Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding, internalization, and degradation of tissue-type plasminogen activator (t-PA) were studied in a rat hepatoma (Novikoff) cell line. Binding of t-PA to specific saturable high affinity binding sites (Kd = 12 nM, 54,000 sites/cell) was followed by internalization and degradation and did not require a functional active site. The catabolism of t-PA was not inhibited by an excess of urokinase-type plasminogen activator (u-PA), and t-PA bound to Novikoff membranes was not complexed to PAI-1, suggesting a mechanism independent of PAI-1. Additionally, a mannose receptor is not involved since t-PA binding was not influenced by an excess of mannose, galactose, ovalbumin, or EDTA. Furthermore, the degradation of t-PA was not influenced by 10 mM 6-aminohexanoic acid, a lysine analogue. The t-PA receptor binds to and can be eluted from wheat germ agglutinin-Sepharose. Cross-linking of t-PA with partially purified receptor and ligand blot analysis, suggest that t-PA binds to two proteins, a principal one of 55 kDa and a minor one of 43 kDa. Novikoff cells are able also to bind (Kd = 1.4 nM, 25,000 sites/cell) and degrade u-PA. The binding was inhibited by pro-u-PA and the amino-terminal fragment of u-PA, but not by an excess of t-PA. The u-PA receptor, but not the t-PA receptor, was removed by treatment with phosphatidylinositol-specific phospholipase C. Our results show that the clearance receptor for t-PA on Novikoff cells is different from the mannose receptor and the PAI-1-dependent receptor described in other cells. The rat hepatoma cells are thus a good model to study the PAI-1 independent hepatocyte-specific clearance of t-PA.
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PMID:Demonstration of a specific clearance receptor for tissue-type plasminogen activator on rat Novikoff hepatoma cells. 131 32

We investigated the effects of polymyxin B (PMB), an antibiotic that binds to endotoxins, on the uptake and degradation of low density lipoproteins (LDLs) in HepG2 cells, a highly differentiated human hepatoma cell line. The results showed that PMB very effectively enhanced the binding, internalization, and degradation of LDL in HepG2 cells. The PMB-mediated enhancement of LDL uptake was not dependent on the LDL receptor-mediated pathway, as blockage of the LDL receptor by use of a monoclonal anti-LDL receptor antibody had no effect on the PMB-mediated cellular processing of LDL and PMB-mediated enhancement of LDL uptake did not cause an increase in cholesterol esterification. In addition, chloroquine and colchicine, which inhibit lysosomal degradation and cellular endocytosis, respectively, diminished PMB-enhanced degradation of LDL, indicating that PMB mediates uptake through a pathway similar to the LDL receptor-mediated pathway. The PMB-mediated uptake of LDL was sensitive to treatment with phospholipase C and pronase and was dependent on the presence of Ca2+. PMB caused similar changes in human skin fibroblasts, bovine smooth muscle cells, and bovine endothelial cells, which suggests that PMB-enhanced LDL uptake is a general cellular phenomenon. Our results thus indicate that PMB increases cellular catabolism of LDL through an endocytotic pathway not involving the LDL receptors.
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PMID:Polymyxin B enhances low density lipoprotein catabolism in hepatic and extrahepatic cells. 131 96

The identification of free glycoinositol phospholipids (GPIs) following biosynthetic labeling with [3H]glucosamine in cultured cells has been reported by several laboratories. We applied this procedure to two of the cell types used in these studies, H4IIE hepatoma cells and isolated hepatocytes, but were unable to detect a [3H]glucosamine-containing lipid that met any of the criteria for GPIs, including sensitivity to phosphatidylinositol-specific phospholipase C (PIPLC) or GPI-specific phospholipase D. Part of the difficulty in radiolabeling a GPI by this procedure was the rapid metabolic conversion of [3H]glucosamine to galactosamine and neutral or anionic derivatives. A PIPLC-sensitive radiolabeled lipid was detected only after 16 h of labeling. The water-soluble fragments released from this lipid by PIPLC corresponded largely to myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate, products expected from PIPLC cleavage of phosphatidylinositol or lyso-phosphatidylinositol. In an alternative approach that we introduce here, free GPIs in lipid extracts from rat liver plasma membranes were labeled by reductive radiomethylation. This procedure, which radiomethylates primary and secondary amines, has been shown to label a glucosamine residue adjacent to inositol in all GPIs characterized to date. The labeled extracts were fractionated by two-dimensional thin-layer chromatography, and a cluster of polar labeled lipids were assigned as GPIs based upon the following observations. 1) They were cleaved by PIPLC, 2) after hydrolysis in 6 N HCl, both radiomethylated glucosamine and a glucosamine-inositol conjugate were identified by cation exchange chromatography, and 3) hydrolysis in 4 M trifluoroacetic acid generated a fragment consistent with glucosamine-inositol-phosphate. These results illustrate new criteria for the identification of GPIs. The labeled GPIs also contained radiomethylated ethanolamine, another component found in GPI anchors of proteins and in mature lipid precursors of GPI anchors, suggesting that the liver plasma membrane GPIs retained considerable structural homology to GPI anchors.
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PMID:Identification of glycoinositol phospholipids in rat liver by reductive radiomethylation of amines but not in H4IIE hepatoma cells or isolated hepatocytes by biosynthetic labeling with glucosamine. 132 29

Antineoplastic ether lipids have entered phase I clinical trial and, although their mechanism of action remains unclear, it is widely believed that the plasma membrane is the primary cellular drug target. In the present study the hypothesis was tested that metabolism of ether lipids acts as a detoxification process. [31P]-nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of the ether lipid SRI 62-834 (SRI) and the phosphate ester hexadecylphosphocholine (HPC) in the presence of both isolated phospholipases C and D and post-mitochondrial rat liver homogenate. Both SRI and HPC were slowly metabolised by phospholipase D to their alkyl phosphates and choline, and the alkyl phosphates were subsequently metabolised by phosphatase to yield the alcohols and inorganic phosphate. These studies failed to detect any metabolism of either SRI or HPC by phospholipase C, and the metabolism of platelet-activating factor (PAF) by this enzyme was not inhibited by the addition of either compound. The cytotoxicity of SRI, the related compound HPC and their metabolites was determined in vitro using three cell lines. Cytotoxicity was measured by analysis of cell growth kinetics, MTT assay and lactate dehydrogenase release. Closely similar results were obtained in the JB1 rat hepatoma cell line, in the non-transformed BL8 rat hepatocyte cell line, and in A549 human lung adenocarcinoma cells. SRI was the most toxic of the compounds analysed, the concentration required to produce 50% toxicity or growth inhibition (IC50) being 6-9 microM. The putative metabolite of SRI, 2,2'-bis(hydroxymethyl)tetrahydrofuran, and the known metabolites [2'-(octadecyloxymethyl)tetrahydrofuran-2'-yl]methyl phosphate and 2-hydroxymethyl-2-octadecyloxymethyltetrahydrofuran exhibited IC50 values of > 200, > 100 and 40-70 microM, respectively, consistent with metabolic detoxification. HPC was more cytotoxic (IC50, 37 microM) than its phosphate metabolite (IC50, 140 microM), but its toxicity was similar to that of its metabolite hexadecanol (IC50, 28 microM), suggesting that only the former metabolic route leads to detoxification.
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PMID:Is metabolism an important arbiter of anticancer activity of ether lipids? Metabolism of SRI 62-834 and hexadecylphosphocholine by [31P]-NMR spectroscopy and comparison of their cytotoxicities with those of their metabolites. 145 Dec 37

The metabolism of phosphatidylinositol was studied in normal quiescent hepatocytes, hepatocellular carcinomas induced by single dose of diethylnitrosamine, followed by 2-acetylaminofluorene and partial hepatectomy (Solt-Farber model), and in an established hepatoma cell line, JB1. The JB1 hepatoma cell line and hepatocellular carcinomas demonstrated a 4- to 5-fold higher rate of turnover of [3H]-inositol and [3H]-glycerol than the control hepatocytes. Significantly, elevated levels of second messengers inositol 1,4,5-trisphosphate and sn-1,2-diacylglycerol were noted in hepatic tumor cells within 4 hr of labeling with precursor molecules, whereas no detectable level of 3H-labeled inositol trisphosphate was noted in quiescent hepatocytes, even after incubation with 10 mM LiCl for 30 min. Approximately 2.5-fold higher specific activities of a guanine nucleotide and Ca+2 dependent phosphatidylinositol 4,5-bisphosphate specific phospholipase C were detected in the hepatocellular carcinoma cells. The cellular location of the phospholipase C activity was also different, being membrane bound in hepatocytes and equally distributed between cytosolic and membrane factions in the hepatomas. These data are consistent with the hypothesis that the enhanced production of diacylglycerol and inositol 1,4,5-trisphosphate in hepatocellular carcinomas may be due to the activation of a guanine nucleotide dependent phosphatidylinositol 4,5-bisphosphate specific phospholipase C. These data are the first to compare phosphoinositide turnover in normal liver and hepatic tumor cells and suggest that the sustained levels of second messengers is closely associated with the transformation and enhanced growth rate in hepatic tumor cells.
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PMID:Altered levels of phosphoinositide metabolites and activation of guanine-nucleotide dependent phospholipase C in rat hepatic tumors. 164 43

Monoclonal antibodies were isolated following immunization with the HBsAg and alpha-fetoprotein-secreting human hepatoma PLC/PRF/5 ("Alexander") cell line. Three antibodies (K-PLC1, K-PLC2 and K-PLC3) showed evidence of carcinoma-associated reactivity by indirect immunofluorescence. Antibodies K-PLC2 and K-PLC3 reacted only with PLC/PRF/5 cells, but not with any other normal or malignant cell type tested, including the Hep/G2 hepatoma cell line. The reactivity of these antibodies was not removed by absorption with homogenates of either normal liver or a primary hepatocellular carcinoma. These results suggest that K-PLC2 and K-PLC3 identify PLC/PRF/5 idiospecific determinants. Following surface iodination of PLC/PRF/5 cells, immunoprecipitation and analysis on polyacrylamide gels, these specific determinants were found to be of 200,000 and 76,000 daltons, respectively. On the other hand, antibody K-PLC1, although unreactive by immunofluorescence on the majority of normal cell types, including those of lymphoid organs and bone marrow liver cells and most epithelia, was weakly positive on some normal ductal secretory epithelia and was positive on vascular endothelium. However, K-PLC1 reacted strongly with all carcinoma specimens tested, and with most carcinoma-derived cell lines, indicating a large increase in K-PLC1 antigen expression by epithelial cells after malignant transformation. Absorption of K-PLC1 with normal liver homogenate had no affect, but absorption with a hepatocarcinoma homogenate abolished its activity. The K-PLC1 antigen could not be immunoblotted or immunoprecipitated and resolved on polyacrylamide gels; yet it showed the properties of a phospholipid, namely resistance to proteases, extractability with organic solvents and sensitivity to phospholipase C.
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PMID:Human hepatocellular carcinoma: cross-reactive and idiotypic antigens associated with malignant transformation of epithelial cells. 243 1

In the present investigation we compared the glycoprotein DPP IV from rat liver and Morris hepatoma 7777 by means of biochemical and immunological methods. For that purpose nine monoclonal anti-DPP IV-antibodies recognizing four different epitopes and a monospecific anti-DPP IV-antiserum were applied. In the homogenates of both tissues a plasma membrane-bound and a soluble form were detected. The immunological cross-reactivity of both forms was demonstrated with the antiserum and the monoclonal antibodies against the epitopes A, B and C while epitope D was restricted to liver plasma membrane. Differences of the distinct DPP IV forms were exhibited in the molecular weights, isoelectric points and peptide maps. In the hepatoma homogenate only 10% of DPP IV activity was found compared to normal liver but the ratio of soluble to membrane-bound form is higher in the hepatoma than in the liver. The fractionation of the homogenates into different cell components revealed for the liver a continuous increase of DPP IV activity from the endoplasmic reticulum fractions to the Golgi apparatus and finally to the plasma membranes. By contrast, in hepatoma the flow from the Golgi apparatus to plasma membrane was greatly reduced. The loss of DPP IV from the surface of cultured hepatoma cells was concomitant with a decrease of cell-substratum adhesion. DPP IV was found to be inserted into the liver plasma membrane by two different mechanisms, a phospholipase C-sensitive and a papain-sensitive one. In the hepatoma the phospholipase C-sensitive anchorage was not expressed. Besides liver and hepatoma the distribution of DPP IV was characterized in various rat organs by enzyme activity, histochemistry and immunohistochemistry with the anti-DPP IV-antibodies.
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PMID:Biochemical properties of dipeptidyl peptidase IV in liver and hepatoma plasma membranes. 248 27

The author reviews the problem of the pattern of lipid peroxidation in cancer cells with special reference to a comparison between normal liver cells and hepatomas both transplanted and induced by diethylnitrosamine. It is stated that the loss of lipid peroxidation is proportional to the degree of de-differentiation of hepatoma cells. During carcinogenesis, however, the loss is already evident at the stage of preneoplastic nodules. A common feature of all tumors, independently of the extent of the loss of peroxidation in basal conditions, is the lack of further stimulation by ADP/iron or by ascorbate/iron. As regards the reasons for the decline in lipid peroxidation, they are certainly not unique. An important cause is the low activity of the enzymes of the monooxygenase microsomal chain. Another very important one is the change in lipid composition of membranes, with a marked decrease in polyunsaturated fatty acids, which are the main substrate for lipid peroxidation. It has been shown that enrichment of membranes of hepatomas with arachidonic acid results in restoration of stimulation of peroxidation by ascorbate/iron, but not with ADP/iron. The last type of stimulation mostly reflects the behaviour of the monooxygenase chain, whereas ascorbate/iron-induced stimulation does not require the presence of an efficient cytochrome P450-chain. Another cause for decreased lipid peroxidation in tumors is the increased rigidity of membranes, due to the large increase in cholesterol content: this prevents to some extent the influx of oxygen inside the membranes. Yet another cause is the presence of increased amounts of antioxidants in both cytosol and membranes. The main toxic product of lipid peroxidation, 4-hydroxynonenal, has been found to elicit several actions at extremely low concentrations. In fact, 4-hydroxynonenal stimulates chemotaxis of polymorphonuclear leukocytes, stimulates plasma membrane adenylate cyclase, stimulates plasma membrane guanylate cyclase, and stimulates phospholipase C. The last three enzymes involve the action of G-proteins. The effect of the aldehyde is present at less than micromolar concentrations, which may occur inside the cells in certain conditions. Moreover, at concentrations from 10(-6) to 10(-7) M, the aldehyde is able to block oncogene c-myc expression in the human erythroleukemic K562 cell line, which at the same time becomes able to express the gamma-globin gene. These facts are discussed with reference to a possible biological meaning of the loss of lipid peroxidation in tumors.
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PMID:Lipid peroxidation and cancer: a critical reconsideration. 251 Mar 83

Transcription of the low-density lipoprotein receptor (LDL-R) gene in the human monocytic leukemic cell line THP-1 and in the human hepatocarcinoma cell line Hep-G2 is regulated by second messengers of the diacylglycerol-protein kinase C (DAG-PKC), inositol 1,4,5-triphosphate-Ca2+, and cyclic AMP pathways. Exogenous phospholipase C (which releases DAG and inositol 1,4,5-triphosphate), PKC activators (phorbol esters and DAG), Ca2+ ionophores, and a cyclic AMP analog all transiently induced accumulation of LDL-R mRNA. The effects of these three signal-transducing pathways were to a large extent additive. Furthermore, PKC stimulation effected an increase in LDL binding, which suggested that the increase in LDL-R mRNA resulted in an increase in functional cell surface receptor activity. These results suggest that uptake of cholesterol by these cells is under control of both intracellular cholesterol levels and external signals.
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PMID:Involvement of second messengers in regulation of the low-density lipoprotein receptor gene. 254 77

Conditioned medium from Reuber H-35 or Fao hepatoma cells contains autocrine factors that both stimulate DNA synthesis and activate acetyl-coenzyme A (CoA) carboxylase in serum-deprived Fao cells. The factor(s), which appears within 4 h of serum-free culture, also increases the cell number and the mitotic index. The effects of the conditioned medium are insulinomimetic, both with respect to stimulation of DNA synthesis and acetyl-CoA carboxylase activity. However, no induction of tyrosine aminotransferase activity or stimulation of aminoisobutyric acid uptake is seen in response to the conditioned medium. Insulin over a 4-h period does not increase the concentration of DNA synthesis stimulating activity that is observed in the medium. This activity is dialyzable and is resistant to acid treatment or to heating to 60-100 degrees C and to trypsin digestion; it is not extracted with chloroform/methanol nor adsorbed by charcoal or by a C18 reverse-phase column. Fractionation of the conditioned medium derived from Reuber H-35 hepatoma cells by gel filtration chromatography reveals two low molecular weight (less than 1000) compounds that both stimulate DNA synthesis in Fao hepatoma cells. The larger compound (peak I) also stimulates the activity of acetyl-CoA carboxylase. The stimulatory effects of the peak I compound are destroyed by nitrous acid deamination, periodate oxidation, and methanolysis. Biosynthetic labeling studies indicate the probable presence of glucosamine, galactose, and perhaps phosphate in the peak I-activating material. No significant incorporation of either myoinositol or mannose into the active material has been observed. These data, taken together, are consistent with a glycan structure for this autocrine factor, which bears strong resemblance to similar insulinomimetic factors generated in BC3H1 myocytes and H-35 hepatoma cells in response to insulin and on digestion of membranes with a phosphatidylinositol-specific phospholipase C. Further characterization of this factor may provide insight into different pathways of insulin action and could provide a strategy to check autocrine-stimulated tumor growth.
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PMID:An autocrine factor from Reuber hepatoma cells that stimulates DNA synthesis and acetyl-CoA carboxylase. Characterization of biologic activity and evidence for a glycan structure. 289 65


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