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 role of phosphatidylinositol-specific phospholipase C (PIase C) in a) the enigmatic phosphatidylinositol (PI) turnover and b) in our understanding of membrane enzyme-PI interactions is the subject matter of this article. PIase C is present in both procaryotes and eukaryotes. This enzyme is considered to be involved in the cells PI breakdown which occurs in response to several external stimuli. Recent information on the physical properties, Ca2+ requirement, cellular localization and modulation of the activity of PIase C of mammalian systems can help to evaluate the PI turnover from a new angle. Existing evidence suggests that Ca2+-dependent PI breakdown is probably mediated through the cytosolic and particulate PIase C while a Ca2+ independent pathway is catalyzed by a lysosomal enzyme. Apparently PI turnover may be operating through more than one mechanism. The association of this phenomenon with a membrane receptor event linked with "Ca2+ gating" may have to be reconsidered. Modulation of the PIase C activity by unsaturated amphiphiles or the presence of this enzyme in different physico-chemical forms could be a potential regulatory feature. Hydrolysis of membrane PI of a number of cells and tissues by the bacterial PIase C has been shown to cause substantial release of acetylcholinesterase, alkaline phosphatase and 5'-nucleotidase in free, soluble form. Other membrane enzymes, e.g., alkaline phosphodiesterase I, L-leucyl-beta naphthyl amidase and Ca2+ or Mg2+ ATPase are not affected. These results indicate a specific interaction between PI and certain enzymes in membranes. The chemical nature of this linkage, whether it is covalent or non-covalent, has also been explored and has provided intriguing insight into this phenomenon. New findings also indicate that hydrolysis of PI by PIase C also can cause modifications in membrane-enzyme activities, e.g., adenylate cyclase.
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PMID:Minireview. Phosphatidylinositol specific phospholipases C. 708 67

Prostasomes are human prostate derived organelles that were isolated from both prostatic fluid and seminal plasma for the present study. Specific activities were determined for prostasome membrane-associated enzymes, alkaline phosphatase (ALP), 5'-nucleotidase (5'NT), and alkaline phosphodiesterase I (APD). The mode of their membranous anchoring was studied by treatment of prostasomes with phosphoinositol-specific phospholipase C (PIPLC) and different detergents. A substantial amount of ALP (50%) and 5'NT (31%) was released by incubation of prostasomes with 2 U/ml of PIPLC contrary to the small amount of APD (12%) released by the same treatment. After PIPLC treatment, the enzymes were recovered in the aqueous phase after phase repartition in Triton X-114 indicating that PIPLC removed the hydrophobic domain converting the enzymes from membrane-linked to aqueous soluble forms. Octyl glycoside was the most efficient one among different detergents to solubilize the enzymes from the prostasome membrane. Both ALP and 5'NT were resistant to the treatment with Triton X-100 and Triton X-114. These results suggest that ALP, 5'NT, and APD are more or less extensively linked to the prostasome membrane via a glycophosphoinositide anchor.
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PMID:Association of some hydrolytic enzymes with the prostasome membrane and their differential responses to detergent and PIPLC treatment. 763 87

Seminal plasma separated from freshly ejaculated bull semen contains vesicles with a 5'-nucleotidase activity incorporated as an ectoenzyme anchored by glycosyl phosphatidylinositol (GPI). After its extraction from bull seminal plasma vesicles, the protein was purified and reconstituted into hen egg yolk lecithin liposomes obtained through prolonged dialysis of buffered n-octylglucoside detergent solutions of lipid, protein and various effectors against detergent-free solutions. Gel filtration experiments showed that the enzyme incorporated into liposomes in a dimeric form with its two subunits linked by disulfide bridges. In the presence of reduced glutathione, the protein dissociated into monomers and failed to incorporate into liposomes. Electron spin resonance (ESR) experiments, performed with liposomes containing electron spin labels localized at the hydrophilic lipid headgroups (5-doxyl stearic acid) or in the hydrophobic lipid hydrocarbon chains (16-doxyl stearic acid), demonstrated that the incorporation of 5'-nucleotidase resulted in the immobilization of the spin probes. Furthermore, the spectral parameters obtained before and after treatment of 5'-nucleotidase-containing liposomes with phosphatidylinositol-specific phospholipase C (PI-PLC) indicated that the liposome membrane bilayer did not contain protein segments. This supports the well-known ecto-localization of 5'-nucleotidase and rules out a previously reported possibility of a proteic transmembrane anchoring of the enzyme.
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PMID:Reconstitution of 5'-nucleotidase of bull seminal plasma in spin-labeled liposomes. 770 50

1. Alkaline phosphodiesterase I release from two tumor cell lines, KB III or AH-130 cells, by the action of phosphatidylinositol-specific phospholipase C (PIPLC) of Bacillus thuringiensis was studied. 2. A significant amount of alkaline phosphodiesterase I was released from both the cell suspension and homogenate of KB III cells, but not from AH-130 cells. 3. The release of the enzyme from KB III cells was dependent on, or proportional to, the reaction time and the PIPLC or cell concentrations. 4. Alkaline phosphatase and 5'-nucleotidase were also released from KB III cells, while gamma-glutamyl transpeptidase and dipeptidyl peptidase IV were not solubilized. The enzyme release by the action of PIPLC was suppressed when purified anti-PIPLC antibody was added to the reaction mixture. This suggests that the enzyme release must be due to the direct action of PIPLC on KB III cells. 5. The alkaline phosphodiesterase I released from KB III cells had a mol. wt of 240,000 and was activated by Mg2+, but strongly inhibited by EDTA and thiol reagents and by 5'-nucleotide-containing compounds. Although KB III cells were derived from Homo sapiens tumor, the released alkaline phosphodiesterase I appeared to be very similar to enzymes obtained from normal tissues of Rattus norvegicus.
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PMID:Alkaline phosphodiesterase I release from eucaryotic plasma membranes by phosphatidylinositol-specific phospholipase C. III. The release from tumor cells. 790 75

A polyclonal rabbit antibody against 5'-nucleotidase purified from bull seminal plasma was used to localize the antigen on bovine spermatozoa. Spermatozoa taken from the ampulla of the vas deferens showed strong immunofluorescence at the anterior rim of the head portion. Evaluation of spermatozoa prepared from different segments of the seminal pathway indicated the presence of the antigen already in rete testis and epididymal spermatozoa. On cryostat sections of testis tissue a positive immunoreaction was found in the anterior head portion of elongated spermatids, but not in earlier forms of sperm development. This distribution corresponded with the enzyme activity and results of Western blotting in extracts of testicular and epididymal spermatozoa. Immunoelectron microscopy of ampullary spermatozoa using antibody detection with gold-labelled anti-rabbit IgG showed a clear-cut labelling of the plasma membrane in the acrosome region. Treatment of ampullary spermatozoa with 0.1% Triton X-100 did not completely remove the immunoreactive material from the acrosome, showing a very stable linkage of the protein to the plasma membrane. Treatment with phospholipase C from Bacillus thuringiensis, however, removed immunoreactive material from the plasma membrane, indicating its binding by a phosphoinositol anchor. Our findings show that endogenous 5'-nucleotidase is present on the plasma membrane covering the anterior head portion of bovine spermatozoa and indicate specialized functions during the acrosomal reaction. Soluble enzyme derived from seminal vesicle secretion covers the whole sperm surface during emission, but is not covalently bound. It provides generalized enzyme activity to the sperm surface in addition to the specialized area of the sperm head.
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PMID:Distribution of endogenous and exogenous 5'-nucleotidase on bovine spermatozoa. 792 8

Incubation of pig kidney microvillar membranes with Bacillus thuringiensis or Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) resulted in the release of a number of glycosyl-phosphatidylinositol (GPI)-anchored hydrolases, including alkaline phosphatase (EC 3.1.3.1), amino-peptidase P (EC 3.4.11.9), membrane dipeptidase (EC 3.4.13.19), 5'-nucleotidase (EC 3.1.3.5) and trehalase (EC 3.2.1.28). Of these five ectoenzymes only for membrane dipeptidase was there a significant (approx. 100%) increase in enzymic activity upon release from the membrane. Maximal activation occurred at a PI-PLC concentration 10-fold less than that required for maximal release. In contrast solubilization of the membranes with n-octyl beta-D-glucopyranoside had no effect on the enzymic activity of membrane dipeptidase. A competitive e.l.i.s.a. with a polyclonal antiserum to membrane dipeptidase indicated that the increase in enzymic activity was not due to an increase in the amount of membrane dipeptidase protein. Although PI-PLC cleaved the GPI anchor of the affinity-purified amphipathic form of pig membrane dipeptidase there was no concurrent increase in enzymic activity. In the absence of PI-PLC, membrane dipeptidase in the microvillar membranes hydrolysed Gly-D-Phe with a Km of 0.77 mM and a Vmax. of 602 nmol/min per mg of protein. However, in the presence of a concentration of PI-PLC which caused maximal release from the membrane and maximal activation of membrane dipeptidase the Km was decreased to 0.07 mM while the Vmax. remained essentially unchanged at 624 nmol/min per mg of protein. Overall these results suggest that cleavage by PI-PLC of the GPI anchor on membrane dipeptidase may relax conformational constraints on the active site of the enzyme which exist when it is anchored in the lipid bilayer, thus resulting in an increase in the affinity of the active site for substrate.
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PMID:Activation of the glycosyl-phosphatidylinositol-anchored membrane dipeptidase upon release from pig kidney membranes by phospholipase C. 798 Apr 26

The specific activity of phosphatidylinositol 4,5-bisphosphate phospholipase C (PIP2-PLC) and inositol 1,4,5-trisphosphate monophosphatase (IP3-MP) involved in phosphoinositide catabolism was found to be significantly lower in the total homogenate of four human lymphoblastoid cell lines, HSB-2, MOLT-4, CEM and JURKAT, than in resting and activated peripheral blood mononuclear cells, ranging from 0.8 to 3.2 and from 1.3 to 3.7 nmol/min/mg for PIP2-PLC and IP3-MP, respectively. In PHA-stimulated cells, the specific activities were enhanced 25 and 35% respectively over the values (8.02 and 7.83 nmol/min/mg, respectively) measured in resting peripheral blood mononuclear cells. After centrifugation on discontinuous sucrose gradient of cell homogenate, PIP2-PLC and IP3-MP activities were found to be predominantly associated with the cytosol fraction (> 69%) in HSB-2 and MOLT-4 cells, with a distribution similar to that found in PHA-stimulated and in resting lymphocytes. In CEM cells, about half of the total activity remained in this fraction, while in JURKAT lymphoblastic cells more than 45% of the total activity was recovered in the high-density membrane fraction (d = 1.20-1.25), the soluble PIP2-PLC and IP3-MP activity accounting for only 13 and 25%, respectively. Conversely, in less differentiated leukemic cells HSB-2 and MOLT-4, conspicuous activity of the ectoenzymes 5'-nucleotidase (5'-NT) and gamma-glutamyltransferase (gamma-GT) was recovered in the soluble fraction. Growing leukemic cells at a distinct level of differentiation have a general reduction in activity but a characteristic distribution of enzymes involved in the transmission of signals usually targeting the cell surface.
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PMID:Subcellular localization of inositide enzymes in established T-cell lines and activated lymphocytes. 809 39

The ability of epidermal growth factor, insulin or guanosine thiotriphosphate to induce the release of two glycosyl-phosphatidylinositol-linked proteins from isolated human placental syncytiotrophoblast plasma membrane vesicles was investigated. Epidermal growth factor induced the ATP-dependent release of a fraction of syncytiotrophoblast plasma membrane placental alkaline phosphatase, whereas no release was detected following insulin treatment. This effect of epidermal growth factor was apparent at 30 min but not at 5 min. Guanosine thiotriphosphate stimulated the release of a small amount of syncytiotrophoblast plasma membrane placental alkaline phosphatase and appeared to have an additive effect when applied together with epidermal growth factor. Guanosine thiodiphosphate did not induce phosphatase release, but partially inhibited the epidermal growth factor response. 28.7% of syncytiotrophoblast plasma membrane 5'-nucleotidase was solubilized using glycosyl-phosphatidylinositol-specific phospholipase C. However, unlike placental alkaline phosphatase, no detectable release of 5'-nucleotidase was observed following treatment of syncytiotrophoblast plasma membrane vesicles with epidermal growth factor or guanosine thiotriphosphate. These results indicate (i) the presence of at least two placental alkaline phosphatase-releasing pathways in syncytiotrophoblast plasma membrane vesicles, and (ii) the presence of subpopulations of glycosyl-phosphatidylinositol-linked proteins sensitive to growth factor-induced release.
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PMID:Growth factor-induced release of placental alkaline phosphatase from human syncytiotrophoblast membranes. 818 14

In our study, 5'-nucleotidase was released from bovine liver by the treatment with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and purified to a homogeneous state by concanavalin A-Sepharose and (diethylaminoethyl)-Toyopearl column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified 5'-nucleotidase were then cleaved by cyanogen bromide (CNBr), and then inositol phosphoglycan-containing C-terminal peptides (IPG peptides) were separated by C18 reverse-phase liquid chromatography and analyzed by peptide sequencer, amino acid analyzer, gas chromatography (GC), and GC-mass spectrometry (MS). Ser523 of the amino acid sequence deduced from 5'-nucleotidase cDNA [Suzuki et al. (1993) J. Biochem. (Tokyo) 113, 607-613] is revealed to be the C-terminal amino acid to which a glycosylphosphatidylinositol is anchored. Separated peaks of CNBr-cleaved IPG peptides were then analyzed by electron spray ionization (ESI)-MS. Eight different molecular weight (MW) species of CNBr-cleaved IPG peptides were detected. Three fractions of CNBr-cleaved IPG peptides were separately treated by trypsin, and trypsinized IPG peptides were purified by C18 reverse-phase liquid chromatography. Finally, five different MW species of trypsinized IPG peptides (1629.4, 1752.7, 1791.8, 1832.8, and 1994.5) were detected by ESI-MS. Together with sequential exoglycosidase treatment and quantitative analysis of sugar moieties by GC and GC-MS, microheterogeneity in the structures of these five glycosylphosphatidylinositol (GPI) anchor species was determined. The common core structure was ethanolamine phosphate-mannose-mannose-mannose(-ethanolamine phosphate)-glucosamine-myoinositol phosphate. Variations observed in additional mannose, N-acetylhexosamine, and ethanolamine phosphate moieties form this heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Microheterogeneity in glycosylphosphatidylinositol anchor structures of bovine liver 5'-nucleotidase. 830 28

A glycosylphosphatidylinositol (GPI)-anchored protein, 5'-nucleotidase [EC 3.1.3.5], was released from the membrane of bovine liver by use of phosphatidylinositol-specific phospholipase C (PI-PLC) of Bacillus thuringiensis and purified by several column chromatographies to a homogeneous state. The purified protein has an apparent molecular mass of 61 kDa, as estimated by SDS-polyacrylamide gel electrophoresis. From the partial amino acid sequence of a tryptic peptide, mixed oligonucleotides were synthesized and used to screen a lambda gt11 liver cDNA library, and one positive clone, pE1, was isolated. Since the insert of the clone lacked the NH2-terminal coding region, another lambda gt11 liver cDNA library was screened by using a synthetic probe corresponding to the 5' region of the insert of pE1. Three additional cDNA clones were obtained. Sequencing of these cDNAs revealed an open reading frame that encodes a 574-residue polypeptide with a calculated mass of 63,084 Da. The predicted structure showed two highly hydrophobic stretches at both ends of the protein, like those of rat and human 5'-nucleotidases. The NH2-terminal 26 residues comprise a signal peptide and the COOH-terminal hydrophobic stretch may serve as a signal for the posttranslational GPI modification. An expression vector of the cDNA, pSVNT, was constructed in a mammalian expression vector pSVL and the 5'-nucleotidase activity was transiently expressed in COS-1 cells. The expressed activity was about 8 times higher than the pSVL-transfected control activity. PI-PLC released 45% of the transiently expressed 5'-nucleotidase activity, indicating that the cDNA isolated here encodes this enzyme expressed as a GPI-anchored protein.
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PMID:Purification and cDNA cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells. 834 Mar 54


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