Expressed sequence tags identify human isologs of the ARF-dependent phospholipase D.

By searching into Expressed Sequence Tags databases (dbEST) using Blast X algorithm software and a plant phospholipase D as template, we have identified a cDNA from human brain (Z45777) which encodes for a protein similar to the amino acid region 743-929 of the human phospholipase D1 (PLD1), and a cDNA from human liver (R93485) which encodes for a protein similar to region 815-932 of PLD1. Sequence comparison between cloned phospholipases showed the presence of 3 conserved amino acid sequences: AFVGGIDLAYGRWD (box A), IIGSANINDRS (box B), and YIYIENQFFI (box C). Phylogenic analysis indicated that the cDNA from brain and liver encoded for human isologs of PLD1.

Transacylase-mediated alkylacyl-GPC synthesis and its hydrolysis by phospholipase D occur in separate cell compartments in the human neutrophil.

Subcellular localizations of CoA-independent transacylase and phospholipase D enzymes have been investigated in human neutrophils performing a two-step gradient system to separate plasma membranes from internal membranes and from the bulk of granules. The internal membranes were constituted by endoplasmic reticulum and by a subpopulation of specific and tertiary granules. The enzymes activities were assayed in vitro on gradient fractions using exogenous substrates.

Involvement of vicinal dithiols in differential regulation of fMLP and phorbol ester-activated phospholipase D in stimulated human neutrophils.

In the human neutrophil, the fMLP-activated phospholipase D (PLD) was entirely calcium and tyrosine kinase dependent, but protein kinase C independent. An opposite regulation was observed with phorbol ester PMA, since the phospholipase D activity was mostly calcium insensitive, tyrosine kinase independent, but protein kinase C dependent. The arsenical compound, phenylarsine oxide (PAO), which reacts with vicinal sulhydryl groups, activated twofold at one minute the PMA stimulated-PLD activity, whereas it inhibited half of the fMLP-activated PLD after a time lag of 30 sec.

Phosphatidylcholine turnover in activated human neutrophils. Agonist-induced cytidylyltransferase translocation is subsequent to phospholipase D activation.

Phosphatidylcholine synthesis and degradation are tightly regulated to assure a constant amount of the phospholipid in cellular membranes. The chemotactic peptide fMLP and the phorbol ester, phorbol 12-myristate 13-acetate, are known to stimulate phosphatidylcholine degradation by phospholipase D in human neutrophils. fMLP alone triggered phosphatidylcholine breakdown into phosphatidic acid, but did not stimulate phosphatidylcholine synthesis or activation of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase.

Reversible translocation of cytidylyltransferase between cytosol and endoplasmic reticulum occurs within minutes in whole cells.

Addition of oleic acid to Krebs II cells induced a rapid incorporation of [3H]choline into phosphatidylcholine, since 500 microM of the fatty acid stimulated choline incorporation by 5-fold over the control after 5 min of incubation. In fact, a noticeable increase in phosphatidylcholine labelling could be monitored immediately after 1 min of cell incubation with [3H]choline, at which time 50% of cytosolic cytidylyltransferase activity (EC 2.7.

Subcellular localization of enzymes related to PAF metabolism in Krebs-II ascites cells: evidence for the lack of intracellular PAF transfer activity.

We have investigated various steps in the metabolism of Platelet-Activating Factor (PAF-acether or PAF) at the subcellular level in Krebs-II ascites cells. Microsomes contained an active acetyltransferase located on a heavy-rough domain of the endoplasmic reticulum quite rich in ribosomes, as monitored by [3H]uridine labelling, and which displayed a very high density across the Percoll gradient. This membrane domain, which was separated from all other cellular organelles including peroxisomes, also contained a membrane-bound acetylhydrolase with similar activity as the acetyltransferase.

Cytidylyltransferase translocation onto endoplasmic reticulum and increased de novo synthesis without phosphatidylcholine accumulation in Krebs-II ascite cells.

Addition of oleic acid to Krebs-II cells stimulated by 9-fold [3H]choline incorporation into choline glycerophospholipids without affecting the selective incorporation of the precursor into diacyl subclass (90% of total [3H]choline glycerophospholipids). The total activity of cytidylyltransferase (E.C.

Modulation of CTP:phosphocholine cytidylyltransferase translocation by oleic acid and the antitumoral alkylphospholipid in HL-60 cells.

Short time effect of oleate and 1-O-alkyl-2-O-methyl-rac-glycero-3-phosphocholine (AMGPC) on choline incorporation into phosphatidylcholines were studied in HL-60 cells. The non lytic concentration of 50 microM oleate induced a three-fold increase in [3H]choline incorporation into phosphatidylcholine. This stimulation was accompanied by a translocation of the CTP:phosphocholine cytidylyltransferase (EC 2.

PAF-acether transfer activity in HL-60 cells is induced during differentiation.

Platelet-activating factor is a proinflammatory lipid active at subnanomolar concentrations. The intermembrane transfer of a biologically active PAF analog has been previously demonstrated in macrophages. Here we demonstrate that the specific activity of this transfer activity increases when HL-60 cells are induced to differentiate by treatment with dimethyl sulfoxide, dibutyryl cAMP or phorbol diester.

Differential activation by fMet-Leu-Phe and phorbol ester of a plasma membrane phosphatidylcholine-specific phospholipase D in human neutrophil.

Signal transduction involving phosphatidylcholine hydrolysis has been investigated in human neutrophils (PMN) after in situ generation of [3H]alkylacyl-sn-glycero-3-phosphocholine ([3H]alkylacyl-GPC) by cell incubation with [3H]alkylacetyl-GPC. When PMN were stimulated with the chemotactic peptide N-formyl-Met-Leu-Phe(fMLP) or phorbol myristate acetate (PMA) in the presence of cytochalasin B, both 1-O-alkyl-2-acyl-sn-glycero-3-phosphate (PA) and 1-O-alkyl-2-acyl-sn-glycerol (AAG) were generated. On addition of the agonists in the presence of ethanol, phosphatidylethanol (PEt) [corrected] was formed with a concomitant decrease in PA and AAG.

Subcellular localization of phospholipids and enzymes involved in PAF-acether metabolism.

The biosynthesis of platelet-activating factor (PAF-acether or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) through the remodeling pathway was investigated at the subcellular level in two different cell lines. In human neutrophils, plasma membrane was isolated not only from granules, but also from internal membranes related to endoplasmic reticulum. Interestingly, the latter exhibited enhanced acetyltransferase upon neutrophil stimulation with ionophore A23187.

Characterization of plasma membranes from A431 cells, isolated by self-generating Percoll gradient: a rapid isolation procedure to obtain plasma membranes with functional epidermal growth factor receptors.

Plasma membranes have been isolated from the human epidermoid carcinoma cell line A431 by a rapid fractionation of lysate on Percoll density gradient at pH 9.6. Endoplasmic reticulum, lysosomes and mitochondria sedimented at the bottom of gradient whereas plasma membranes focused at low density, as shown with specific markers.

Intracellular processing of cytidylyltransferase in Krebs II cells during stimulation of phosphatidylcholine synthesis. Evidence that a plasma membrane modification promotes enzyme translocation specifically to the endoplasmic reticulum.

After a 3-h incubation of Krebs II ascitic cells in the presence of phospholipase C from Clostridium welchii under nonlytic conditions, the incorporation of [3H] choline into phosphatidylcholine was increased 1.7-fold as compared to untreated cells. The total amounts of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were unchanged up to 3 h of incubation.

Evidence that biosynthesis of platelet-activating factor (paf-acether) by human neutrophils occurs in an intracellular membrane.

Human polymorphonuclear leukocytes (PMN) were incubated in the absence or presence of the calcium ionophore A23187 (6 microM) for 10 min at 37 degrees C. They were then lysed by nitrogen cavitation and fractionated using Percoll gradients. Three major fractions of increasing density corresponding to plasma membrane, intracellular membranes and secretory granules were detected using [3H]concanavalin A, NADH-dehydrogenase and beta-D-glucuronidase as respective markers.

Carrier-mediated choline uptake by Krebs II ascites cells.

Krebs II ascites cells have a low affinity uptake system for choline (Km = 36 microM, Vm = 76 nmol/min per 2 X 10(8). Choline entered the cells and was rapidly phosphorylated (95% of total intracellular soluble label). Trans acceleration of labeled choline from cells preloaded with radiolabeled choline and postincubated in the presence of unlabeled choline indicates that choline transport in Krebs II ascites cell is carrier mediated.

An in vitro study of hemicholinium-3 on phospholipid metabolism of Krebs II ascites cells.

With [Me-14C]choline as marker and after separation of choline and phosphocholine by ion-exchange column chromatography or thin layer chromatography on alumina, it is shown that 40 microM hemicholinium-3 (HC-3) inhibits the cytosolic choline-kinase of rat liver and Krebs cells. This inhibition is competitive (Km different, Vm similar) in the first case and mixed in the second (Km and Vm different). Despite this general inhibition of the phosphocholine formation, the synthesis of phosphatidylcholine (PC) by post-nuclear supernatants of rat liver and Krebs cells is different when tested with HC-3.