Ectoenzyme release from rat liver and kidney by phosphatidylinositol-specific phospholipase C

Ectoenzyme release from rat liver and kidney by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis was studied. Alkaline phosphatase and 5'-nucleotidase were released from rat kidney slices to extents of up to 60% and 30%, respectively. Release of alkaline phosphatase was observed at lower amounts of PI-specific phospholipase C than that of 5'-nucleotidase. Both enzymes were more easily released from microsomal fractions or free cells. From kidney cells, alkaline phosphatase was released without cell lysis, and more than 80% release of alkaline phosphatase was observed at 3.8% hydrolysis of PI. Isoelectric focusing profiles of alkaline phosphatase released by PI-specific phospholipase C were significantly different from the control in the cases of both rat liver and kidney. Lubrol-solubilized alkaline phosphatase was eluted at the void volume of a Toyopearl HW-55 column, while the enzyme obtained by further treatment with PI-specific phospholipase C was eluted in the lower-molecular-weight region corresponding to 100,000-110,000 daltons. Furthermore, Lubrol-solubilized phosphatase became more thermostable on treatment with PI-specific phospholipase C.     

ARE CELLS RESCUED FROM ‘LOW DENSITY DEATH’ BY CO‐OPERATION BETWEEN PHOSPHOLIPASES C AND D?

Cells of the ciliate Tetrahymena thermophila die when transferred at low density to a lipid-free nutritionally complete medium. This death is prevented and they will start to proliferate if protein kinase C is activated and this activation is sustained. We propose that this takes place in two stages. Firstly, the phospholipase C pathway beginning with and specific for phosphatidylinositol leads to the formation of diacylglycerol and inositol tris -phosphate. Diacylglycerol activates protein kinase C, and inositol tris -phosphate via Ca(2+)phospholipase D (PLD). Secondly, the protein kinase C response can now be sustained by diacylglycerol produced by phospholipase D, using phosphatidylcholine and phosphatidylserine as substrates. Should this switching from PI-specific phospholipase C (PLC) to phospholipase D fail, then the cell will die in the course of milliseconds during the minutes following inoculation.     

Properties of bovine erythrocyte acetylcholinesterase solubilized by phosphatidylinositol-specific phospholipase C1

The properties of acetylcholinesterase solubilized from bovine erythrocyte membrane by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis or with a detergent, Lubrol-PX, were studied. The activity of Lubrol-PX-solubilized acetylcholinesterase was broadly distributed in the fractions having Ve/Vo = 1.0-2.0 in gel filtration on a Sepharose 6B column. The intermediary fractions (Ve/Vo = 1.3-1.7) were collected as "the middle active Sepharose 6B eluate" and characterized on the basis of enzymology and protein chemistry. When this eluate was treated with PI-specific phospholipase C, the major activity peak was obtained in the later fractions with Ve/Vo = 1.75-2.0 on the same column chromatography. Lubrol-solubilized and phospholipase C-treated acetylcholinesterase preparations were different in the thermostability, the elution profiles of chromatography on Mono Q, butyl-Toyopearl and phenyl-Sepharose columns, and the affinity to phospholipid micelles. On treatment with PI-specific phospholipase C, Lubrol-solubilized acetylcholinesterase became more thermostable. The phospholipase C-treated enzyme was eluted at lower NaCl concentration from the Mono Q column than the Lubrol-solubilized enzyme. The most important difference was observed in the hydrophobicity of these two enzyme preparations. The Lubrol-solubilized enzyme shows high affinity to phospholipid micelles and hydrophobic adsorbents such as butyl-Toyopearl and phenyl-Sepharose. However, this hydrophobicity was lost when acetylcholinesterase was solubilized from bovine erythrocyte membrane by PI-specific phospholipase C. The presence of myo-inositol was confirmed in the purified preparation of acetylcholinesterase by gas chromatography (GC)-mass spectrometry (MS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of antibodies against the coenzyme pyrrolequinoline quinone

Rat intestinal epithelial cells were isolated and the activity of the enzyme diacylglycerol lipase (DG lipase, EC 3.1.1.3) was investigated. When cells were treated with Escherichia coli heat-stable toxin (ST) liberation of endogenous glycerol and fatty acids was observed. The enzyme responsible for this effect could be demonstrated to be a DG lipase by using specific substrates. It was found that the activity of DG lipase was increased 5–6-fold with the substrates diolein and 1,2-dioleyl-rac-glycerol and triolein being neutral lipid insensitive to DG lipase. ST had no direct effect on the DG lipase. The enzyme DG lipase was activated via a chain reaction due to the hydrolysis of phosphatidylinositol (PI) by the enzyme PI-specific phospholipase C stimulated by ST.     

Insulin rapidly increases glycerol-3-phosphate-acyltransferase activity in rat adipocytes.

Glycerol-3-phosphate acyltransferase (G3PAT) was activated by insulin in intact rat adipocytes within 1 min: this activation persisted for 10 min, and was due to a decrease in the Km of the enzyme. The addition of insulin to control adipocyte membranes also increased G3PAT activity, and this effect was mimicked by phosphatidylinositol-specific phospholipase C. Cytosol fractions from insulin-treated adipocytes stimulated G3PAT activity of control membranes, suggesting that a soluble mediator is released during insulin action, possibly through activation of a PI-specific PLC.     

Ethanol is a potent stimulator of phosphatidylcholine breakdown in cultured rat hepatocytes.

Addition of ethanol (17 to 340 mM) to cultured rat hepatocytes stimulated the breakdown of phosphatidylcholine phospholipases D and C as measured by an increase in the rate of release of choline and phosphocholine into the medium. The effects of ethanol were mimicked by propanol, dimethylsulfoxide and to a lesser extent methanol. The magnitude of the stimulation seen with ethanol was equivalent to and additive to that produced by glucagon vasopressin, norepinephrine, A23187 or PMA. In contrast, ethanol (340 mM) stimulated PI-specific phospholipase C activity by less than 20%. An equivalent stimulation of PC-specific phospholipase D and C was seen with as little as 20 mM ethanol and a 100% increase was seen with 340 mM ethanol. Ethanol did not significantly affect the ability of vasopressin, norepinephrine, ATP or A23187 to stimulate PI-specific phospholipase C. It is concluded that while ethanol is only a weak stimulator of PI-specific phospholipase C, it is a potent stimulator of phosphatidylcholine breakdown in rat hepatocytes.     

Enzymatic removal of alkaline phosphatase from renal brush-border membranes. Effect on phosphate transport and on phosphate binding

Brush-border membrane vesicles prepared from rabbit kidney cortex were incubated at 37 degrees C for 30 min with phosphatidylinositol-specific phospholipase C. This maneuver resulted in a release of approx. 85% of the brush-border membrane-linked enzyme alkaline phosphatase as determined by its enzymatic activity. Transport of inorganic [32P]phosphate (100 microM) by the PI-specific phospholipase C-treated brush-border membrane vesicles was measured at 20-22 degrees C in the presence of an inwardly directed 100 mM Na+ gradient. Neither initial uptake rates, as estimated from 10-s uptake values (103.5 +/- 6.8%, n = 7 experiments), nor equilibrium uptake values, measured after 2 h (102 +/- 3.4%) were different from controls (100%). Control and PI-specific phospholipase C-treated brush-border membrane vesicles were extracted with chloroform/methanol to obtain a proteolipid fraction which has been shown to bind Pi with high affinity and specificity (Kessler, R.J., Vaughn, D.A. and Fanestil, D.D. (1982) J. Biol. Chem. 257, 14311-14317). Phosphate binding (at 10 microM Pi) by the extracted proteolipid was measured. No significant difference in binding was observed between the two types of preparations: 31.0 +/- 9.37 in controls and 29.8 +/- 8.3 nmol/mg protein in the proteolipid extracted from PI-specific phospholipase C-treated brush-border membrane vesicles. It appears therefore that alkaline phosphatase activity is essential neither for Pi transport by brush-border membrane vesicles nor for Pi binding by proteolipid extracted from brush-border membrane. These results dissociate alkaline phosphatase activity, but not brush-border membrane vesicle transport of phosphate, from phosphate binding by proteolipid.     

Activation of calcium transport in synaptosomes by phosphatidylinositol-specific phospholipase C

The isotope labeling method was used to study the influence of phospholipases C of different origin and specificity on Ca2+ accumulation in rat brain synaptosomes. It was found that phospholipases C specific to phosphatidylinositides (PI) stimulate Ca2+ transport into synaptosomes, while non-specific phospholipase C, which hydrolyzes different membrane lipid fractions, decreases the Ca2+ content in synaptosomes. It is supposed that the stimulating effect of PI-specific phospholipases C is determined by the activation of PI metabolism, which results in an increase in the content of some PI metabolism products serving as Ca2+ ionophores in synaptosomal membranes. The inhibition of Ca2+ uptake by synaptosomes treated with non-specific phospholipase C is thought to result from partial disruption of synaptosomal membranes.     

The TRPM7 channel is inactivated by PIP(2) hydrolysis

TRPM7 (ChaK1, TRP-PLIK, LTRPC7) is a ubiquitous, calcium-permeant ion channel that is unique in being both an ion channel and a serine/threonine kinase. The kinase domain of TRPM7 directly associates with the C2 domain of phospholipase C (PLC). Here, we show that in native cardiac cells and heterologous expression systems, G alpha q-linked receptors or tyrosine kinase receptors that activate PLC potently inhibit channel activity. Numerous experimental approaches demonstrated that phosphatidylinositol 4,5-bisphosphate (PIP(2)), the substrate of PLC, is a key regulator of TRPM7. We conclude that receptor-mediated activation of PLC results in the hydrolysis of localized PIP(2), leading to inactivation of the TRPM7 channel.     

Regulation of arachidonic acid release by enzyme(s) of rat brain cortex

Brain cortex membranes labeled with [14C]arachidonic acid were used as the source of substrate and enzyme for the assay of arachidonic acid (AA) liberation. A significant amount of AA was released Ca2(+)-independently, mainly from phosphatidic acid, polyphosphoinositides and phosphatidylserine. Quinacrine, inhibitor of phospholipase A2 (PLA2), suppressed AA release by 60% and neomycin, inhibitor of phospholipase C (PLC) by about 30%. Both inhibitors applied together have an additive effect. Physiological calcium level elevated AA liberation by 50%, whereas 2 mM calcium enhanced this process by a further 30%. Carbachol, exclusively in the presence of calcium, activated AA release selectively from phosphatidylinositol and diglycerides. We suggest that Ca2(+)-independent PLA2 and PLC play an important role in AA liberation, and that physiological increments of calcium may have serious implications.     

Regulation of eosinophil function by phosphatidylinositol-specific PLC and cytosolic PLA(2)

We examined the regulatory role of cytosolic phospholipase A(2) (cPLA(2)) and phosphatidylinositol (PI)-specific phospholipase C (PLC) in the degranulation of human eosinophils and leukotriene (LT) C(4) synthesis. Activation with formyl-Met-Leu-Phe + cytochalasin B (fMLP/B) caused a time-dependent release of eosinophil peroxidase (EPO) and LTC(4), which was inhibited by pertussis toxin. By immunoblotting, eosinophil PLC-beta2 and -gamma2 isoforms were identified, and PLC activation was measured as a function of inositol 1,4,5-trisphosphate concentration. Stimulated release of EPO and intracellular Ca(2+) concentration was inhibited by ET-18-OCH(3), a PI-PLC inhibitor, whereas trifluoromethylketone (TFMK), a cPLA(2) blocker, had no inhibitory effect. Both TFMK and ET-18-OCH(3) attenuated stimulated arachidonate release and LTC(4) secretion, suggesting that activation of both PLC and cPLA(2) is essential for LTC(4) synthesis caused by fMLP/B. The structurally unrelated protein kinase C inhibitors bisindolylmaleimide, Ro-31-8220, and Go-6976 all blocked fMLP/B-induced EPO release but not LTC(4) secretion. 1,2-bis(2-Aminophenoxy)ethane-N,N,N',N'- tetraacetic acid acetoxymethyl ester, an intracellular Ca(2+) chelator, suppressed both EPO release and LTC(4) secretion. We found that fMLP/B-induced LTC(4) secretion from human eosinophils is regulated by PI-PLC through calcium-mediated activation of cPLA(2). However, cPLA(2) does not regulate eosinophil degranulation.     

Action of phosphatidylinositol specific phospholipase C on platelets: nonlytic release of acetylcholinesterase, effect on thrombin and PAF induced aggregation

Phosphatidylinositol (PI) specific phospholipase C treatment of rabbit platelets caused 95% release of acetylcholinesterase in the supernatant and 4 to 6% hydrolysis of membrane PI in 2 min. Under these conditions there was no cell lysis as monitored by lack of lactate dehydrogenase activity in the medium. The phospholipase C had no activity towards phosphatidylinositol-4- phosphate and phosphatidylinositol-4,5-bis phosphate. Platelets pretreated with the phospholipase C responded normally to thrombin and platelet activating factor. It is concluded that acetylcholinesterase exists in specific interaction with PI in platelet membranes. Further, the membrane protein release phenomenon caused by the PI-specific phospholipase C did not effect the physiological responsiveness of platelets. Possible implications of these findings to the linkage between PI and membrane enzyme are also discussed.     

Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.     

Cell surface sialylation and fucosylation are regulated by the cell recognition molecule L1 via PLCγ and cooperate to modulate embryonic stem cell survival and proliferation

Cell surface glycosylation patterns are markers of cell type and status. However, the mechanisms regulating surface glycosylation patterns remain unknown. Using a panel of carbohydrate markers, we have shown that cell surface sialylation and fucosylation are upregulated in L1-transfected embryonic stem cells (L1-ESCs). Consistently, the mRNA levels of sialyltransferase ST6Gal1 and ST3Gal4, and fucosyltransferase FUT9 were significantly increased in L1-transfected ESCs. Activation of L1 signaling promoted cell survival and inhibited cell proliferation. ShRNAs knocking down FUT9, ST6Gal1 and ST3Gal4 blocked these effects. A phospholipase Cγ (PLCγ) inhibitor and shRNA reduced ST6Gal1, ST3Gal4 and FUT9 mRNA levels in the L1-ESCs. Thus, embryonic stem cell surface sialylation and fucosylation are regulated via PLCγ by L1, with which they cooperate to modulate cell survival and proliferation.     

Inositol phospholipid metabolism in Arabidopsis. Characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C

Phosphoinositides (PIs) constitute a minor fraction of total cellular lipids in all eukaryotic cells. They fulfill many important functions through interaction with a wide range of cellular proteins. Members of distinct inositol lipid kinase families catalyze the synthesis of these phospholipids from phosphatidylinositol. The hydrolysis of PIs involves phosphatases and isoforms of PI-specific phospholipase C. Although our knowledge of the roles played by plant PIs is clearly limited at present, there is no doubt that they are involved in many physiological processes during plant growth and development. In this review, we concentrate on inositol lipid-metabolizing enzymes from the model plant Arabidopsis for which biochemical characterization data are available, namely the inositol lipid kinases and PI-specific phospholipase Cs. The biochemical properties and structure of characterized and genome-predicted isoforms are presented and compared with those of the animal enzymes to show that the plant enzymes have some features clearly unique to this kingdom.     

CGDEase, a Pseudomonas fluorescens protein of the PLC/APase superfamily with CDP-ethanolamine and (dihexanoyl)glycerophosphoethanolamine hydrolase activity induced by osmoprotectants under phosphate-deficient conditions

A novel enzyme, induced by choline, ethanolamine, glycine betaine or dimethylglycine, was released at low temperature and phosphate from Pseudomonas fluorescens (CECT 7229) suspensions at low cell densities. It is a CDP-ethanolamine pyrophosphatase/(dihexanoyl)glycerophosphoethanolamine phosphodiesterase (CGDEase) less active on choline derivatives, and inactive on long-chain phospholipids, CDP-glycerol and other NDP-X compounds. The reaction pattern was typical of phospholipase C (PLC), as either phosphoethanolamine or phosphocholine was produced. Peptide-mass analyses, gene cloning and expression provided a molecular identity for CGDEase. Bioinformatic studies assigned it to the PLC branch of the phospholipase C/acid phosphatase (PLC/APase) superfamily, revealed an irregular phylogenetic distribution of close CGDEase relatives, and suggested their genes are not in operons or conserved contexts. A theoretical CGDEase structure was supported by mutagenesis of two predicted active-site residues, which yielded essentially inactive mutants. Biological relevance is supported by comparisons with CGDEase relatives, induction by osmoprotectants (not by osmotic stress itself) and repression by micromolar phosphate. The low bacterial density requirement was related to phosphate liberation from lysed bacteria in denser populations, rather than to a classical quorum-sensing effect. The results fit better a CGDEase role in phosphate scavenging than in osmoprotection.     

Detection of a phosphatidylinositol-specific phospholipase C at the surface of Swiss 3T3 cells and its potential role in the regulation of cell growth

The metabolism and intracellular distribution of a fluorescent analog of phosphatidylinositol (PI), 1,2-[oleoyl,N-(6-[(7-nitrobenz-2-oxa-1,3-diazo-4-yl) aminocaproyl)]-PI (C6-NBD-PI), was examined in monolayer cultures of Swiss 3T3 cells following its insertion into the plasma membrane. Evidence is presented that the exogenously supplied C6-NBD-PI was hydrolyzed by a calcium-dependent PI-specific phospholipase C (PI-PLC) at the external cell surface and that this PI-specific phospholipase C may play a role in the density-dependent inhibition of cell growth: (i) When confluent monolayer cultures were incubated with C6-NBD-PI for 60 min at 7 degrees C, the lipid spontaneously transferred to the cells, and prominent labeling of intracellular membranes was observed. Lipid extraction and analysis demonstrated that more than 60% of the fluorescent lipid in these cells was fluorescent diacylglycerol (DAG). However, when the corresponding fluorescent analogs of phosphatidylcholine or phosphatidylethanolamine were used, the fluorescent lipids readily transferred to cells, but no hydrolysis to fluorescent DAG occurred. (ii) Both intracellular labeling and hydrolysis of C6-NBD-PI to -DAG were inhibited in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. (iii) When myo-[2-3H]inositol-labeled C6-NBD-PI was incubated with cells, [3H]inositol phosphate was released into the incubation medium, but no water-soluble 3H-labeled products were found associated with the cells. (iv) The level of C6-NBD-PI hydrolysis increased dramatically with increasing density of 3T3 cells in monolayer culture.     

Constitutive activation of A(3) adenosine receptors by site-directed mutagenesis

The objective of this study was to create constitutively active mutant human A(3) adenosine receptors (ARs) using single amino acid replacements, based on findings from other G protein-coupled receptors. A(3) ARs mutated in transmembrane helical domains (TMs) 1, 3, 6, and 7 were expressed in COS-7 cells and subjected to agonist radioligand binding and phospholipase C (PLC) and adenylyl cyclase (AC) assays. Three mutant receptors, A229E in TM6 and R108A and R108K in the DRY motif of TM3, were found to be constitutively active in both functional assays. The potency of the A(3) agonist Cl-IB-MECA (1-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide) in PLC activation was enhanced by at least an order of magnitude over wild type (EC(50) 951 nM) in R108A and A229E mutant receptors. Cl-IB-MECA was much less potent (>10-fold) in C88F, Y109F, and Y282F and mutants or inactive following double mutation of the DRY motif. The degree of constitutive activation was more pronounced for the AC signaling pathway than for the PLC signaling pathway. The results indicated that specific locations within the TMs proximal to the cytosolic region were responsible for constraining the receptor in a G protein-uncoupled conformation.