Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A rapid, inexpensive method for the separation of 5-1-isoleucyl[14C] angiotensin II (A-II) from its various metabolites has been devised. A-II was extracted from tissues with absolute methanol (recovery 96%) and paper chromatographed in a butanol-acetic acid-water (18:2:5) medium for two ascents at 60 degrees C. The resulting RF for A-II of 0.45 was then compared with the RF values of three A-II metabolites produced by enzymatic degradation of the 14C-A-II and [14C]isoleucine. Trypsin degradation produced the [14C]hexapeptide metabolite, chymotryptic degradation produced the [14C]tetrapeptide metabolite and carboxypeptidase A degradation produced the [14C]heptapeptide. Increases in temperature produced a continuous increase in RF values for all the substances examined but the resolution decreased above 60 degrees C. Similarly, increases in the temperature caused the appearance of secondary peaks with some but not all peptides. The tryptic digest (hexapeptide) and the chymotryptic digest (tetrapeptide) are apparently acid- and heat-stable under the experimental conditions. All of the peptides examined failed to produce secondary peaks when heated at neutral pH. The method was used to study the tissue distribution of 14C-A-II after intravenous injection.
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PMID:Rapid paper chromatographic separation of [14C] angiotensen II from some metabolites: application to organ distribution. 3 36

The enzyme carboxypeptidase A has been extensively studied and is thus a good candiate for chemical models and imitation. A model system was constructec which combined two of the three catalytic functional groups of the enzyme together with the appropriate substrate group, and cooperative effective catalysis was demonstrated. However, the mechanism which the model system used in enzyme-like conditions was unexpected, and this led to a study of the enzyme itself. Carboxypeptidase A was studied by examination of oxygen-18 exchange reactions and the ability of the enzyme to substitute only lytic agents, such as methanol, forthe water which is normally used. The result of these studies is proposed mechanism of action of this enzyme which accommodates the large amount of information already available, and which indicates that this enzyme uses a mechanism similar to that found in the model system. Approaches have also been made to an artificial enzyme which could hydrolyse amides. Cyclodextrin has been functionalized with two imidazole groups placed on opposite sides of the cavity. This material catalyses the hydrolysis of an amide, which binds to the cyclodextrin cavity and then is hydrolysed with the assistance of one imidazole in its basic form and the other one in its protonated form, acting together in a way reminiscent of the cooperation of some of the functional groups of hydrolytic enzymes.
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PMID:Studies on enzyme models and on the enzyme carboxypeptidase A. 24 79

The generation of slow reacting substance (SRS) from ionophore A23187-stimulated rat peritoneal mast cells was enhanced by arachidonic acid (AA). This SRS generation was inhibited by 5,8,11,14-eicosatetraynoic acid (ETYA), an acetylenic analogue of AA and an inhibitor of both fatty acid cyclooxygenase and lipoxygenase. Indomethacin, a fatty acid cyclooxgenase inhibitor, had an enhancing effect upon SRS generation. This suggests SRS generation occurred through an ETYA sensitive step--perhaps a lipoxygenase. Radiolabel from [14C]-AA was incorporated into SRS with comigration of radioactivity and bioreactivity in silicic acid and thin layer chromatographies. Upon silicic acid chromatography, the active principle was eluted in the methanol fraction. Two-dimensional thin layer chromatography revealed chromatographic separation from other known spasmogenic substances and phospholipids. Mast cell SRS was found to display physiochemical properties similar to those of rat basophilic leukemia cell SRS, namely: that mast cell SRS generation was 1) enhanced by arachidonic acid; 2) inhibited by ETYA but not by indomethacin; 3) incorporation of [14C]-AA into the active principle; and 4) similar behavior during purification in silicic acid and thin layer chromatographies.
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PMID:Slow reacting substance (SRS) from ionophore A23187-stimulated peritoneal mast cells of the normal rat. II. Evidence for a precursor role of arachidonic acid and further purification. 37 31

The effect of viscosity on the rate of catalysis of carboxypeptidase A has been tested. By use of the tripeptide carbobenzoxy-l-alanyl-l-alanyl-l-alanine [Z(L-Ala)3] as substrate, it was shown that most of the effect on the hydrolysis rate caused by the presence of 30 or 40% methanol or glycerol in aqueous solution can be ascribed to a contribution of viscosity to the catalytic rate constant, kcat. Arrhenius plots of kcat in 30 and 40% glycerol or methanol are linear and almost parallel. When the rate constants are "corrected" for the viscosity of various media, the difference between the various Arrhenius plots is considerably reduced; it vanishes, within experimental error, when the effect of the dielectric constant of the solutions is taken into account as well. It is proposed that the viscosity of the medium can influence the rate-limiting step of the enzymic reaction, which is the rate of transitions over the energy barrier preceding product formation. According to the suggested mechanism, the enzyme--substrate complex can overcome this energy barrier by viscosity-dependent structural fluctuations. The quantitative agreement between the theory and the experimental results suggests that (a) due to the temperature dependence of the viscosity of the solution, the potential energy barrier of the reaction is about 5 kcal/mol lower than the observed activation energy and (b) information about the structural flexibility of the complex can be obtained by kinetic measurements.
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PMID:Viscosity-dependent structural fluctuations in enzyme catalysis. 42 12

Tropomyosin digested with carboxypeptidase A [EC 3.4.12.2] (CTM) shows a lower viscosity than the undigested protein in solution. From the relation between the viscosity decrease and the amount of amino acids liberated from the carboxyl terminus during this digestion, it is inferred that loss of the tri-peptide-Thr-Ser-Ile from the C-terminus is responsible for the decrease in viscosity. The secondary structure of -TM was not affected by the digestion according to circular dichroic measurements. The viscosity of CTM did not increase in methanol-water mixtures, whereas that of tropomyosin increased markedly. These results indicate that polymerizability was lost upon the removal of a small peptide from the C-terminus without change in the secondary structure. A decrease in the viscosity of tropomyosin solutions was observed on the addition of CTM, indicating that CTM interacts with intact tropomyosin. The dependence of the viscosity decrease on the amount of CTM showed that CTM binds tropomyosin in a one-to-one ratio as a result of end-to-end interaction. Since paracrystals having a 400 A repeated band structure could be grown in the presence of Mg ions at neutral pH, side-by-side interactions in CTM molecules remain intact, even though polymerizability is lost. The disc gel electrophoretic pattern showed that troponin could bind to CTM, but no increase in viscosity due to the complex was observed in solution. That is, the C-terminal part of tropomyosin is not required for the formation of the complex. The amount of CTM bound to F-actin was less than half of that bound to undigested tropomyosin, and could be reduced to one-tenth by a washing procedure. In the presence of troponin, however, the amount recovered to the level of tropomyosin normally bound to F-actin. Therefore, it is concluded that troponin is bound in the middle of the tropomyosin molecule and strengthens the binding of tropomyosin to F-actin.
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PMID:Properties of non-polymerizable tropomyosin obtained by carboxypeptidase A digestion. 100 69

Rat mast cell granules and plasma membrane fractions were obtained by homogenization of highly purified rat mast cells and isolation in a Percoll gradient and a sucrose gradient, respectively. Immunostaining of rat mast cells, granules and plasma membrane fractions was performed with mouse monoclonal antibody M6764 which was produced against the crude membrane fractions of the neural tubes. Rat mast cells and granules were immunostained with the monoclonal antibody, but not the plasma membrane fractions. The granules fixed with glutaraldehyde-paraformaldehyde showed ring-like forms. Chloroform-methanol treatment did not effect the staining of rat mast cells and granules with the monoclonal antibody. Western blotting analysis of rat mast cells and granules with the monoclonal antibody showed broad protein bands ranging from 100 to 250 kD.
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PMID:Rat mast cell granules reacting with a mouse monoclonal antibody M6764 which recognizes the same epitope of a mouse monoclonal antibody HNK-1. 170 1

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. Diphosphoinositide (DPI) synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32P] ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of polyamines, spermine and spermidine, to the granules caused an increase of DPI synthesis, which can be catalyzed by PI kinase. This effect of polyamines in the DPI synthesis was in a dose-dependent manner and maximal effects were observed at 1 mM spermine and 10 mM spermidine, respectively.
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PMID:Polyamines stimulate the phosphorylation of phosphatidylinositol in rat mast cell granules. 216 49

Enhanced phospholipid methylation has been suggested to be an obligatory process in IgE-dependent stimulus-secretion coupling in human lung mast cells. Our studies with mast cell-enriched lung preparations do not support this hypothesis, demonstrating no increased 3H-methyl radiolabeling of chloroform/methanol-extracted lipids or chromatographically separated phospholipids accompanying anti-IgE-dependent histamine secretion. Inhibitors of transmethylation, 3-deazaadenosine, and homocysteine thiolactone inhibited histamine secretion by both anti-IgE and calcium ionophore A23187, reflecting a requirement of secretion for overall integrity of cellular transmethylation. These agents induced small increases in cAMP concentration which are considered to make at most a minor contribution to this inhibition. The inability of methylation inhibitors to diminish anti-IgE-dependent increases in lung mast cell cAMP levels would suggest that not only does phospholipid methylation have no role in histamine secretion but also it does not participate in the activation of adenylate cyclase by this stimulus.
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PMID:IgE-dependent activation of human lung mast cells is not associated with increased phospholipid methylation. 245 37

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. DPI synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32P] ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HC1 and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of PMA to the granules caused an increase of DPI synthesis, which can be catalysed by PI kinase. Neither an inactive phorbol ester, 4-alpha-phorbol-12, 13-didecanoate, nor dimethyl sulfoxide (DMSO) used as a solvent for PMA had any effect. The effect of PMA in the DPI synthesis was dose-dependent and maximal effects were observed at 10-100 ng/ml. Dose-response curves of the effects of PMA in DPI synthesis in the granules corresponded to those of other biochemical effects of PMA in rat mast cells, such as mediator release mediated through the activation of protein kinase C. These results suggest that PMA may directly affect PI kinase or indirectly regulate its activity in rat mast cell granules.
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PMID:[Effect of phorbol myristate acetate (PMA) on diphosphoinositide (DPI) synthesis in rat mast cell granules]. 254 22

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. Diphosphoinositide (DPI) synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32] ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of polyamines, spermine and spermidine to the granules caused an increase in DPI synthesis, which can be catalyzed by PI kinase. This effect of polyamines on DPI synthesis in the rat mast cell granules was dose-dependent and maximal effects were observed at 1 mM spermine and 10 mM spermidine respectively. When the effect of 1 mM spermine on 32P incorporation into DPI in rat mast cell granules was investigated serially, 32P incorporation into DPI in rat mast cell granules incubated with spermine for 15 min was enhanced significantly (p less than 0.05) compared with that in the granules in the absence of spermine.
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PMID:[Polyamines stimulate the phosphorylation of phosphatidylinositol in rat mast cell granules]. 255 94


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