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)

Two polyclonal antisera were raised in rabbits to the phospholipase C-solubilized forms of pig renal dipeptidase (EC 3.4.13.11) and pig aminopeptidase P (EC 3.4.11.9). These antisera were purified and shown to cross-react with other glycosyl-phosphatidylinositol (G-PI)-anchored proteins isolated from pig, human and trypanosomes. The epitopes involved in this cross-reactivity were characterized by Western-blot analysis after mild acid or nitrous acid treatment of the G-PI-anchored proteins and by a competitive e.l.i.s.a. with other G-PI-anchored proteins and individual components of the anchor structure. These studies revealed that the primary epitope for both antisera is the inositol 1.2-(cyclic)monophosphate that is formed on phospholipase C cleavage of the intact G-PI anchor. Other minor epitopes, such as phosphoethanolamine, probably involve side-chain modifications to the core anchor structure that may be species-specific.
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PMID:Characterization of antibodies to the glycosyl-phosphatidylinositol membrane anchors of mammalian proteins. 170 60

Subcellular fractionation of pig kidney cortex revealed that aminoacylase I (EC 3.5.1.14, N-acyl-L-amino-acid aminohydrolase) is predominantly a soluble enzyme with only 0.5% of the total activity being recovered in the membrane fraction. The aminoacylase I activity associated with the membrane preparations displayed neither rapid release following incubation with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis nor the distinctive differential pattern of detergent solubilization which was seen with glycosyl-phosphatidylinositol-anchored proteins (renal dipeptidase, alkaline phosphatase). When fractionated by phase separation in Triton X-114, integral membrane proteins of kidney microvillar membranes partitioned predominantly (greater than 90%) into the detergent-rich phase. In contrast, only 3.7% of aminoacylase I activity associated with microvillar membranes partitioned into the detergent-rich phase. Aminoacylase I activity of pig kidney would therefore appear to be a hydrophilic protein in nature and is not, as suggested previously, a G-PI-anchored integral membrane protein.
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PMID:Aminoacylase I is not a glycolipid-anchored ectoenzyme in pig kidney. 182 88

Renal dipeptidase (EC 3.4.13.11) has been purified from human kidney cortex by affinity chromatography on cilastatin-Sepharose following solubilization with either n-octyl-beta-D-glucopyranoside or bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). Phase separation in Triton X-114 revealed that the detergent-solubilized form was amphipathic and retained the glycosyl-phosphatidylinositol membrane anchor whereas the phospholipase solubilized form was hydrophilic. Both forms of the enzyme existed as a disulphide-linked dimer of two identical subunits of Mr 59,000 each. The glycosyl-phosphatidylinositol anchor of purified human renal dipeptidase was hydrolysed by a range of bacterial PI-PLCs and by a plasma phospholipase D. Mild acid treatment and nitrous acid deamination of the hydrophilic form revealed that the cross-reacting determinant, characteristic of the glycosyl-phosphatidylinositol anchor, was due exclusively to the inositol 1,2-cyclic phosphate ring epitope. The N-terminal amino acid sequences of the amphipathic and hydrophilic forms were identical, locating the membrane anchor at the C-terminus. The N-terminal sequence of human renal dipeptidase showed a high degree of similarity with that of the pig enzyme, and enzymic deglycosylation revealed that the difference in size of renal dipeptidase between these two species is due almost entirely to differences in the extent of N-linked glycosylation.
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PMID:Characterization of the glycosyl-phosphatidylinositol-anchored human renal dipeptidase reveals that it is more extensively glycosylated than the pig enzyme. 213 35

The chemical properties of human renal dipeptidase (hrDP) purified from the membrane fraction of kidney have been characterized. When treated with phosphatidylinositol-specific phospholipase C, hrDP was released from renal membrane fractions. After digestion with trypsin, carboxyl-terminal peptide was isolated employing anhydrotrypsin-agarose column chromatography and reversed-phase high performance liquid chromatography. The amino acid sequence of the peptide was identified at positions 363-369 in the primary structure deduced from the cDNA sequence (Adachi, H., Tawaragi, Y., Inuzuka, C., Kubota, I., Tsujimoto, M., Nishihara, T., And Nakazato, H. (1990) J. Biol. Chem. 265, 3992-3995). Further examination of the chemical composion of the peptide showed that it contained, respectively, 2, 1, 5, 1, and 1 mol of ethanolamine, glucosamine, mannose, inositol, and phosphate in addition to amino acids. These results suggest that the mature hrDP molecule lacks the carboxyl-terminal hydrophobic peptide extension predicted from the cDNA sequence and is anchored at Ser369 via glycosylphosphatidylinositol to the membrane. To characterize further the action of the enzyme, we have established expression systems for both secretory and membrane anchored forms of hrDP using COS-1 cells and found that both recombinant forms were as active as natural enzyme. Our expression system made it possible to prepare large amounts of soluble enzyme, and will contribute toward elucidation of the physiological roles of the enzyme.
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PMID:Identification of membrane anchoring site of human renal dipeptidase and construction and expression of a cDNA for its secretory form. 216 7

Clones expressing renal dipeptidase (EC 3.4.13.11) have been isolated from a pig kidney cortex cDNA library after employing the polymerase chain reaction technique to amplify a region of the dipeptidase cDNA. The complete primary sequence of the enzyme has been deduced from a full length cDNA clone. This predicts a protein of 409 amino acids, a cleavable N-terminal signal sequence of 16 residues and two N-linked glycosylation sites. At the C-terminus of the predicted sequence is a stretch of mainly hydrophobic amino acids which is presumed to direct the attachment of the glycosyl-phosphatidylinositol membrane anchor. Expression of the mRNA for pig renal dipeptidase in Xenopus laevis oocytes led to the production of a disulphide-linked dimeric protein of subunit Mr 48,600 which was recognized by a polyclonal antiserum raised to renal dipeptidase purified from pig kidney cortex. Bacterial phosphatidylinositol-specific phospholipase C released renal dipeptidase from the surface of the oocytes and converted the amphipathic detergent-solubilized form of the dipeptidase to a hydrophilic form, indicating that Xenopus laevis oocytes can process expressed proteins to their glycosyl-phosphatidylinositol anchored form.
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PMID:cDNA cloning and expression in Xenopus laevis oocytes of pig renal dipeptidase, a glycosyl-phosphatidylinositol-anchored ectoenzyme. 217 7

Renal dipeptidase (dehydropeptidase-I, EC 3.4.13.11) was released from pig kidney membrane preparations by treatment with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus and Bacillus thuringiensis and a phospholipase C preparation from Bacillus cereus to a similar extent as alkaline phosphatase. Endopeptidase-24.11 and aminopeptidase N were not released by this treatment. After treatment of the membrane fraction with the S. aureus phospholipase C the dipeptidase was converted from an amphipathic to a hydrophilic form, as deduced from phase-separation experiments in Triton X-114. It is concluded that renal dipeptidase is anchored to the microvillar membrane by covalently attached phosphatidylinositol.
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PMID:Renal dipeptidase is one of the membrane proteins released by phosphatidylinositol-specific phospholipase C. 282 7

Renal dipeptidase (EC 3.4.13.11) was solubilized from pig kidney microvillar membranes with bacterial phosphatidylinositol-specific phospholipase C and then purified by affinity chromatography on cilastatin-Sepharose. The enzyme was apparently homogeneous on SDS/polyacrylamide-gel electrophoresis with an Mr of 47,000. Immunohistochemical analysis of the distribution of the dipeptidase showed it to be concentrated in the brush-border region of the proximal tubules in close association with endopeptidase-24.11) (EC 3.4.24.11). The purified dipeptidase was shown to contain 1 mol of inositol/mol and to possess the cross-reacting determinant characteristic of the glycosyl-phosphatidylinositol membrane-anchoring domain. The glycoprotein nature of renal dipeptidase was confirmed by chemical and enzymic deglycosylation. These results establish renal dipeptidase as a glycosyl-phosphatidylinositol-anchored ectoenzyme of the microvillar membrane.
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PMID:Ectoenzymes of the kidney microvillar membrane. Affinity purification, characterization and localization of the phospholipase C-solubilized form of renal dipeptidase. 293 Apr 55

We present data indicating that aminoacylase I (EC 3.5.1.14) from porcine kidney and 'renal dipeptidase' (EC 3.4.13.11) are closely related. We show that, in situ, a considerable fraction of aminoacylase activity ist attached to membranes. Incubation of washed microsomal membranes with phospholipase C from B. cereus results in the rapid solubilization of aminoacylase I, suggesting that aminoacylase--as shown for renal dipeptidase before--bears a glycolipid 'membrane anchor'. In agreement with this assumption, purified aminoacylase was found to contain myo-inositol, a characteristic component of phosphatidylinositol-anchored membrane proteins. A reexamination of the molecular mass of purified aminoacylase yielded values (46,000 +/- 2,000 Da by SDS polyacrylamide electrophoresis, 98,000 +/- 5,000 Da by sedimentation equilibrium centrifugation) similar to those reported for renal dipeptidase. The enzymes coelute during most of the procedures applied in the purification of aminoacylase or renal dipeptidase, but can be separated by hydrophobic interaction chromatography. A survey of the literature revealed a series of additional features of aminoacylase I and renal dipeptidase (amino-acid composition, isoelectric points, metal dependence, and more) that are strikingly similar.
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PMID:Further characterization of porcine kidney aminoacylase I reveals close similarity to 'renal dipeptidase'. 322 87

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

Spontaneous enzymic release of renal dipeptidase (RDPase; EC 3.4.13.19), a glycosylphosphatidylinositol (GPI)-linked ectoenzyme, was observed in vitro during incubation of porcine proximal tubules at 37 degrees C. Triton X-114 phase separation of the released RDPase showed that the majority of the enzyme activity partitioned into the aqueous phase, indicating its hydrophilic nature. Immunoblot analyses using an antibody against the cross-reacting determinant (CRD) inositol 1,2-cyclic monophosphate, the epitope formed by phospholipase C (PLC) cleavage of the GPI anchor on a protein, detected the released RDPase. Reprobing the immunoblot with an anti-RDPase serum showed the RDPase band co-migrating with the CRD band. The release of RDPase from the proximal tubules was a Ca(2+)-dependent process and had a pH optimum of 9.0. These results indicate that RDPase is released from the proximal tubules by the action of a distinct endogenous GPI-specific PLC.
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PMID:Endogenous glycosylphosphatidylinositol-specific phospholipase C releases renal dipeptidase from kidney proximal tubules in vitro. 1113 99


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