Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
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Indoleamine 2,3-dioxygenase was purified from rabbit small intestine to apparent homogeneity as judged by polyacrylamide gel electrophoresis and analytical ultracentrifugation. The native enzyme was a monomeric protein of a molecular weight of 41,000 +/- 1,000 with an s020,w value of 3.45 S. It had a relative abundance of hydrophobic amino acids such as valine, leucine, and isoleucine, and contained approximately 5% carbohydrate by weight. The estimated content of sugar residues per mol of enzyme was: galactose, 1.2; mannose, 2.6; N-acetylglucosamine, 5.2; and sialic acid, 0.8. One mole of enzyme had 0.8 mol of protoheme IX as a prosthetic group. However, copper was not detected in a significant amount and the ratio of copper to heme was less than 0.03. EPR spectra of the nitric oxide complex of the ferrous enzyme indicated that a nitrogen atom, possibly in an imidazole group, might be coordinated as the fifth ligand of the heme coenzyme. The anisotropic g values were gx = 2.08, gy = 1.98, and gz = 2.01. A single enzyme protein catalyzed the oxygenative ring cleavage of D- and L-tryptophan, D- and L-5-hydroxytryptophan, tryptamine, and serotonin. In addition, the purified enzyme had a peroxidase activity with guaiacol and potassium iodide as hydrogen donors, but not a catalase activity.
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PMID:Indoleamine 2,3-dioxygenase. Purification and some properties. 2 87

Changes in the structural components of the Streptococcus pyogenes membrane between exponential and early stationary phases of growth are reported. The overall protein composition ranged from 70 to 73% of the dry weight of the membranes, irrespective of the phase of growth from which they were isolated. Amino acid analyses of membranes isolated from streptococci in either the exponential or stationary phase of growth demonstrated that two amino acids, cysteine and tryptophan, were absent. Further analysis of the membrane proteins by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis demonstrated that there were proteins unique to a particular phase of growth as well as differences in the amount of specific proteins from the various growth phases. In addition, membranes isolated from exponential-phase cultures contained a higher percentage of peripheral protein than did stationary-phase membranes. There also appeared to be an increase in the amount of outer surface proteins during this growth phase. The phosphorus content of the membranes increased during the stationary phase of growth, whereas the sugar composition remained constant. The only sugar found under various conditions of growth in any of the strains was glucose. Total fatty acid content and the mole percent composition of various fatty acids did not change in the different phases of growth. However, the mole percent composition of fatty acids in the membranes of various group A streptococci did differ between strains. Therefore, these results provide evidence that the composition of membranes of S. pyogenes does not remain constant throughout the growth phases of the culture.
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PMID:Chemical analysis of changes in membrane composition during growth of Streptococcus pyogenes. 16 Aug 90

L-Tryptophan, 2,3-dioxygenase (EC 1.13.11.11) has been purified to homogenity from L-tryptophan induced Pseudomonas acidovorans (ATCC 11299b) and from L-tryptophan and cortisone induced rat liver. The enzyme from both sources is composed of four subunits and contains two g-atoms copper and two moles heme per mole tetramer. The proteins from the two sources are not identical. Three oxidation states of tryptophan oxygenase have been isolated: (1) fully oxidized, [Cu(II)]2[Ferriheme]2; (2) half reduced, [Cu(i)]2[ferriheme]2; and (3) fully reduced, [Cu(I)]2[ferroheme]2. Catalytic activity is dependent solely on the presence of Cu(I) in the enzyme, the heme may be either ferro or ferri. The presence of Cu(II) in the enzyme results in a requirement for an exogenous reductant, such as ascorbate, in order to elicit enzymic activity. Ligands, such as cyanide and carbon monoxide, can inhibit catalysis by binding to either or to both the copper and heme moieties. Metal complexing agents, such as bathocuproinesulfonate and bathophenanthrolinesulfonate, can inhibit catalysis by binding to Cu(I) resent only in catalytically active enzyme molecules. During catalysis by the fully reduced form of the enzyme, molecular oxygen binds to the heme moieties, while during catalysis by the half reduced form of the enzyme it does not, presumably binding instead to the Cu(I) moieties. Enzymes that catalyze similar reactions have been purified from other sources. Indoleamine 2,3-dioxygenase appears to be a heme protein, but its copper content is unknown. Pyrrolooxygenases appear to be completely different enzymes, although they have not yet been purified to homegeneity.
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PMID:Tryptophan 2,3-dioxygenase: a review of the roles of the heme and copper cofactors in catalysis. 17 84

The catalytic groups, involved in aminoacyl-tRNA formation remain unknown. The isolation and identification of an active covalent complex between the enzyme and substrate is an essential step in understanding the reaction mechanism. We identified and isolated the covalent complex of tryptophanyl-tRNA synthetase (EC 6.1.1.2) and tryptophane which was able to aminoacylate the tRNATrp in the absence of ATP. In beef pancreas tryptophanyl-tRNA synthetase preparations, isolated by the previously described method, a tightly bound tryptophan was revealed which could not be removed by charcoal treatment, by gel-filtration and by replacement with the excess of typtamine, a competitive inhibitor of tryptophane. This tightly bound tryptophane is able to exchange rapidly and specifically with radioactive tryptophane allowing to obtain [14C]tryptophane-tryptophanyl-tRNA synthetase complex. After the reaction of this complex with NH2OH at neutral pH tryptophanyl hydroxamate is formed proving the activated state of the tryptophane in the initial complex with the enzyme. No nucleotide impurites were noticed in the enzyme preparation; the complex is stable at denaturation. A conclusion is made that the tryptophanyl-tRNA synthetase isolated by our method is a tryptophanyl-enzyme. The tryptophanyl residue could be specifically transferred to tRNATrp in the absence of other substrates of the reaction, the efficiency of the transfer does not exceed 25%. The content of the covalently bound tryptophane never exceeds 1 mole per mole of the dimeric enzyme. The total content of tryptophane in the forms of tryptophanyl-enzyme and tryptophanyl adenylate enzyme complex equals 2 moles per mole of the enzyme. The tryptophanyl-enzyme is destroyed during incubation with AMP or with pyrophosphate. The role of the tryptophanyl-enzyme as a possible intermediate in the course of aminoacylation of tRNATrp is discussed.
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PMID:[Tryptophanyl tRNA synthetase: isolation and characteristics of the tryptophanyl-enzyme]. 20 77

Inhibitor-1 is a protein which inhibits phosphorylase phosphatase only when it has been phosphorylated by cyclic-AMP-dependent protein kinase [Huang, F. L. and Glinsmann, W. H. (1976) Eur. J. Biochem. 70, 419--426]. Inhibitor-1 was purified by a heat treatment at 90 degrees C, precipitation with ammonium sulphate, chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and finally rechromatography of the phosphorylated protein on DEAE-cellulose, The protein was purified 4000-fold and 1.5 mg per 1000 g muscle was obtained in seven days corresponding to an overall yield of 15-20%. The purified protein was in a state approaching homogeneity as judged by the criteria of polyacrylamide-gel electrophoresis and ultracentrifugal analysis. The concentration of inhibitor-1 in vivo was calculated to be 1.5 micron, which is at least as high as the concentration of phosphorylase phosphatase. The amino acid composition of inhibitor-1 showed several unusual features. Glutamic acid and proline accounted for nearly one third of the residues, tyrosine, tryptophan and cysteine were absent, and the content of aromatic amino acids was very low. The molecular weight measured by sedimentation equilibrium centrifugation was 19200 and by amino acid analysis was 20800. These values were lower than the mol. wt 26000 determined empirically by gel electrophoresis in the presence of sodium dodecyl sulphate, and much lower than the apparent molecular weight of 60000 estimated by gel filtration on Sephadex G-100. The gel filtration behaviour, stability to heating at 100 degrees C and amino acid composition suggest that inhibitor-1 may possess little ordered structure. The phosphorylated from of inhibitor-1 contained close to one molecule of covalently bound phosphate per mole of protein, which is consistent with the previous finding of a unique decapeptide sequence at the site of phosphorylation, Ile-Arg-Arg-Arg-Arg-Pro-Thr(P)-Pro-Ala-Thr- [Cohen, P., Rylatt, D. B. and Nimmo, G. A. (1977) FEBS Lett. 76, 182-186].the phosphorylated form of inhibitor-1 inhibited phosphorylase phosphatase activity (0.02U) by 50% at a concentration of only 7.0 nM in the standard assay, but the phosphorylated decapeptide was 1000-2000 times less effective as an inhibitor.
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PMID:The regulation of glycogen metabolism. Purification and characterisation of protein phosphatase inhibitor-1 from rabbit skeletal muscle. 20 44

When dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B, MB 1428, is treated with approximately a 5 mol ratio of N-bromosuccinimide (NBS) to enzyme at pH 7.2 and assayed at the same pH, there is a 40% loss of activity due to the modification of 1 histidine residue and possibly 1 methionine residue before oxidation of tryptophan occurs. The initial modification is accompanied by a shift of the pH for maximal enzymatic activity from pH 7.2 to pH 5.5 Upon further treatment with N-bromosuccinimide, the activity is gradually reduced from 60 to 0% as tryptophan residues become oxidized. An NBS to enzyme mole ratio of approximately 20 results in 90% inactivation of the enzyme. When the enzyme is titrated with NBS in 6 M guanidine HCl, 5 mol of tryptophan react per mol of enzyme, a result in agreement with the total tryptophan content as determined by magnetic circular dichroism. The 40% NBS-inactivated sample posses full binding capacity for methotrexate and reduced triphosphopyridine nucleotide, and the Km values for dihydrofolate and TPNH are the same as for the native enzyme. After 90% inactivation, only half of the enzyme molecules bind methotrexate, and the dissociation constant for methotrexate is 40 nM as compared to 4 nM for native enzyme in solutions of 0.1 M ionic strength, pH 7.2 Also, TPNH is not bound as tightly to the modified enzyme-methotrexate complex as to the unmodified enzyme-methotrexate complex. Circular dichroism studies indicate the 90% NBS-inactivated enzyme has the same alpha helix content as the native enzyme but less beta structure, while the 40% inactivated enzyme is essentially the same as the native enzyme. Protection experiments were complicated by the fact that NBS reacts with the substrates and cofactors of the enzyme. Although protection of specific residues was not determined, it was clear that TPNH was partially protected from NBS reaction when bound to the enzyme, and the enzyme, and the enzyme was not inactivated by NBS until the TPNH had reacted.
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PMID:Effect of N-bromosuccinimide modification on dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli. Activity, spectrophotometric, fluorescence and circular dichroism studies. 23 91

Uridine nucleosidase (EC 3.2.2.3) was purified from commercial bakers' yeast to homogeneity, as judged by a single band observed on polyacrylamide disc gel electrophoresis. The molecular weight of the enzyme, estimated by gel filtration, was approximately 32,500. Polyacrylamide electrophoresis in 0.2% sodium dodecyl sulfate showed the presence of two apparently identical subunits of 17,000 molecular weight. The amino acid composition indicated a large excess of glutamic acid and aspartic acid over other amino acid residues and a very low content of tyrosine and tryptophan. Th SH groups analysis performed with 5,5'-dithiobis (2-nitrobenzoic acid) on thenative protein as well as in the presence of 1% sodium dodecyl sulfate showed the existence of one sulfhydryl group per mole of enzyme. Uridine nucleosidase is active on uridine and 5-methyluridine (ribosylthymine) resulting inactive toward all other pyrimidine and purine nucleosides tested. The Km values for uridine and 5-methyluridine were 0.86 x 10(-3) M and 1.66x10--3M, respectively. The optimal pH is around 7.0. The isoelectric point is 5.1. Among a variety of compounds tested only ribose and glucose 6-phosphate were inhibitory and Ki values were 7.2 mM and 0.19 mM, respectively. Furthermore, ribosylthymine competitively inhibited the hydrolysis of uridine. The type of all inhibitions was competitive and the n' values of the Hill plots were near 1. The effect of temperature on the enzyme activity plotted accoring to Arrhenius gave a value of E = 4740 cal per mole. The enzyme in 100 mM phosphate, pH = 7.0, is stable at 4 degrees for 15 days without any loss of activity.
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PMID:Bakers' yeast uridine nucleosidase. Purification, composition, and physical and enzymatic properties. 23 97

A new double-labelling procedure for amino acid analysis which requires only routine chromatographic equipment is described. When 1-fluoro-2,4-dinitro[3H]benzene is reacted with a mixture of 14C-labelled amino acids followed by reaction with the same 14C-labelled amino acid mixture diluted with an unlabelled sample of amino acids, the 3H:14C ratio in the resulting 2,4-dinitrophenyl (DNP) amino acid derivatives of the diluted sample will be increased in proportion to the quantity of unlabelled amino acid in the diluted sample. This procedure gave reliable results when applied to the known proteins insulin and lysozyme. The procedure is most advantageous when applied to amino acids which are unstable during acid hydrolysis or present in low molar fractions. When applied to the analysis of the bacteriorhodopsin in Halobacterium cutirubrum, this procedure showed the presence of one histidine residue and four tryptophan residues per mole protein but no cystine or cysteine; in general, the analyses obtained were consistent with those originally reported by Oesterhelt, D. and Stoeckenius, W. (1971) (Nature (London) New Biol. 233, 149-152) for bacteriorhodopsin of H. halobium.
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PMID:A new double-labelling procedure for determination of amino acid composition: application to bacteriorhodopsin. 66 97

The ATP-energy transducing system in membranes of Escherichia coli is inhibited by dicyclohexylcarbodiimide. The protein component of this complex with which carbodiimides covalently react to inhibit function was previously identified by labeling wild type and dicyclohexylcarbodiimide-resistant mutants with dicyclohexyl[14C]carbodiimide (Fillingame, R. H. (1975) J. Bacteriol. 124, 870-883). This specific carbodiimide-reactive protein has now been purified. The protein was extracted from the membrane with chloroform:methanol and chromatographed on DEAE-cellulose and hydroxypropyl Spehadex G-50 in this sulvent mixture. The resultant 700-fold purification yielded a protein that was homogeneous on dodecyl sulfate-acrylamide gel electrophoresis and virtually free of phospholipid. It remained soluble in neutral chloroform:methanol throughout the purification procedure. The amino acid composition of the purified protein was extraordinary in that only 16% of the amino acids present could be considered polar. Histidine, serine, cysteine, and tryptophan were not found. Abnormally high contents of methionine, glycine, alanine, and leucine were present. One mole of lysine and threonine were found/mole of dicyclohexyl[14C]carbodiimide bound. The minimum molecular weight based on the amino acid composition was 8400. The specific carbodiimide-reactive protein has also been purified without prior modification by dicyclohexylcarbodiimide. The unmodified protein eluted from DEAE-cellulose at a higher salt concentration than the dicyclohexylcarbodiimide-modified form, which suggested that the reaction with the carbodiimide neutralized the negative charge. Only one-third of the total carbodiimide-reactive protein in the membrane was modified by dicyclohexylcarbodiimide under conditions which maximally inhibited adenosine triphosphatase activity. These results rais the possibility that the carbodiimide-reactive protein may be present as an oligomer in the energy-transducing complex. The purification of the unmodified carbodiimide-reactive protein should permit assessment of tis biological function, particularly its role in the protein-translocation process that is catalyzed by this energy-transducing complex.
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PMID:Purification of the carbodiimide-reactive protein component of the ATP energy-transducing system of Escherichia coli. 78 71

A low molecular weight protein found in the soluble extract of bovine adrenal medulla, and having a high affinity for calcium ions has been purified to apparent homogeneity. The purification requires three steps, including ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100. The protein was homogeneous by the criteria of both standard and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sedimentation velocity analysis, and NH2-terminal analysis. The calcium-binding protein is very acidic and has an isoelectric point of 4.27. Aspartic and glutamic acid together account for 30% of the total amino acid composition. The ultraviolet absorption spectrum reveals a A280/A260 ratio of 0.83 and shows discrete maxima at 258, 264, 269, 278, and 284 nm. Two moles of calcium are bound per mole of protein. The apparent Kp is approximately 20 muM. The molecular weight was found to be 16,000 +/- 1,000 by both sodium dodecyl sulfate gel electrophoresis and sedimentation equilibrium ultracentrifugation. The protein was found to consist of a single polypeptide chain by the criteria of tryptic peptide mapping and NH2-terminal analysis. Amino acid analysis revealed the absence of tryptophan, single residues of cysteine and histidine, and 2 residues of tyrosine. The protein was void of carbohydrate and phosphate. The structural similarities and possible functional correlation between adrenal medulla calcium-binding protein and troponin-C from muscle tissue are discussed.
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PMID:Purification and characterization of a troponin-C-like protein from bovine adrenal medulla. 81 60


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