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)

A polyclonal antiserum raised to the phospholipase C-solubilized form of membrane dipeptidase (EC 3.4.13.11) purified from human kidney was found to cross-react with unrelated trypanosomal and porcine glycosyl-phosphatidylinositol anchored proteins. Those antibodies recognising the cross-reacting determinant (CRD) were isolated by chromatography on a column of immobilized phospholipase C-solubilized porcine aminopeptidase P (EC 3.4.11.9), and the epitopes involved in the recognition were then characterized by immunoelectrophoretic blot analysis and by a competitive ELISA. The phospholipase C-solubilized forms of human and porcine membrane dipeptidase, porcine aminopeptidase P and trypanosome variant surface glycoprotein were recognised by the anti-CRD antiserum, and this recognition was abolished by prior treatment of the proteins with either mild acid or nitrous acid. In contrast, the detergent-solubilized, membrane-forms of human and porcine membrane dipeptidase were not recognised. Of a range of components of the glycosyl-phosphatidylinositol anchor, only inositol 1,2-cyclic monophosphate and the insulin-mimetic disaccharide, glucosaminyl-1,6-inositol 1,2-cyclic monophosphate, inhibited in the micromolar range the binding of the anti-CRD antiserum to immobilized porcine aminopeptidase P. These results indicate that the major epitope recognised by this anti-CRD antiserum is the inositol 1,2-cyclic monophosphate formed on phospholipase C cleavage of the glycosyl-phosphatidylinositol anchor.
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PMID:Characterization of an antibody to the cross-reacting determinant of the glycosyl-phosphatidylinositol anchor of human membrane dipeptidase. 767 86

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

Sheep lung dipeptidase was released from a plasma membrane preparation by digestion with a phosphatidylinositol-specific phospholipase C. The dipeptidase was purified to homogeneity using affinity chromatography followed by high performance liquid chromatography. The NH2-terminal sequence was determined and employed to prepare a probe to clone the c-DNA of the enzyme. The primary structure of sheep lung dipeptidase, deduced from the c-DNA exhibited a high homology to kidney dipeptidases cloned from other animal species. Northern analysis detected the m-RNA of the dipeptidase in lung, kidney, and intestinal tissues of the sheep.
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PMID:Molecular cloning of sheep lung dipeptidase: a glycosyl phosphatidylinositol-anchored ectoenzyme that converts leukotriene D4 to leukotriene E4. 805 66

The complement of brush border hydrolases provides an excellent model system for study of the structure, topology and assembly of plasma membrane proteins. Among the peptidases of the renal brush border are a number of glycosylphosphatidylinositol (GPI)-anchored proteins, especially membrane dipeptidase and aminopeptidase P. Affinity purification protocols have led to the isolation of homogeneous forms of these proteins and membrane dipeptidase has been cloned and expressed in Xenopus oocytes and Cos-1 cells. The core glycan structures of both human and porcine dipeptidase have been determined, allowing direct comparisons of the GPI anchors on the same protein in different species. Aminopeptidase P has been compared in the brush borders of pig kidney and intestine and may well be anchored in distinct ways in the two tissues. Immunological cross-reactivity of polyclonal antibodies to these two proteins has revealed the phospholipase C-cleaved GPI anchor as the common epitope and defined those components of the anchor important for recognition. These antibodies are also proving useful in characterizing GPI-derived mediators that may be involved in cell signalling processes. These abundant ectopeptidases offer a number of advantages for studies of the biochemistry of mammalian GPI anchors.
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PMID:Structural and immunological studies of GPI-anchored brush border hydrolases. 808 Dec 54

Membrane dipeptidase (EC 3.4.13.19) enzyme activity that is inhibited by cilastatin has been detected in pancreatic zymogen granule membranes of human, porcine and rat origin. Immunoelectrophoretic blot analysis of human and porcine pancreatic zymogen granule membranes with polyclonal antisera raised against the corresponding kidney membrane dipeptidase revealed that the enzyme is a disulphide-linked homodimer of subunit mass 61 kDa in the human and 45 kDa in the pig. Although membrane dipeptidase was, along with glycoprotein-2, one of the only two major components of carbonate high pH-washed membranes, no enzyme activity or immunoreactivity was detected in the zymogen granule contents. Digestion with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and subsequent recognition by antibodies specific for the cross-reacting determinant, revealed that membrane dipeptidase in human and porcine pancreatic zymogen granule membranes is glycosyl-phosphatidylinositol-anchored. Membrane dipeptidase was released from the pancreatic zymogen granule membranes by an endogenous hydrolase, and the released form migrated as a disulphide-linked dimer on SDS/PAGE under non-reducing conditions. Under reducing conditions it migrated with the same apparent molecular mass as the membrane-bound form, and was still a substrate for bacterial PI-PLC. Treatment of kidney microvillar membranes with phospholipase A2 resulted in the release of membrane dipeptidase in a form that demonstrated electrophoretic and cilastatin-Sepharose binding properties identical to those of the endogenously released form of the enzyme from zymogen granule membranes. These results indicate that the glycosyl-phosphatidylinositol anchor on the pancreatic membrane dipeptidase is cleaved by an endogenous hydrolase, probably a phospholipase A, and that this cleavage may promote the release of the protein from the membrane.
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PMID:Identification of membrane dipeptidase as a major glycosyl-phosphatidylinositol-anchored protein of the pancreatic zymogen granule membrane, and evidence for its release by phospholipase A. 916 51

Membrane dipeptidase (MDP; EC 3.4.13.19) enzymic activity that was inhibited by cilastatin has been detected on the surface of 3T3-L1 cells. On differentiation of the cells from fibroblasts to adipocytes the activity of MDP increased 12-fold. Immunoelectrophoretic blot analysis indicated that on adipogenesis the increase in the amount of MDP preceded the appearance of GLUT-4. MDP on 3T3-L1 adipocytes was anchored in the bilayer by a glycosyl phosphatidylinositol (GPI) moiety as evidenced by its release into the medium in a hydrophilic form on treatment of the cells with bacterial phosphatidylinositol-specific phospholipase C and the appearance of the inositol 1,2-cyclic monophosphate cross-reacting determinant. Incubation of 3T3-L1 adipocytes with either insulin or the sulphonylurea glimepiride led to a rapid concentration- and time-dependent release of MDP from the cell surface. The hydrophilic form of MDP released from the cells on stimulation with insulin was recognized by antibodies against the inositol 1,2-cyclic monophosphate cross-reacting determinant, indicating that it had been generated by cleavage of its GPI anchor through the action of a phospholipase C.
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PMID:Insulin stimulates the release of the glycosyl phosphatidylinositol-anchored membrane dipeptidase from 3T3-L1 adipocytes through the action of a phospholipase C. 929 Nov 28

The GPI-anchored membrane dipeptidase is the major peptidase activity of the secretory granule membrane in the exocrine pancreas. The enzyme is also found in the granule content and in pancreatic secretions. Immunocytochemical localization confirmed its location in the granule membrane and in the acinar cell apical plasma membrane. In the endoplasmic reticulum and Golgi, membrane dipeptidase was strictly membrane-bound. There was no membrane dipeptidase in duct cells. The release of membrane dipeptidase from the membrane starts in the immature granule. To identify the mechanism responsible for its release, secretions were collected from cannulated conscious pig under basal conditions and atropine perfusion. The latter treatment caused complete inhibition of protein secretion but had a negligible effect on membrane dipeptidase activity in the secretions. In secretions, membrane dipeptidase partitioned into the detergent-rich phase on phase separation in Triton X-114, whereas treatment with bacterial phosphatidylinositol-specific phospholipase C caused the peptidase to partition into the aqueous phase, indicating that the secreted enzyme could come from shedding of membrane fragments at the apical surface or via the action of a previously characterized phospholipase A activity.
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PMID:Membrane dipeptidase in the pig exocrine pancreas. Ultrastructural localization and secretion. 963 43

Leukotriene D4 (LTD4) is one of the slow-reacting substances of anaphylaxis and is reported to have a diverse response including the mediation of glomerular nephritis. However, little is known about the functions of LTD4 and its mechanisms of action in primary cultured rabbit renal proximal tubular cells (PTCs). The purpose of this study is to investigate the effect of LTD4 on Na+ uptake and its related signal transduction pathways in PTCs. LTD4 (>10(-9) M) significantly inhibited the Na+ uptake after 15 min (in nmol/mg protein: controls 431.7+/-11.4 vs. LTD4 (10(-9) M) 355.0+/-23.6; p<0. 05); and its effect was blocked by MK-571 (10(-6) M), a leukotriene receptor antagonist, in PTCs. Preincubation with cilastatin, a renal dipeptidase inhibitor, and polyclonal antibody against renal dipeptidase potentiated the inhibitory effect of LTD4 on Na+ uptake. SQ 22536 (10(-6) M), an adenylate cyclase inhibitor, and the myristoylated protein kinase A inhibitor amide 14-22 (PKI; 10(-5) M) blocked the effect of LTD4 on Na+ uptake (in nmol/mg protein: LTD4 349.9+/-18.5 vs. SQ 22536+LTD4 476.5+/-22.0 and PKI+LTD4 440.3+/-19. 3; p<0.05), and LTD4 induced an increase in cyclic adenosine monophosphate (cAMP), suggesting the involvement of cAMP in the inhibition of Na+ uptake. In addition, U 73122 (10(-6) M) and neomycin (10(-4) M), phospholipase C (PLC) inhibitors, W-7 (10(-4) M), a calmodulin antagonist, and bisindolylmaleimide I, a protein kinase C (PKC) inhibitor, blocked the LTD4-induced inhibition of Na+ uptake, strongly suggesting involvement of the PLC-PKC signal pathways in the effect of LTD4. LTD4 significantly increased [Ca2]i by 49+/-7% as compared with baseline. TMB-8 (10(-5) M) and BAPTA/AM (10(-5) M), intracellular calcium mobilization blockers, completely blocked the LTD4-induced inhibition of Na+ uptake (in nmol/mg protein: LTD4 347.6+/-19.0 vs. TMB-8+LTD4 436.4+/-22.3 and BAPTA/AM+LTD4 419.9+/-14.3; p<0.05); however, EGTA (1 mM), a calcium chelator, partially blocked the LTD4-induced inhibition of Na+ uptake. In conclusion, LTD4-induced inhibition of Na+ uptake may be involved in both cAMP and PLC-PKC signal pathways in PTCs. In addition, Ca2+, which comes from the intracellular Ca2+ mobilization, is primarily responsible for the LTD4-induced inhibition of Na+ uptake.
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PMID:Leukotriene D4 inhibits Na+ uptake through cAMP and PLC pathways in primary cultured renal proximal tubular cells. 1039 8

It was previously hypothesised that the requirements for glycosyl-phosphatidylinositol (GPI) anchoring in mammalian cells and parasitic protozoa are similar but not identical. We have investigated this by converting the GPI cleavage/attachment site in porcine membrane dipeptidase to that found in the trypanosomal variant surface glycoprotein 117 and expressing the resulting mutants in COS-1 cells. Changing the entire (omega), (omega)+1 and (omega)+2 triplet in membrane dipeptidase from Ser-Ala-Ala to Asp-Ser-Ser resulted in efficient GPI anchoring of the mutant proteins, as assessed by cell-surface activity assays and susceptibility to release by phosphatidylinositol-specific phospholipase C. Immunoelectrophoretic blot analysis with antibodies recognising epitopes either side of the native (omega) residue in porcine membrane dipeptidase, and expression of a mutant in which potential alternative cleavage/attachment sites were disrupted, indicated that alternative GPI cleavage/attachment sites had not been used. These results indicate that the requirements for GPI anchoring between mammalian and protozoal cells are not as different as previously suggested, and that rules for predicting the probability of a sequence acting as a GPI cleavage/attachment site need to be applied with caution.
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PMID:Comparison of the glycosyl-phosphatidylinositol cleavage/attachment site between mammalian cells and parasitic protozoa. 1065 64

There are many reports on acute renal failure (ARF) after ingestion of grass carp bile (CB; Ctenopharyngodon idellus). Renal dipeptidase (RDPase; EC 3.4.13.19) is a glycosylphosphatidylinositol-anchored ectoenzyme within the renal proximal tubules (PTs) and is proposed as a diagnostic enzyme of renal disease. We examined the release of RDPase following treatment with CB and various nitric oxide (NO) related compounds in porcine PTs. The RDPase release from PTs was inhibited by CB in a concentration-dependent manner and was also inhibited by sodium nitroprusside (direct NO donor) and L-arginine (NO synthase substrate) in the tested range (0-12 mM). CB-treated (0. 1 mg/ml) PTs showed a decreased RDPase activity in comparison with the control group. This inhibition was blocked by 2 mM L-NAME (NO synthase inhibitor) and U73122 (inhibitor of phosphatidylinositol-specific phospholipase C) in a concentration-dependent manner. Eel bile (0-0.1 mg/ml), used as the control, did not significantly affect the RDPase release from PTs. The NO concentration was observed as nitrite, the degradation product of the NO metabolism, increased in proportion to CB and L-arginine. The increase of nitrite to 151.5% by CB treatment (0.1 mg/ml) was blocked by 2 mM L-NAME (95.5%). When the phospholipase C pathway was blocked by 10 and 20 microM U73122, the nitrite generation decreased to 122.7 and 89.4%, respectively. These results strongly suggest that NO generation and the phospholipase C pathway affect the RDPase release from the PTs and that they may be involved in the development of ARF in vivo following CB ingestion. The release of RDPase from PTs could be a useful tool not only for this CB-caused ARF, but also for the elucidation of other biochemical mechanisms.
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PMID:Grass carp (Ctenopharyngodon idellus) bile may inhibit the release of renal dipeptidase from the proximal tubules by nitric oxide generation. 1076 13


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