfer of phosphocholine to alkylacetyl-sn-glycerol (alkylacetyl-G) via a putative de novo pathway involving a dithiothreitol-insensitive cholinephosphotransferase. However, the relevance of the de novo pathway in the biosynthesis of PAF depends on the existence of enzymes that can directly synthesize alkylacetyl-G from 1-alkyl-2-lyso-sn-glycero-3-P (alkyllyso-GP) or some other source. In this study, we demonstrated that microsomal preparations of rat spleen can synthesize alkylacetyl-GP by an alkyllyso-GP:acetyl-CoA acetyltransferase and that this intermediate is subsequently dephosphorylated by an alkylacetyl-GP phosphohydrolase to generate alkylacetyl-G. The properties of alkyllyso-GP:acetyl-CoA acetyltransferase were characterized under conditions where the contaminating activity of alkylacetyl-GP phosphohydrolase was minimal; this was accomplished by inhibiting the phosphohydrolase with the addition of sodium vanadate and sodium fluoride to the assay mixtures and incubating at relatively low temperatures (23 degrees C). Alkyllyso-GP:acetyl-CoA acetyltransferase had a pH optimum of 8.4 at 23 degrees C and was located in the microsomal fraction. The apparent Km for acetyl-CoA under these conditions was 226 microM and the optimal concentration of alkyllyso-GP ranged between 16 and 25 microM. Based on pH optima, substrate inhibition studies, and sensitivities to preincubation temperatures of the microsomes, it appears that alkyllyso-GP:acetyl-CoA acetyltransferase differs from the acetyltransferase responsible for the transfer of acetate from acetyl-CoA to alkyllyso-GPC to form PAF. A variety of tissues had high activities of alkyllyso-GP:acetyl-CoA acetyltransferase, which indicates that this pathway is operational in many cell types. Our results document the existence of a complete de novo biosynthetic pathway for the assembly of PAF, and this route could be responsible for maintaining physiological levels of platelet activating factor for normal cell function.

134. Precipitating and steroid-releasing antiserum against the estrogen receptor

A gene, mf3, encoding glycosyltransferase in glycoglycerophospholipid (GGPL; GGPL-I and GGPL-III) biosynthesis in Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, modified codon usage, and expressed in Escherichia coli. The mf3 gene consists of an open reading frame of 1221 bp encoding 406 amino acids. The mf3 gene product, Mf3, has 27% amino acid homology with glycosyltransferase of Borrelia burgdorferi but no homology to genes of other Mycoplasma species in the GenBank database. The reaction product of Mf3 using 1,2-dipalmitoilglycerol and UDP-glucose as substrates showed a specific sodium adducted ion at m/z 753, which corresponded to glucopyranosyl dipalmitoilglycerol as determined by MALDI-TOF mass spectrometry. Furthermore, in the reaction product by Mf3 and Mf1 which was a cholinephosphotransferase and previously cloned from M. fermentans PG18, an ion at m/z 896 corresponding to GGPL-I was detected by mass spectrometry. The product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem MS analysis of protonated molecules at m/z 896. From these results, mf3 was identified as a glycosyltransferase. It was suggested that glucose transfer and phosphocholine transfer to 1,2-dipalmitoylglycerol are involved in the GGPL biosynthesis pathway of M. fermentans PG18.

Does de novo synthesis of lysophosphatidylcholine occur in rat lung microsomes?

Incubation of rat lung microsomes with CDP[ Me- 14C]choline resulted in formation of radioactive lysophosphatidylcholine and phosphatidylcholine. Evidence is provided which suggests that lysophosphatidylcholine formation cannot be ascribed completely to phospholipase A degradation of phosphatidylcholine. Lysophosphatidylcholine production can be stimulated by addition of monoacylglycerol or diacylglycerol. It is suggested that diacylglycerol is partly hydrolyzed to monoacylglycerol and subsequently converted to lysophosphatidylcholine. A direct transfer of phosphocholine from CDPcholine to monoacylglycerol is demonstrated by equimolar incorporation of 1(3)-[9,10- 3H 2]palmitoylglycerol and phospho[ Me- 14C]choline into lysophosphatidylcholine.