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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The membrane penicillinase of Bacillus licheniformis 749/C has been demonstrated to be a phospholipoprotein. The homogeneous enzyme gives a positive reaction for phosphorous and for unsaturated fatty acids, has a molecular weight of 33,000 in contrast to 29,000 for the exoenzyme, and contains 8 to 9 additional residues of aspartate or asparagine, 4 to 5 of serine, 7 of glutamate or glutamine, and 4 to 5 of glycine per mole. The COOH-terminal sequence of both membrane and exoenzymes is -Met-Asn-Gln-Lys-COOH; hence the extra peptide portion present in the membrane enzyme is not attached to the COOH-terminus of the exoenzyme. Procedures which readily detected the lysine residue at the NH2 terminus of the exoenzyme did not yield a positive test with the membrane form. The NH2 terminus of the membrane enzyme may be blocked by or linked to the phospholipid. A procedure for the preparation of membrane penicillinase on a large scale and an improved method for purification of the exoenzyme have been developed.
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PMID:Purification of plasma membrane penicillinase from Bacillus licheniformis 749/C and comparison with exoenzyme. 0 71

Methioninase of Pseudomonas putida was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, with a specific activity 270-fold higher than that of the crude extract. 1. The purified enzyme had an S20,w of 8.37, a molecular weight of 160,000, and an isoelectric point of 5.6. 2. A break in the Arrhenius plot was observed at 40 degrees and the activation energies below and above this temperature were 15.5 and 2.97 kcal per mole, respectively. 3. In addition to L-methionine, various S-substituted derivatives of homocysteine and cysteine could serve as substrates. D-Methionine, 2-oxo-4-methylthiobutanoate, and related non sulfur-containing amino acids were inert. Equimolar formation of alpha-ketobutyrate and CH3SH was observed with methionine as a substrate. 4. In addition to the protein peak at 278 nm, two absorption maxima were observed at 345 and 430 nm at pH 7.5. Hydroxylamine removed the enzyme-bound pyridoxal phosphate, resulting in almost complete resolution with the concomitant disappearance of both peaks. Reconstruction of the treated enzyme could be achieved by addition of the cofactor; the Km value was calculated to be 0.37 muM. 5. The reported purified enzyme should be designated as L-methionine methanethiollyase (deaminating).
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PMID:Purification and characterization of methioninase from Pseudomonas putida. 0 40

The trypsin and chymotrypsin inhibitor from chick peas (CI) is stable in HCl 0.001 M -- 0.01 M and in KOH 0.01 M -- 0.05 M even after 24 h. Increased KOH concentrations decrease considerably the inhibitory activity already after 1 h. Maleyation and succinylation of the inhibitor resulted in almost full loss of its trypsin-inhibitory activity but had no effect on the chymotrypsin-inhibitory activity. A series of modifications directed towards tyrosyl residues showed that iodination influenced only the chymotrypsin-inhibitory activity; however, nitration and arsanilation affected not only the chymotrypsin-inhibitory activity but also the trypsin-inhibitory activity. Treatment of the inhibitor with CNBr and chloramine T resulted only in a decrease in the chymotrypsin-inhibitory activity indicating that the only methionine is involved in the chymotrypsin-inhibitory activity. When CI-fragment A, previously treated with trypsin at pH 3.75, was further treated with carboxypeptidase B, a release of three lysyl residues per mole protein was found. CI was separated by equilibrium chromatography on SP-Sephadex column into two isoinhibitors, CII and CIII, respectively. Both inhibited trypsin and chymotrypsin with the same specific activity as CI. They differed from each other only in a glutamyl, aspartyl, glycyl and alanyl residue.
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PMID:Trypsin and chymotrypsin inhibitor from chick peas. Selective chemical modifications of the inhibitor and isolation of two isoinhibitors. 4 22

Specific modification of the monomeric fraction III of ferri-hemoglobin from insect larvae Chironomus thummi thummi (Hb CTT) was studied on histidyl residues His-G19 (pK 4,8), His-E5 (pK 7,3) and Met-H22 at different pH using iodacetamide and spin label 2,2,6,6-tetramethyl-4-bromacethyl-piperidin-1-oxyl, an analogue of bromacetate. The analysis of the products of carboxymethylation (CM) showed that at pH 5,0 two products of modification CM-(His-G19)-Hb CTT, and CM-(Met-H22)-Hb CTT were obtained. In the case of modification at pH 7,2 with a spin label dicarboxymethylatid product CM-(His-G19)-CM (His-E5)-Hb CTT is obtained. In all products the degree of modification was one spin label per mole protein. Based on the data on the primery and tertiary structures Hb CTT and the results of the investigation, different reactivity of His-G19 and His-E5, as well as the cause of the absence of the product of carboxymethylation on His-G2 have been discussed. By analizing the absorption spectra of carboxymethylated derivatives of hemoglobin in the ultraviolet and visible region, as well as from the pH dependence curves of the absorption at Soret band in the interval pH 5,5-11,5 it has been shown that carboxymethylation of His-G19 and His E5 is not accompanied by any substantial disturbance of the structures of aquous-complexes Hb CTT. Modification of Met-H22 leads to strong changes in the absorption spectrum and to the absence of pH dependence of the absorption at Soret band, which indicates a change in the aquous-complexes Hb CTT structure.
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PMID:[Selected carboxymethylation of ferri-hemoglobin from insect larvae Chironomus thummi thummi]. 17 63

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

1. Mucosal amino acid uptake by pig proximal colon, measured independently for fourteen different amino acids each used at a concentration of 1 mM, ranged from 0.6 to 8.6 n-mole. cm(-2). min(-1) in the new-born to 0 to 0.3 n-mole. cm(-2). min(-1) in the 2-day-old animal. Long chain amino acids entered the mucosa of new-born pig proximal colon much more readily than did short chain amino acids.2. Glycine was used extensively to inhibit the uptake of other neutral amino acids. The degree of maximal inhibition produced depended on the amino acid used. The relative inability of glycine to inhibit the uptake of long chain amino acids suggested that these compounds could cross the brush border on a carrier inaccessible to glycine. The glycine-sensitive uptake remained more or less constant for all amino acids tested (1-2 n-mole.cm(-2).min(-1)); the glycine-insensitive uptake varied from 0 to 7 n-mole.cm(-2).min(-1) (glycine and methionine respectively).3. It is suggested that at least two mechanisms exist for the entry of neutral amino acids into pig proximal colon, one showing specificity for hydrophobic amino acids and the other having broad specificity. The mechanism responsible for the uptake of long chain essential amino acids predominates over the less specific mechanism.4. These results are discussed in relation to previous work carried out on the rabbit ileum where two similar systems for neutral amino acid entry have been shown to be present. Both tissues transport hydrophobic amino acids on their own specific carrier at approximately the same rate; the ability of the pig colon to transport amino acids on the broad specificity carrier is eight times less than in the rabbit ileum. The possibility is raised that this system is subject to regulation.
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PMID:Different mechanisms for neutral amino acid uptake by new-born pig colon. 43 36

1. The influx of [3H]gamma-aminobutyric acid ([3H]GABA) into isolated rat superior cervical ganglia has been measured by radioassay, supplemented by autoradiography. Ganglia were incubated in oxygenated Krebs solution at 25 degrees C, containing 10 microM-amino-oxyacetic acid. Under these conditions more than 95% of accumulated tritium was unmetabolized [3H]GABA. 2. Ganglionic radioactivity increased linearly with incubation time, to yield an intracellular fluid/extracellular fluid concentration ratio (Ci/Co) of about 200 after 6 hr in 0.5 microM-external [3H]GABA. 3. Uptake showed saturation with an apparent transport constant (KT) of 6.8 microM and maximum influx velocity (Jmaxi) of 7 mumole 1. cell fluid-1- min-1. 4. The influx rate at Co = 0.5 microM was unaltered by raising intracellular GABA from 0.2 to 1 mM. 5. Influx velocity increased with temperature (5--35 degrees C) in a monotonic manner with an apparent activation energy of 14 kcal mole-1. 6. Concentrative uptake was depressed by reducing external [Na+] with ouabain, by raising [K+]o above 20 mM, or by removing external Cl-. Uptake was not particularly sensitive to Ca2+ or Mg2+ ions. 7. Utake of [3H]GABA (0.5 microM) was inhibited by beta-guanidinopropionic acid (apparent KI, 28 microM), beta-alanine (KI, 55 microM), gamma-amino-beta-hydroxybutyric acid (KI, 220 microM), beta-amino-n-butyric acid (KI, 708 microM), 3-aminopropanesulphonic acid (KI, 832 microM) and taurine (KI greater than 1 mM). Uptake was not depressed by 1 mM-glycine, alpha-alanine, leucine, serine, methionine or alpha-amino-iso-butyric acid. 8. Radioactively labelled methionine, leucine, glycine, serine, beta-alanine and taurine (concentrations less than or equal to 5 microM) were also taken up by ganglia. Of these, only uptake of beta-alanine and taurine were significantly depressed by 1 mM-GABA. 9. Autoradiographs confirmed that [3H]GABA and [3H] beta-alanine were taken up predominantly into extraneuronal sites (presumed to be neuroglial cells). Methionine, leucine, glycine and serine showed preferential accumulation in neurones. Neuronal uptake of leucine was not prevented by inhibiting protein synthesis. 10. Calculations of net fluxes from unidirectional tracer fluxes suggest that the sympathetic glial cells are capable of promoting net uptake of GABA at external concentrations above 1 microM.
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PMID:[3H]gamma-Aminobutyric acid uptake into neuroglial cells of rat superior cervical sympathetic ganglia. 50 28

Beta-conglycinin consisting of six major isomers (designated B1- to B6-conglycinin) was dissociated and fractionated on columns of DEAE- and CM-Sephadex in buffers containing 6 M urea. Three major (alpha, alpha' and beta) and one minor (gamma) subunits were isolated and further characterized by gel electrophoresis and gel electrofocusing. Gel electrophoresis in urea and in sodium dodecyl sulfate, and gel filtration in 6 M guanidine hydrochloride gave a molecular weight of 57 000 for alpha, alpha' subunits; and 42 000 for beta and gamma subunits. The isoelectric points of the isolated subunits, measured by disc gel electrofocusing, were as follows: alpha, 4.90; alpha', 5.18; beta, 5.66-6.00. On gel electrofocusing, beta subunit showed four microheterogeneous components; three of them comprised 95% of the total beta subunit. Leucine and valine were the N-terminal amino acids of beta and alpha alpha' subunits, respectively. The isolated subunits contained mannose and glucosamine in varying quantities. Two carbohydrate moieties were calculated for one mole of alpha, alpha' subunits; and one carbohydrate moiety for the beta subunit. Considerable similarity in the amino acid composition of alpha and alpha' subunits was observed. The beta subunit was devoid of cysteine and methionine; and in comparison with alpha, alpha' subunits, had a higher content of hydrophobic amino acids. The isolated subunits exhibited antigen-antibody reaction with antisera to the native beta-conglycinin. Each of them was partglycinins. The alpha and alpha' subunits were in addition identical with each other and with B5-, B6-conglycinins. They were immunologically unrelated with beta subunit. The recovery of immuno-properties from the individual subunits may be attributed to the reconstruction of the three-dimensional structure upon removal of denaturing reagents.
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PMID:Beta-conglycinin from soybean proteins. Isolation and immunological and physicochemical properties of the monomeric forms. 55 58

The 3-alpha-hydroxysteroid dehydrogenase and the 3-beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni were purified to homogeneity by polyaerylamide gel electrophoresis using the following stages: DEAE cellulose chromatography, affinity chromatography on oestrone-aminocaproate sepharose and Sephadex gel filtration. The pure 3-alpha-hydroxysteroid dehydrogenase was completely devoid of 3-beta-hydroxysteroid dehydrogenase activity but could oxidize estradiol 17-beta at an appreciable rate. This activity accounts for about 40 per cent of the total 17-beta-estradiol dehydrogenase of the crude bacterial extract. Affinity labelling of pure 3-alpha-hydroxysteroid dehydrogenase was carried out using 5-beta-pregnane 3,20-dione-12-alpha-iodoacetate and 5-alpha-androstane 3-one-17-beta-bromoacetate. With both reagents, inactivation was obtained only in the presence of coenzyme, the substrate protected against inactivation and the enzyme was fully inhibited with covalent binding of 1 mole of reagent per mole of subunit suggesting an active site directed inhibition. Histidine and methionine were identified as the labelled aminoacid residues.
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PMID:Characterisation of an associate 17-beta-hydroxysteroid dehydrogenase activity and affinity labelling of the 3-alpha-hydroxysteroid dehydrogenase of Pseudomonas testosteroni. 60 95

An unusual class of wheat germ tRNAs has been isolated which completely lacks ribothymidine (rT) and contains an unmodified uridine in its place. We discuss here the isolation, identification and properties of these tRNAs. The rT-lacking tRNAs of wheat germ are essentially limited to the glycine isoacceptors (a minimum of five identifiable species), three threonine and at least, one tyrosine tRNA. All tRNAs were obtained 70-100% pure by chromatographic methods, and were detected by their ability to be methylated by E. coli rT-forming uracil methyltransferase with methyl-labeled S-adenosyl-L-methionine (SAM) as the methyl donor. In vitro methylation of each of the tRNAs resulted in the formation of 1 mole of rT per mole of tRNA. In the one case analyzed in detail (tRNA1Gly), all of the rT was found to be located at the 23rd position from the 3' end of the tRNA molecule. Following complete digestion of four highly purified glycine isoacceptors (tRNAGly1,4,5,6) to nucleosides and subsequent periodate oxidation and 3H potassium borohydride reduction, all were found to contain an unusually high level of 5-methylcytidine (m5C) (3-4 residues per molecule), and all contained no rT. The possible correlation between the presence of m5C and the absence of rT is discussed. All of the chromatographically purified glycine tRNAs function in a wheat germ cell-free protein synthesizing system and polymerize glycine in response to either poly G or poly (G, U).
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PMID:Wheat germ tRNAs containing uridine in place of ribothymidine: a characterization of an unusual class of eukaryotic tRNAs. 65 15


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