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PubMed1987

Possible involvement of the phospholipases in the mitogenic actions of prolactin (PRL) on Nb2 node lymphoma cells.

The possible role of the phospholipase enzymes in the prolactin stimulation of mitogenesis in Nb2 node lymphoma cells was investigated. Two phospholipase inhibitors including quinacrine and alpha-para-dibromoacetophenone (BPB) were employed. Quinacrine at concentrations of 1-5 microM attenuated the magnitude of the PRL stimulation of cell division; at concentrations of 10 microM and above quinacrine abolished the PRL response. BPB at concentrations of 1-10 microM also inhibited the mitogenic effect of PRL in a concentration response fashion. The polyunsaturated fatty acid arachidonic acid partially reversed the inhibitory effects of these drugs. In further studies, exogenously added phospholipase C at concentrations of 5-50 ng/ml was found to potentiate the mitogenic effect of prolactin when prolactin was employed at a concentration that evoked a half-maximal response. By itself, however, phospholipase C had no effect on the rate of cell division. Phospholipase A2 either by itself or in the presence of prolactin was without effect.

Acetophenones↗

PubMed1979

In vitro actions of some antibiotics on phospholipases.

The effects of some antibiotics on activities of phospholipase A2, B and C were investigated in vitro. Tetracyclines, macrolides, chloramphenicol and carbenicillin inhibited the activity of Crotalus adamanteus phospholipase A2 towards phospholipids of egg-yolk emulsions. When the ability to inhibit the activity of Penicillium notatum phospholipase B towards mixed micelles of phosphatidylcholine and Triton X-100 was investigated, polymyxin B was found to be inhibitory while chloramphenicol and carbenicillin were found to stimulate the activity of the phospholipase. The activity of Bacillus cereus phospholipase C towards the mixed micelles was inhibited by bleomycin, oleandomycin and chloramphenicol.

Carbenicillin↗

PubMed2001

Purification, sequencing, and phylogenetic analyses of novel Lys-49 phospholipases A(2) from the venoms of rattlesnakes and other pit vipers.

Basic phospholipase A(2) homologs with Lys49 substitution at the essential Ca(2+)-binding site are present in the venom of pit vipers under many genera. However, they have not been found in rattlesnake venoms before. We have now screened for this protein in the venom of rattlesnakes and other less studied pit vipers. By gel filtration chromatography and RP-HPLC, Lys49-phospholipase-like proteins were purified from the venoms of two rattlers, Crotalus atrox and Crotalus m. molossus, and five nonrattlers, Porthidium nummifer, Porthidium godmani, Bothriechis schlegelii, Trimeresurus puniceus, and Trimeresurus albolabris. Their N-terminal amino acid sequences were shown to be characteristic for this phospholipase subfamily. The purified basic proteins from rattlesnakes caused myonecrosis and edema in experimental animals. We have also cloned the cDNAs and solved the complete sequences of four novel Lys49-phospholipases from the venom glands of C. atrox, P. godmani, B. schlegelii, and Deinagkistrodon acutus (hundred-pace). Phylogenetic analyses based on the amino acid sequences of 28 Lys49-phospholipases separate the pitviper of the New World from those of the Old World, and the arboreal Asiatic species from the terrestrial Asiatic species. The implications of the phylogeny tree to the systematics of pit vipers, and structure-function relationship of the Lys49-phospholipases are discussed.

Amino Acid Substitution↗

PubMedDate not supplied

Phospholipase activity in Cryptococcus neoformans.

Phospholipases have only been detected in a few fungi and yeasts, in particular in Candida albicans. Secreted phospholipases are considered by some researchers to be a potential factor of virulence and pathogenicity in C. albicans. Twenty-three Cryptococcus neoformans strains were tested in order to observe phospholipase production. Twenty-two of the 23 strains tested were able to produce phospholipases, and the ratio diameter of the colony to total diameter of the colony plus zone of precipitation (Pz) ranged between 0.271 and 0.949. C. neoformans, just like C. albicans, can be divided on the basis of the Pz into different strains according to their virulence and pathogenicity. There also appeared to be a correlation between the phospholipase production and the size of the capsule in the strains isolated from AIDS patients. For this reason, further studies on C. neoformans phospholipase activity would be useful in evaluating the virulence of different strains.

AIDS-Related Opportunistic Infections↗

PubMedDate not supplied

Transient desensibilization of isolated hepatocytes against phalloidin by treatment with phospholipase A.

The development of typical protrusions in isolated hepatocytes after incubation with phalloidin was prevented by phospholipase A (from bee venom). When cells were preincubated with low concentrations of phospholipase A and the enzyme was removed by washing, the number of cells affected by 10 microgram phalloidin/ml was markedly reduced. If the pretreated cells were allowed to recover after removal of phospholipase, the sensitivity to phalloidin returned to nearly normal values. Transient treatment of hepatocytes with sublytic concentrations of phospholipase A did not destroy cell membranes, whereas 5-fold higher concentrations of the enzyme produced large protrusions quite different from those appearing during phalloidin poisoning. These findings suggest that phosphatides are needed for the recognition of phalloidin by liver cells. A series of marker enzymes were analysed in isolated plasma membranes from rat liver after treatment with phospholipase A. Changes in the activities of K+ Na+-ATPase and of p-nitrophenylphosphatase were observed. Other membranal enzymes were not markedly influenced. The inhibitory effect of phospholipase A on the phalloidin response is discussed in context of earlier findings suggesting an evident role of a membranal protein for the recognition of phalloidin.

Animals↗

PubMed1995

Bradykinin stimulates phospholipase D in PC12 cells by a mechanism which is independent of increases in intracellular Ca2+.

These experiments were designed to learn the role of bradykinin induced changes in intracellular Ca2+ in the activation of phospholipase D activity in PC12 cells. Ionomycin at a concentration of 0.1 microM caused an increase in intracellular Ca2+ comparable to bradykinin, but had no effect on phospholipase D activity. Carbachol, ATP, and thapsigargin also increased intracellular Ca2+ but had no effect on phospholipase D activity. Increases in intracellular Ca2+ may be a necessary but not a sufficient factor in the activation of phospholipase D. To investigate this issue, the bradykinin induced increase in intracellular Ca2+ was blocked by preincubating the cells in Ca(2+)-free media plus EGTA or in media containing the intracellular Ca2+ chelator BAPTA/AM. These preincubations completely blocked the bradykinin induced increase in intracellular Ca2+ but only attenuated the bradykinin mediated activation of phospholipase D. Physiological increases in intracellular Ca2+ apparently do not mediate the effect of bradykinin on phospholipase D.

Adenosine Triphosphate↗

PubMed1995

Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification.

Alkaline phosphatase activity was released up to 100% from the membrane by incubating the rat osseous plate membrane-bound enzyme with phosphatidylinositol-specific phospholipase C. The molecular weight of the released enzyme was 145,000 on Sephacryl S-300 gel filtration and 66,000 on PAGE-SDS, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyse PNPP, ATP and pyrophosphate. The hydrolysis of ATP and PNPP by phosphatidylinositol-specific phospholipase C-released enzyme exhibited 'Michaelian' kinetics with K0.5 = 70 and 979 microM, respectively. For pyrophosphate, K0.5 was 128 microM and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K0.5 = 1.5 mM) and zinc ions were a powerful noncompetitive inhibitor (Ki = 6.2 microM) of phosphatidylinositol-specific phospholipase C-released enzyme. Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40 degrees C. However, with increasing temperature from 40-60 degrees C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08 x 10(-4) min-1 to 0.684 min-1. Treatment of phosphatydilinositol-specific phospholipase C-released alkaline phosphatase with Chellex 100 depleted to 5% its original PNPPase activity. Magnesium (K0.5 = 29.5 microM), manganese (K0.5 = 5 microM) and cobalt ions (K0.5 = 10.1 microM) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K0.5 = 653 microM) was less effective (only 26%) and occurred with site-site interactions (n = 0.7). Zinc ions had no stimulatory effects. The possibility that the soluble form of the enzyme, detected during endochondral ossification, would arise by the hydrolysis of the Pl-anchored form of osseous plate alkaline phosphatase is discussed.

Alkaline Phosphatase↗

PubMed1977

ATPase and phosphatase activities from human red cell membranes: II. The effects of phospholipases on Ca2+-dependent enzymic activities.

Treatment of human red cell membranes with pure phospholipase A2 results in a progressive inactivation of both Ca2+-dependent and (Ca2+ + K+)-dependent ATPase and phosphatase activities. When phospholipase C replaces phospholipase A2, Ca2+-dependent ATPase activity and Ca2+-dependent phosphorylation of red cell membranes are lost, while Ca2+-dependent phosphatase activity is enhanced and its apparent affinity for Ca2+ is increased about 20-fold. Activation of Ca2+-dependent phosphatase following phospholipase C treatment was not observed in sarcoplasmic reticulum preparation. Phospholipase C increases the sensitivity of the phosphatase to N-ethylmaleimide but has little effect on the kinetic parameters relating the phosphatase activity to substrate and cofactors, suggesting that no extensive structural disarrangement of the Ca2+-ATPase system has occurred after incubation with phospholipase C.

Adenosine Triphosphatases↗

PubMed1991

Platelet-derived growth factor activates phospholipase D and chemotactic responses in vascular smooth muscle cells.

Platelet-derived growth factor (BB dimer; PDGF-BB) stimulates a mitogenic response in A-10 vascular smooth muscle cells. In addition, PDGF-BB stimulates phospholipase D activity against phosphatidylcholine in A-10 cells. This response was observed as a rapid metabolism of phosphatidylcholine to phosphatidate and choline; a subsequent metabolism generates sustained levels of diacylglycerol. The accumulation of phosphatidylethanol, a transphosphatidylation product of phospholipase D, was obvious in PDGF-treated cells. PDGF-BB also stimulates a chemotactic response in A-10 cells. The concentrations of PDGF-BB required to stimulate mitogenesis, phospholipase D activity and chemotaxis are similar. This finding shows that PDGF induces a variety of cellular responses and suggests that these responses may share common metabolic pathways. That conception was tested by investigating the activity of the different PDGF dimers. PDGF-AA had little or no activity in A-10 cells for any of the responses measured. PDGF-AB and PDGF-BB were equally potent in stimulating mitogenic responses. However, the AB heterodimer was only half as active as PDGF-BB with respect to activation of phospholipase D and chemotactic responses. These results demonstrate that PDGF stimulates phospholipase D in vascular smooth muscle cells. In addition, the data indicate that different PDGF dimers can transduce varying signals and suggest a link between the mechanisms by which PDGF-BB activates phospholipase D and the chemotactic response.

Animals↗

PubMed1994

Angiotensin II-mediated stimulation of phospholipase D in rabbit kidney proximal tubule cells.

The present study was undertaken to demonstrate whether or not angiotensin II activates a phospholipase D in rabbit kidney proximal tubule cells. By measuring the formation of [3H]phosphatidic acid and [3H]phosphatidylethanol, we elucidate the direct stimulation of phospholipase D by angiotensin II. Angiotensin II leads to a rapid increase in [3H]phosphatidic acid and [3H]diacylglycerol, and [3H]phosphatidic acid formation preceded the formation of [3H]diacylglycerol. This result suggests that some phosphatidic acid seems to be formed directly from phosphatidylcholine by the action of phospholipase D, not from the action of diacylglycerol kinase on the diacylglycerol. In addition, the other mechanisms by which phospholipase D is activated was examined. We have found that phospholipase D was activated by extracellular calcium ion. It has also been shown that angiotensin II may activate phospholipase D through protein kinase C-independent pathway.

Angiotensin II↗

PubMed2003

Association of a polymorphism of the phospholipase D2 gene with the prevalence of colorectal cancer.

Phospholipase D plays an important role in transmembrane signaling in a variety of cell types and its activity is increased in certain cancers, suggesting that it also contributes to tumorigenesis. A C-->T transition at nucleotide 1814 of the human phospholipase D(2) gene, which results in a Thr-->Ile substitution at amino acid 577, was noted in the GenBank database. The relationship of this polymorphism to the prevalence of cancer of the esophagus, stomach, colon-rectum, lung, and breast in Japanese was investigated in a case-control study. The genotype of the phospholipase D(2) gene was determined by the polymerase chain reaction with confronting two-pair primers. Multivariate logistic regression analysis with adjustment for age, gender, and smoking status revealed that the frequency of the T allele of the 1814C-->T polymorphism was significantly higher in individuals with colorectal cancer than in controls. A significant association of the polymorphism with the prevalence of colorectal cancer was found in analyses assuming either dominant (TT+CT vs. CC) or additive (CT vs. CC) effects of the T allele, but the T allele was not associated with the prevalence of esophageal, gastric, lung, or breast cancer. The activities of phospholipase D in cell lysates or membrane fractions did not differ between cells transfected with cDNAs encoding the Thr-577 or Ile-577 variants of phospholipase D(2). These results suggest that the phospholipase D(2) gene is a susceptibility locus for colorectal cancer in Japanese individuals, although a functional effect of the 1814C-->T (Thr577Ile) polymorphism was not detected.

Colorectal Neoplasms↗

PubMed2007

Phospholipases and their industrial applications.

Phospholipids are present in all living organisms. They are a major component of all biological membranes, along with glycolipids and cholesterol. Enzymes aimed at modifying phospholipids, namely, phospholipases, are consequently widespread in nature, playing very diverse roles from aggression in snake venom to signal transduction and digestion in humans. In this review, we give a general overview of phospholipases A1, A2, C and D from a sequence and structural perspective and their industrial application. The use of phospholipases in industrial processes has grown hand-in-hand with our ability to clone and express the genes in microbial hosts with commercially attractive amounts. Further, the use in industrial processes is increasing by optimizing the enzymes by protein engineering. Here, we give a perspective on the work done to date to express phospholipases in heterologous hosts and the efforts to optimize them by protein engineering. We will draw attention to the industrial processes where phospholipases play a key role and show how the use of a phospholipase for oil degumming leads to substantial environmental benefits. This illustrates a very general trend: the use of enzymes as an alternative to chemical processes to make products often provides a cleaner solution for the industrial processes. In a world with great demands on non-polluting, energy saving technical solutions--white biotechnology is a strong alternative.

Animals↗

PubMed2003

Determination of the phospholipase activity of patatin by a continuous spectrophotometric assay.

Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. This protein has been reported to serve as a storage protein and also to exhibit lipid phospholipase activity. This paper describes a simple continuous spectrophotometric method for assaying patatin phospholipase activity. The procedure is based on a coupled enzymatic assay using [1,2-dilinoleoyl] PC as the phospholipase substrate and lipoxygenase as the coupling enzyme. In the procedure developed in this work, lipoxygenase oxidizes the linoleic acid released by the phospholipase activity of patatin. This activity can then be followed spectrophotometrically by recording the increase in absorbance at 234 nm that results from the formation of the corresponding hydroperoxide from linoleic acid by the action of lipoxygenase. The optimal assay concentrations of patatin and lipoxygenase were established. Phospholipase activity varied with pH, reaching its optimal value at pH 9.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3 mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (Vm = 9.8 x 10(-3) micromol/min x microg protein, Km = 7.8 microM, Vm/Km = 1.3 min(-1) x microg protein). This method proved to be specific since there was no activity in the absence of patatin. It also had the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase.

Carboxylic Ester Hydrolases↗

PubMed1986

Chinese hamster ovary cells depend on exogenous lipids to survive phospholipase C treatment.

Chinese hamster ovary cells were maintained in culture medium supplemented with delipidated serum to make them dependent on nonlipid components for lipid synthesis. Growth in lipid-free medium resulted in an increased flux through the CDP-choline pathway for phosphatidylcholine synthesis. The increased flux appeared to be mediated by the CTP:phosphocholine cytidylyltransferase because cellular phosphocholine levels decreased in cells grown in lipid-free medium, and both cell-free cytidylyltransferase activity and membrane-associated cytidylyltransferase activity increased in cells grown in lipid-free medium. Chinese hamster ovary cells maintained in culture medium supplemented with complete serum can grow at nearly normal rates in the presence of phospholipase C for many generations, even though the treatment enhances turnover of cellular phosphatidylcholine (R. Sleight and C. Kent (1983) J. Biol. Chem. 258, 824-830). The phospholipase C treatment, however, was toxic to cells maintained in medium supplemented with delipidated serum. Lysophosphatidylcholine protected cells from phospholipase toxicity, but did not support growth. The rate of utilization of lysophosphatidylcholine for phosphatidylcholine synthesis was about 8% of the turnover rate for phosphatidylcholine, and was not increased by phospholipase C treatment. Reconstitution of the medium with fractionated serum lipids showed that the cells required specific neutral lipids, namely, fatty acids plus cholesterol, in order to grow in the presence of phospholipase C. Either oleate or cholesterol (up to 80 microM) alone did not allow growth in phospholipase C, but a combination of these two lipids supported growth effectively.

Animals↗

PubMed1986

Diacylglycerol metabolism in phospholipase C-treated mammalian cells.

Treatment of cultured cells with phospholipase C causes increased rates of hydrolysis of cellular phosphatidylcholine and increased rates of incorporation of choline into phosphatidylcholine. The fate of the diacylglycerol produced by the phospholipase C hydrolysis was examined in two cell lines, Chinese hamster ovary and HeLa. In the former cells, turnover of the glycerol moiety of phosphatidylcholine was not enhanced by phospholipase C treatment, indicating that the phospholipase C-generated diacylglycerol was recycled into new phosphatidylcholine. In HeLa cells, turnover of the glycerol backbone of phosphatidylcholine was enhanced by phospholipase C treatment, and the increased rate of turnover of the glycerol moiety was similar to that of the phosphate moiety. Thus, the fate of diacylglycerol generated at the plasma membrane was demonstrated to differ in these two cell lines. Incorporation of precursors of diacylglycerol into phosphatidylcholine was not enhanced by phospholipase C treatment in either cell line.

Cell Line↗

PubMed1990

Production of phosphatidylethanol by phospholipase D phosphatidyl transferase in intact or dispersed pancreatic islets: evidence for the in situ metabolism of phosphatidylethanol.

To determine if phospholipase D is present in intact adult islets, we took advantage of the fact that, in the presence of ethanol, this enzyme generates phosphatidylethanol via transphosphatidylation. Extracts of cells prelabeled with [14C]arachidonate, [14C]myristate, or [14C]stearate were analyzed via three TLC systems; the identify of phosphatidylethanol was further confirmed via incorporation of [14C]ethanol into the same phospholipid bands. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate stimulated phosphatidylethanol (to 603% of basal by 60 min) both in intact adult islets and in dispersed neonatal islet cells. A nonphorbol activator of protein kinase C (mezerein) also stimulated phospholipase D, whereas a phorbol which does not activate protein kinase C (4 alpha-phorbol-12,13-didecanoate) was virtually inactive. The effects of the active phorbol ester or of mezerein were reduced by the protein kinase C inhibitor H-7 and were virtually eliminated by prior down-regulation of that enzyme. In addition, a calcium-selective ionophore (ionomycin) or fluoroaluminate also activated the islet phospholipase D. When accumulation of phosphatidylethanol (labeled with any of three fatty acids) was induced by a preincubation in the presence of ethanol plus agonist, which then were removed, phosphatidylethanol declined by 34-47% over a subsequent 60-min incubation. Thus, while phosphatidylethanol is relatively stable metabolically, it is detectably degraded (a variable overlooked in previous studies). In the absence of ethanol, stimulated islet cells generated phosphatidic acid, although such hydrolysis was less evident than transphosphatidylation. Ethanol provision distinguished phosphatidate formed via phospholipase D (inhibition, via phosphatidylethanol formation) from that due predominantly to phospholipase C (phosphatidate not inhibited). In view of our recent findings that phosphatidic acid (or exogenous phospholipase D) has potent insulinotropic effects, this pathway could play a role in stimulus-secretion coupling; conversely, stimulation of transphosphatidylation at the expense of hydrolysis could contribute to the inhibition of secretion caused by ethanol.

Aging↗

PubMed1976

A phospholipase A2 with anticoagulant activity. II. Inhibition of the phospholiped activity in coagulation.

An anticoagulant factor with phospholipase A2 activity has been isolated from Vipera berus venom. Phospholipase activity was studied on platelet phospholipid and on brain cephalin. The venom factor showed a potent anticoagulant activity: 1 mug impaired the clotting of 1 ml of citrated recalcified platelet-poor plasma. The anticoagulant inhibited clotting by antagonism to phospholipid. The antagonism constant (Kan = 6.8-10(-9) M) demonstrated the high affinity of the inhibitor for phospholipid. As with other phospholipases A2, the venom factor was thermoresistant but very sensitive to photo-oxidation. Both activities (anticoagulant activity and phospholipase activity) were not markedly dissociated by either denaturation or neutralization processes. Slightly different curves of photo-oxidative inactivation of both activities suggested the presence, on the molecule, of two very close sites responsible for phospholipase and anticoagulant activities. The inhibitor effect on coagulation was independent of the hydrolysis process. In fact, lysoderivatives and fatty acids, resulting from complete hydrolysis with the venom factor, were as active as the native phospholipids. Moreover phospholipase A2 from other viperidae venom, which did not have anticoagulant activity, produced similarly active lysoderivatives. This showed that the cleavage of the beta-acyl bond does not interfere with the activity of phospholipid. A possible mechanism of clotting inhibition by the venom factor was proposed. Owing to its high affinity for phospholipid, the inhibitor would complex phospholipid at its protein binding site impairing the normal arrangement of coagulation protein factors and, consequently, their activation. The positive charges of the inhibitor (pI = 9.2) could bind with phosphoryl or carboxyl groups of phospholipid, making them unavailable for protein binding. The complex formation involves a loss of dissociating capacity of the enzyme towards its substrate. This required an additional interaction of the inhibitor with a coagulation protein factor. The inhibitor could be removed from the complex by specific antibodies, permitting recovery of normal phospholipid-protein interaction. The role of calcium in the complex has not yet been elucidated. This venom factor affords a useful tool for investigating the phospholipid-clotting protein interaction.

Anticoagulants↗

PubMed1976

Phospholipases A1 and A2 in bovine thyroid.

In both supernatant and sediment of thyroid tissue homogenate phospholipase and lysophospholipase activities were demonstrated. In the supernatant, using 1-acyl-2[1-14C]linoleoyl-sn-glycero-3-phosphorocholine in the presence of sodium taurocholate, phospholipase A1 activity with pH optima at 3.6 and 4.8 and phospholipase A2 activity with pH optima at 3.6 and 5.7 were found. The sediment showed mainly phospholipase A2 activity with a pH optimum at pH 6.5. Lysophospholipase activity (optimum pH 7--8), USING 1-[9,10-(3)H]stearyl-sn-glycero-3-phosphorocholine as a substrate was present in both supernatant and sediment. Enzyme assays performed on subcellular fractions suggest the soluble phospholipases to be of lysosomal origin and the solubilized phospholipase A2 activity of homogenate sediment to be of microsomal origin. Incubations with 3H-14C mixed labelled phosphatidylcholine further confirmed the above observations.

Animals↗