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 Rho(D) antigen of red cell membranes was solubilized using ethylene-diamine tetraacetic acid (EDTA) and 2-mercaptoethanol. The solubilized antigen was partially separated from other solubilized membrane components using molecular filtration. The antigen was treated with various enzymes to learn some of the chemical characteristics. It was found that the activity of the antigen, as measured by hemagglutination inhibition, was not affected by bee venom phospholipase A, Clostridium welchii phospholipase C, calf-intestinal alkaline phosphatase, Vibrio cholerae neuraminidase, pig kidney leucine aminopeptidase, bovine pancreatic carboxypeptidase A, and pig pancreatic carboxypeptidase B. However, the proteolytic enzymes, pronase, trypsin, chymotrypsin and papain, did destroy Rho(D) activity as measured by hemagglutination inhibition. These results indicate that protein is an important part of the active determinant of the Rho(D) antigen. The experiments by other investigators have shown that lipid is important to maintain the Rho(D) activity in the intact membrane; lipid probably helps to maintain the structural conformation of the Rho(D) molecule in its natural environment. The solubilized Rho(D) molecules are apparently not dependent on lipid for their Rho(D) activity.
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PMID:Studies on the characterization of the Rho(D) antigen. 10 79

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.
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PMID:Membrane-bound aminopeptidase P from bovine lung. Its purification, properties, and degradation of bradykinin. 153 67

Pseudorabies virus hemagglutinin was readily adsorbed on mouse erythrocytes at 4, 22, or 37 degrees C, but not on cattle erythrocytes. The adsorbed hemagglutinin could not be eluted from the cells by resuspending in phosphate-buffered saline (PBS), by incubating at 37 or 50 degrees C, or by incubating in the presence of neuraminidase. The receptor on mouse erythrocytes for the hemagglutinin was inactivated by trypsin, but not by neuraminidase, sodium deoxycholate (DOC), potassium periodate (KIO4), dithiothreitol (DTT), 2-mercaptoethanol (2-ME) and formalin. The hemagglutinin was inactivated by trypsin, alpha-amylase, pepsin, DOC, KIO4, and ethylendiamine-tetraacetic acid (EDTA), but not by papain, beta-glucosidase, phospholipase C, neuraminidase, DTT, 2-ME, Tween-80, ethylether, chloroform, trichloro-trifluoroethane, beta-propiolactone and formalin, suggesting that the hemagglutinin active component involved glycoproteins. The hemagglutinin was stable at 37 degrees C for lower temperatures but not at 60 degrees C or higher. The hemagglutinin activity was resistant to ultraviolet irradiation, while the infectivity was very susceptible. The hemagglutinin and the infectivity were readily sedimented by ultracentrifugation at 48,000 x g for 3 hr. In rate zonal centrifugation of the preparation on a sucrose density gradient, the hemagglutination (HA) activity showed a sharp peak at 1.22 g/ml coinciding with the peak of infectivity. The HA activity in the peak fraction seemed to be structually associated with virus particles. After fractionation of the virus by Nonidet P-40, the HA activity was found only in the fraction of the envelope material, indicating that the hemagglutinin is situated in the viral envelop.
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PMID:Physicochemical properties of pseudorabies virus hemagglutinin. 166 85

The potential involvement of vicinal dithiols in the expression of platelet-activating factor (AGEPC)- and A23187-induced alterations in rabbit platelets was explored through the use of phenylarsine oxide (PhAsO) and certain analogous derivatives. PhAsO (As3+) but not phenylarsonic acid (As5+) inhibited markedly at 1 microM concentration the release of arachidonic acid initiated by AGEPC and the ionophore A23187. In contrast, AGEPC-induced phosphatidic acid formation, phosphorylation of 40- and 20-kDa proteins, and Ca2+ uptake from external medium were not inhibited substantially by 1 microM PhAsO. However, these latter metabolic responses to AGEPC were inhibited by PhAsO at higher doses (10 microM). AGEPC- and thrombin-induced platelet aggregation and serotonin secretion also were prevented by PhAsO. The IC50 value of PhAsO was 2.7 +/- 1.2 microM toward AGEPC (5 X 10(-10) M)-induced serotonin release. Further, ATP and cAMP levels in PhAsO-treated platelets were not changed from controls. Interestingly, addition of Ca2+ to platelet sonicates (prepared in EDTA) caused diacylglycerol production and free arachidonic acid formation, even in the presence of 133 microM PhAsO. This would suggest that in the intact platelets PhAsO acted indirectly on phospholipase A2 and/or phospholipase C activities. Finally, a dithiol compound, 2,3-dimercaptopropanol, reversed the inhibition of platelet aggregation and arachidonic acid release effected by PhAsO. On the other hand, a monothiol compound, 2-mercaptoethanol, was not effective in preventing or in reversing the action of PhAsO. These observations suggest that vicinal sulfhydryl residues may be involved in stimulus-induced platelet activation.
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PMID:Potential involvement of vicinal sulfhydryls in stimulus-induced rabbit platelet activation. 282 9

Transmissible gastroenteritis virus was readily adsorbed onto chicken erythrocytes at 4 degrees C. The hemagglutinin thus adsorbed could be eluted from the erythrocytes by incubating in phosphate buffered saline at 37 degrees C. The receptor on chicken erythrocytes for the hemagglutinin was inactivated by neuraminidase and potassium periodate, but not by trypsin, 2-mercaptoethanol and formalin. The hemagglutinin was inactivated by trypsin, papain, pepsin, alpha-amylase, phospholipase C, neuraminidase, formalin, 2-mercaptoethanol, potassium periodate, ethyl ether, chloroform, Tween-80 and beta-propiolactone, but not by sodium deoxycholate and trichlorotrifluoroethane, suggesting that the active component of the hemagglutinin involved glycoproteins. The hemagglutinin was stable at 37 degrees C or lower temperatures but not at 60 degrees C or higher temperatures. The hemagglutinin activity was resistant to ultraviolet irradiation, while the infectivity was very susceptible. The hemagglutinin and the infectivity were readily sedimented by ultracentrifugation at 45,000 x g for 60 minutes. In rate zonal centrifugation of the hemagglutinin preparation on a sucrose density gradient, the hemagglutinin activity showed a sharp peak at 1.19 g/ml coinciding with the peak of infectivity. The activity in the peak fraction seemed to be structurally associated with virus particles.
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PMID:Physicochemical properties of transmissible gastroenteritis virus hemagglutinin. 283 45

An aspartic proteinase associated with human erythrocyte membranes was shown to be responsible for autodegradation of the membrane proteins at pH values below 5.0. When the membrane was treated with phospholipase C (Bacillus cereus) or trypsin, and simply heated at 40 degrees C, the membrane-bound latent enzyme was activated, with this being accompanied by dissociation of the enzyme from the membrane. Divalent cations such as Ca2+ and Mg2+ had an inhibitory effect on the dissociation of the membrane-bound enzyme when preincubated with the membrane. The results indicate that the activation of the membrane-bound enzyme is due probably to perturbation of the normal membrane organization. When the purified enzyme was treated with 10mM 2-mercaptoethanol at 37 degrees C, the enzyme (79-82 kDa) was converted to a low molecular mass form with 42-47 kDa without any loss of activity. With the exception of treatments by thiol-reducing reagents, no conversion was observed by a variety of procedures such as exposure to 1 M NaCl and 0.1% sodium dodecyl sulfate, treatment with trypsin and incubation at pH 3.5 for up to 15 h, indicating that the enzyme consists of two polypeptide chains held together by disulfide bonds.
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PMID:An aspartic proteinase of erythrocyte membranes. Proposed mechanism for activation and further molecular properties. 306 Jan 44

Binding of acetylcholine in the concentration range 1 nM-1 muM was measured by equilibrium dialysis to a particulate preparation of Torpedo electroplax, without or with prior treatment of the tissue with one of three chemical modifying reagents. Significant reduction in binding of acetylcholine resulted after treatment with 1,4-dithiothreitol, p-chloromercuribenzoate, or p-(trimethylammonium)-benzenediazonium fluoroborate. Partial reversal of the reduction in binding occurred when dialysis was performed in the presence of 5,5'-dithiobis-(2-nitrobenzoic acid) or potassium ferricyanide (in the case of treatment with dithiothreitol), and 2-mercaptoethanol (in the case of treatment with p-chloromercuribenzoate). It is concluded that the functional acetylcholine-receptor macromolecule of Torpedo electroplax has disulfide bond(s), sulfhydryl group(s), and one or more of the amino acids vulnerable to diazotization by p-(trimethylammonium)-benzenediazonium fluoroborate. This, plus the effect of phospholipase C (EC 3.1.4.3) in elimination of detectable binding of acetylcholine after electrofocusing, is additional evidence that the functional acetylcholine receptor is a phospholipoprotein or a phospholipid-protein complex, which has a low isoelectric point of 4.5 +/- 0.2, yet is denatured by exposure to low pH for 24 hr. Due to this adverse effect, recovery of binding of acetylcholine after electrofocusing, as detected by equilibrium dialysis or ultrafiltration, is only 23% and, so far, only 6.3-fold purification of functional acetylcholine receptors by this technique is possible. Three or two forms of acetylcholinesterase (EC 3.1.1.7), whose peaks have isoelectric points ranging from 4.3 to 7.2, appear after electrofocusing of Torpedo extracted with 1% Triton X-100 or Lubrol, respectively. The major peak in either preparation has an isoelectric point of 5. Although the peaks of the functional acetylcholine receptors and of acetylcholinesterase of Torpedo electroplax are separable by electrofocusing, it has not been possible to isolate fractions that contain functional receptors but that are free of acetylcholinesterase. The opposite is possible.
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PMID:Characterization and partial purification of the acetylcholine receptor from Torpedo electroplax. 450 55

The Fc receptor activity in placental extracts prepared using EDTA and 2-mercaptoethanol was assayed using an indirect hemagglutination technique with sheep erythrocytes sensitized with rabbit IgG. The agglutinating activity of the extract was not affected by storage at -70 degrees C, by rapid freezing and thawing, by treatment with periodic acid, formaldehyde, neuraminidase, trypsin, pronase, or phospholipase C. Papain abolished the activity, indicating that the receptor is a protein. Reduction and alkylation had no effect on the agglutinating activity, indicating that -S-S-bonds are not important for binding. In the presence of 0.6 M NaCl the agglutinating activity was abolished, indicating that electrostatic interactions are of significance. The solubilized Fc receptor shows so many similarities to the previously studied in situ Fc receptor that they are probably identical.
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PMID:Properties of the solubilized placental receptor for IgG. 621 64

Properties of guinea pig peritoneal macrophage Fc receptor for IgG are described. It was found that the receptor was binding both monomeric and aggregated rabbit IgG. The values of apparent affinity constants were 2.6 +/- 0.6 x 10(8) M-1 and 3.84 +/- x 10(8) m-1 for IgG monomers and aggregates, respectively. The number of monomeric IgG molecules bound was calculated to be 3.3 +/- 0.2 x 10(5) per cell and of aggregated IgG 3.95 +/- 0.12 x 10(5) per cell. When the homologous system: guinea pig IgG2 and guinea pig macrophages was investigated, the affinity constant found was 1.66 +/- 0.45 x 10(8) M-1 and 2.6 +/- 0.24 x 10(5) molecules of IgG were bound per cell. Both, rabbit IgG and, guinea pig IgG2, interacted wih the same receptor binding sites on macrophages. Treatment of macrophages with 2-mercaptoethanol, formaldehyde, and iodoacetamide was without any effect on the IgG binding properties of cells. Periodate, trypsin, pronase, phospholipase C considerably diminished the number of IgG molecules bound to macrophages. Treatment of macrophages with neuraminidase increased the number of IgG molecules bound per cell. The results obtained suggests that both sugar and protein components are important for the IgG binding activity of guinea pig peritoneal macrophages. Studies on the effect of pH, ionic strength, and temperature on the interaction of macrophages with IgG showed that electrostatic interactions are important for binding of IgG to the macrophage Fc receptor.
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PMID:Studies on properties of guinea pig peritoneal macrophage Fc receptor. 744 40

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified 670-fold to apparent homogeneity from rat lung microsomes. The enzyme was solubilized from the membranes using a phosphatidylinositol-specific phospholipase C. The purification scheme also resulted in homogeneous preparations of dipeptidylpeptidase IV (EC 3.4.14.5) and membrane dipeptidase (EC 3.4.13.19). Aminopeptidase P had a subunit molecular weight of 90,000, which included at least 17% N-linked carbohydrate. The molecular weight by gel permeation chromatography varied from 220,000 to 340,000, depending on the conditions used. The amino acid composition was determined and the N-terminal sequence was found to be X1-Gly2-Pro3-Glu4-Ser5-Leu6-Gly7-Arg8-Glu9-As p10-Val11-Arg12-Asp13-X14-Ser15- Thr16-Asn17-Pro18-Pro19-Arg20-Leu21- X22-Val23-Thr24-Ala25-. Aminopeptidase P cleaved the Arg1-Pro2 bond of bradykinin with a kcat/Km of 5.7 x 10(5) s-1 M-1. N-Terminal fragments of bradykinin including Arg-Pro-Pro, but not Arg-Pro, were also cleaved. The enzyme was shown to have four binding subsites (S1, S1', S2'. S3'), the first three of which must be occupied for hydrolysis to occur. Neuropeptide Y and allatostatin I were hydrolyzed at the Tyr1-Pro2 bond and Ala1-Pro2 bond, respectively. The pH optimum for Arg-Pro-Pro cleavage was 6.8-7.5 in most buffers. The enzyme was most stable in the range of pH 7.0-10.5 in the presence of poly(ethylene glycol). NaCl inhibited activity completely at 2 M. Mn2+ had variable effects on activity, depending on its concentration and the substrate used. Various peptides having an N-terminal Pro-Pro sequence were inhibitory. The enzyme was also inhibited by EDTA, o-phenanthroline, 2-mercaptoethanol, dithiothreitol, p-(chloromercuri)benzenesulfonic acid, apstatin, and captopril. The carboxyalkyl angiotensin-converting enzyme inhibitors, ramiprilat and enalaprilat, inhibited activity in the micromolar range only in the presence of Mn2+.
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PMID:Purification and properties of membrane-bound aminopeptidase P from rat lung. 766 81


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