scholarly journals The oxygenation of [3-methyl-3H]desacetoxycephalosporin C [7β-(5-d-aminadipamido)-3-methylceph-3-em-4-carboxylic acid] to [3-hydroxymethyl-3H]desacetylcephalosporin C by 2-oxoglutarate-linked dioxygenases from Acremonium chrysogenum and Streptomyces clavuligerus

1978 ◽  
Vol 173 (3) ◽  
pp. 839-850 ◽  
Author(s):  
M K Turner ◽  
J E Farthing ◽  
S J Brewer
Keyword(s):  
Carboxylic Acid ◽  
Deae Cellulose ◽  
Streptomyces Clavuligerus ◽  
Reducing Agents ◽  
Side Chain ◽  
Acremonium Chrysogenum ◽  
Hydroxymethyl Group ◽  
Methyl Group ◽  
Full Activity

Cell-free extracts of Acremonium chrysogenum and Streptomyces clavuligerus oxidize the 3-methyl group of desacetoxycephalosporin C to a 3-hydroxymethyl group. The enzyme responsible for this reaction in these organisms was purified 20- and 30-fold respectively by chromatography on DEAE-cellulose. The enzymes, which were assayed with [3-methyl-3H]desacetoxycephalosporin C as substrate, have the properties expected of 2-oxoglutarate-linked dioxygenases. They require 2-oxoglutarate, Fe2+ cations and a mixture of reducing agents (dithiothreitol and ascorbate) for full activity. The enzyme from A. chrysogenum, but not that S. clavuligerus, is activated about 10-fold when it is preincubated with a reaction mixture from which either desacetoxycephalosporin C or 2-oxoglutarate is omitted. Fe2+ cations seem to play a key role in this activation. Both enzymes seem highly specific for cephalosporins with the natural 7beta-(5-D-aminoadipamido) side chain and are likely to be responsible for the oxidation of the 3-methylcephem nucleus in vivo.

scholarly journals An adenosine triphosphate-dependent carbamoylphosphate-3-hydroxymethylcephem O-carbamoyltransferase from Streptomyces clavuligerus

1980 ◽  
Vol 185 (3) ◽  
pp. 555-564 ◽  
Author(s):  
S J Brewer ◽  
P M Taylor ◽  
M K Turner
Keyword(s):  
Carboxylic Acid ◽  
Oxygen Atom ◽  
Deae Cellulose ◽  
Streptomyces Clavuligerus ◽  
Wide Range ◽  
Nucleoside Triphosphates ◽  
Phosphate Anions ◽  
Beta 2 ◽  
Methylene Group ◽  
Carbamoyl Group

Cell-free supernatants from cells of Streptomyces clavuligerus (N.R.R.L. 3585), which are actively synthesizing cephamycin C, transfer a carbamoyl group from carbamoylphosphate to a 3-hydroxymethylceph-3-em-4-carboxylic acid nucleus to form a 3-carbamoyloxymethylcephem. This reaction was stimulated by nucleoside triphosphates and by a mixture of Mn2+ and Mg2+ cations. The enzyme responsible was purified 40-fold by batch absorption onto DEAE-cellulose and hydroxyapatite. The purified O-carbamoyltransferase is most active at pH 6.8. It is stabilized by phosphate anions, but is inhibited by PPi anions, (NH4)2SO4 or NaCl. The enzyme is stimulated by ATP, but it is not known whether this nucleotide acts as an effector or as a substrate. Some activity is observed with dATP, but two other analogues of ATP, in which a methylene group replaced the oxygen atom between the alpha- and beta- or the beta- and gamma-phosphorus atoms, inhibit the action of ATP itself. The enzyme synthesizes a wide range of 3-carbamoyloxymethylcephems. The structure of some of these products, for example that of cefuroxime (3-carbamoyloxymethyl-7 beta-[2-(fur-2-yl)-2-syn-methoxyiminoacetamido]ceph-3-em-4-carboxylic acid), was confirmed by their proton-n.m.r. spectra.

scholarly journals Eukaryotic Wobble Uridine Modifications Promote a Functionally Redundant Decoding System

2008 ◽  
Vol 28 (10) ◽  
pp. 3301-3312 ◽  
Author(s):  
Marcus J. O. Johansson ◽  
Anders Esberg ◽  
Bo Huang ◽  
Glenn R. Björk ◽  
Anders S. Byström
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Side Chain ◽  
Side Chains ◽  
Methyl Group ◽  
Wobble Position ◽  
Naturally Occurring

ABSTRACT The translational decoding properties of tRNAs are modulated by naturally occurring modifications of their nucleosides. Uridines located at the wobble position (nucleoside 34 [U34]) in eukaryotic cytoplasmic tRNAs often harbor a 5-methoxycarbonylmethyl (mcm5) or a 5-carbamoylmethyl (ncm5) side chain and sometimes an additional 2-thio (s2) or 2′-O-methyl group. Although a variety of models explaining the role of these modifications have been put forth, their in vivo functions have not been defined. In this study, we utilized recently characterized modification-deficient Saccharomyces cerevisiae cells to test the wobble rules in vivo. We show that mcm5 and ncm5 side chains promote decoding of G-ending codons and that concurrent mcm5 and s2 groups improve reading of both A- and G-ending codons. Moreover, the observation that the mcm5U34- and some ncm5U34-containing tRNAs efficiently read G-ending codons challenges the notion that eukaryotes do not use U-G wobbling.

Methods for the Concentration of Bovine Ac-Globulin (Factor V)

1961 ◽  
Vol 06 (03) ◽  
pp. 435-444 ◽  
Author(s):  
Ricardo H. Landaburu ◽  
Walter H. Seegers
Keyword(s):  
Room Temperature ◽  
Barium Carbonate ◽  
Deae Cellulose ◽  
Reducing Agents ◽  
Factor V ◽  
Isoelectric Precipitation ◽  
Stabilizing Agent ◽  
Bovine Plasma ◽  
The Stability

SummaryAn attempt was made to obtain Ac-globulin from bovine plasma. The concentrates contain mostly protein, and phosphorus is also present. The stability characteristics vary from one preparation to another, but in general there was no loss before 1 month in a deep freeze or before 1 week in an icebox, or before 5 hours at room temperature. Reducing agents destroy the activity rapidly. S-acetylmercaptosuccinic anhydride is an effective stabilizing agent. Greatest stability was at pH 6.0.In the purification bovine plasma is adsorbed with barium carbonate and diluted 6-fold with water. Protein is removed at pH 6.0 and the Ac-globulin is precipitated at pH 5.0. Rivanol and alcohol fractionation is followed by chromatography on Amberlite IRC-50 or DEAE-cellulose. The final product is obtained by isoelectric precipitation.

scholarly journals Effect of side-chain length in lignin model compound on MnO2 oxidation: comparison of oxidations between C6-C2- and C6-C1-type compounds

2021 ◽  
Vol 67 (1) ◽  
Author(s):  
Shirong Sun ◽  
Tomoya Yokoyama
Keyword(s):  
Isotope Effects ◽  
Aromatic Nucleus ◽  
Side Chain ◽  
Direct Oxidation ◽  
Initial Oxidation ◽  
Methyl Group ◽  
Lignin Model Compound ◽  
Oxidation Rates ◽  
Kinetic Isotope ◽  
Lignin Model

AbstractMonomeric C6-C2-type lignin model compounds with a p-hydroxyphenyl (H), guaiacyl (G), syringyl (S), or p-ethylphenyl (E) nucleus (1-phenylethanol derivatives) were individually oxidized by MnO2 at a pH of 1.5 and room temperature. The results were compared with those of the corresponding C6-C1-type benzyl alcohol derivatives obtained in our recent report to examine the effect of the presence of the β-methyl group on the oxidation. The presence decelerated the oxidation regardless of the type of aromatic nucleus, although it did not change the order of the oxidation rates: G > S >> H > E. This deceleration results from the steric factor of the β-methyl group in the C6-C2-type compounds. The MnO2 oxidations of the corresponding C6-C2-type compounds deuterated at their α-(benzyl)positions showed that the magnitudes of the kinetic isotope effects are smaller than those observed in the oxidations of the corresponding C6-C1-type compounds, regardless of the type of aromatic nucleus. These smaller magnitudes suggest that the presence of the β-methyl group shifts the initial oxidation mode of MnO2 from direct oxidation of the benzyl position to one-electron oxidation of the aromatic nucleus. Only the S-type compounds afforded products via degradation of the aromatic nuclei.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals EVIDENCE THAT THE L-ASPARAGINASE OF GUINEA PIG SERUM IS RESPONSIBLE FOR ITS ANTILYMPHOMA EFFECTS

1963 ◽  
Vol 118 (1) ◽  
pp. 99-120 ◽  
Author(s):  
J. D. Broome
Keyword(s):  
Guinea Pig ◽  
Guinea Pigs ◽  
Serum Proteins ◽  
Deae Cellulose ◽  
Salt Precipitation ◽  
Asparaginase Activity ◽  
Pig Serum

A number of the properties of the L-asparaginase present in guinea pig serum have been examined and shown to be indistinguishable from those of the agent responsible for inhibiting cells of lymphoma 6C3HED in vivo. The patterns of instability of the enzyme to changes in temperature and pH were found to parallel closely those of the antilymphoma agent. L-Asparaginase activity was essentially absent from the serum of newborn guinea pigs and this failed to inhibit 6C3HED cells. On separating guinea pig serum proteins by salt precipitation, electrophoresis, and chromatography on DEAE cellulose, antilymphoma activity was found only in fractions which contained L-asparaginase.

Secondary biosynthesis of aflatoxin B1 in Aspergillus parasiticus

1970 ◽  
Vol 16 (10) ◽  
pp. 959-963 ◽  
Author(s):  
R. W. Detroy ◽  
C. W. Hesseltine
Keyword(s):  
Protein Synthesis ◽  
Aflatoxin B1 ◽  
Aspergillus Parasiticus ◽  
Synthetase Activity ◽  
Macromolecular Synthesis ◽  
High Concentration ◽  
Methyl Group ◽  
Aflatoxin B ◽  
Concentration Levels

The effect of two inhibitors on the formation of aflatoxin B1 synthetase activity in strain NRRL 2999 Aspergillus parasiticus has been studied. Aflatoxin B1 synthesizing activity was measured in vivo by incorporation of the 14C-methionine methyl group into aflatoxin B1. Cycloheximide at a concentration of 150 μg/ml blocks protein synthesis completely. If addition of cycloheximide is made before B1 synthetase appears, no activity accumulates; if added during accumulation, activity is frozen at the level reached at the time of addition. The cycloheximide effect is reversible since morphogenesis, total protein synthesis, and aflatoxin B1 synthetase activity all resume after removal of the inhibitor.DL-p-Fluorophenylalanine partially inhibits aflatoxin B1 synthesis in vivo; however, its effect upon macromolecular synthesis is incomplete even at high concentration levels. Once formed, the aflatoxin synthetase appears to maintain B1 synthesis when further protein synthesis is blocked; i.e., it is not rapidly degraded.

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