bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization and Replication Control of Plasmids from Enterobacter cloacae DF 13
Authors: ZANDVLIET Gerard M. , JANSZ Hendrik S. 
Published: March 2005

It has been shown previously that Enterobacter cloacae DF13 harbours at least five different size classes of plasmids. A 45 × 106‐Mr self‐transmissible R factor determining resistance against tetracyclin, sulfanilamide, streptomycin and chloramphenicol, a 6.0 × 106‐Mr bacteriocinogenic factor without sex factor activity and cryptic plasmids in the size classes of Mr 1.3 × 106, 2.8 × 106 and 8.0 × 106 respectively. The present work deals with the determination of the homogeneity and molecular relationship of 1.3 106‐Mr (mini) and 2.8 × 106‐Mr (midi) cryptic plasmids and the 6.0 106‐Mr (maxi) bacteriocinogenic factor, their kinetics of replication and their replication control in response to inhibition of protein synthesis.It has been shown by reassociation kinetics and agarose gel electrophoresis that the maxi plasmids represent a homogeneous population of molecules without repeating sequences. Although homogeneous in size the midi plasmids show considerable heterogeneity in base sequence. The mini plasmids have been shown by agarose gel electrophoresis to consist of four subclasses with contour lengths of 0.79 μm, 0.69 μm, 0.66 μm and 0.61 μm respectively. There is less than 25 % base sequence homology between maxi, midi and mini plasmids and only partial homology within the mini size subclasses.The replication of the four mini plasmids shows absolute dependency on protein synthesis. The replication of midi and maxi plasmids proceeds in the absence of protein synthesis and is inhibited immediately after relief from arginine starvation. These results suggest that the replication of mini plasmids is under control of a positively acting protein and that of midi and maxi plasmids under control of a negatively acting protein.A common feature of these plasmids is their replication kinetics, which follow the anarchical model, i.e. to maintain a fixed number (n) of plasmids, n replications occur during one cell cycle the plasmids taking part at random.
The Involvement of RNA in the Initiation of DNA Synthesis in Mammalian Cells
Authors: PEARSON Colin K. , DAVIS Peter B. , TAYLOR Alison , AMOS Norma A. 
Published: March 2005

Artifacts were encountered during caesium salt density gradient centrifugation which simulated results expected if newly synthesised DNA is covalently attached to RNA. Newly synthesised DNA (in baby hamster kidney cells, BHK‐21/C13), pulse‐labelled with [3H]thymidine for 10 min at temperatures below 25 °C, banded at a greater buoyant density than mature [14C]DNA (heatdenatured nucleic acids) when centrifuged to equilibrium in caesium chloride gradients. Some of the newly synthesised RNA, labelled with [3H]uridine, banded at a buoyant density slightly greater than the DNA in caesium sulphate gradients. These results were not obtained when nucleic acids were pulse‐labelled at 37 °C, nor when samples were heat‐denatured in the presence of formaldehyde. This suggests that nucleic acids can aggregate during centrifugation; this is discussed in relation to the molecular size of the DNA.Such artifacts can also be produced when centrifuging mixtures of mature (non‐replicating) nucleic acids by appropriate manipulation of experimental conditions. Mature [3H]RNA was shown to aggregrate with mature [14C]DNA when the DNA concentration was greater than 100 μg/ml. Sonicated mature [3H]DNA banded with a mean buoyant density in caesium chloride gradients greater than non‐sonicated mature [14C]DNA. This density difference was prevented by heat denaturation in the presence of formaldehyde.
The Similarity between the Primary Structures of Two Non‐histone Chromosomal Proteins
Authors: WALKER John M. , GOODWIN Graham H. , JOHNS Ernest W. 
Published: March 2005

A comparison has been made of two non‐histone chromosomal proteins, high‐mobility‐group proteins 1 and 2. Peptides produced from the two proteins by various chemical and enzymic cleavages have been compared by polyacrylamide gel electrophoresis and peptide mapping. In addition the cysteine content, C‐terminal sequences and amide content have been determined for both proteins. The results obtained, together with previous results, show that high‐mobility‐group proteins 1 and 2 have closely related primary structures, although obviously being different molecules.When analysed by isoelectric focusing both proteins exhibit complex banding patterns. In the case of high‐mobility‐group protein 1, this could be caused by aggregation, but with high‐mobilitygroup protein 2 there are four distinctive subfractions focusing between pH 7.8 and 8.9.
The Threonine‐Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K‐12
Authors: JACQUES Yannick , TRUFFA‐BACHI Paolo 
Published: March 2005

Aspartokinase I–homoserine dehydrogenase I from Escherichia coli K‐12, a homotetrameric enzyme, dissociates into dimers upon alkaline treatment.Both aspartokinase and homoserine dehydrogenase inactivation, as well as desensitization towards l‐threonine, occur in a multi‐step process. Dithiothreitol stabilizes a dimeric form retaining full activity and sensitivity; l‐homoserine stabilizing another dimeric form devoid of aspartokinase activity and retaining a substantial dehydrogenase activity insensitive towards l‐threonine.A model is proposed showing that dissociation into dimers occurs in a first step, the resulting dimer losing both aspartokinase and homoserine dehydrogenase sensitivity in two subsequent steps involving the formation of intrachain disulfide bonds.
Aldehyde Oxidase and Xanthine Dehydrogenase from Wild‐Type Drosophila melanogaster and Immunologically Cross‐Reacting Material from ma‐1 Mutants
Authors: ANDRES Roger Y. 
Published: March 2005

The pleiotropic effect of the ma‐1 mutation on the enzymes xanthine dehydrogenase and aldèhyde oxidase in Drosophila melanogaster can most readily be explained by assuming that the enzymes share a subunit or cofactor whose synthesis is controlled by the ma‐1 locus. According to this hypothesis a protein or a tightly bound cofactor common to both enzymes should be inactive or missing in the corresponding immunologically cross‐reacting material found in ma‐1 flies. Three of the proteins involved were purified by immunoadsorption: xanthine dehydrogenase, xanthine dehydrogenase cross‐reacting material and aldehyde oxidase.On gel filtration xanthine dehydrogenase and xanthine dehydrogenase cross‐reacting material showed identical molecular weights of 300000 whereas 280000 was found for aldehyde oxidase. All three proteins appear to be dimers. Under dissociating conditions (polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate) a single protein band with a mobility corresponding to one half the native molecular weight was found. There was no evidence of small polypeptide subunits. All three proteins contain FAD, molybdenum and iron, cofactors known to be present in related vertebrate enzymes.Thus the ma‐1 locus apparently does not control the mere incorporation of a polypeptide subunit or one of the prosthetic groups necessary for activity into the enzymes.
The Major Proteins of the Escherichia coli Outer Cell‐Envelope Membrane. Heterogeneity of Protein I
Authors: SCHMITGES Claus J. , HENNING Ulf 
Published: March 2005

One of the major proteins of the Escherichia coli outer cell envelope membrane, protein I, can be separated electrophoretically into protein components la and lb. Strain differences exist regarding presence or absence of component lb and this component can selectively be lost by mutation to resistance against a phage. Both components Ia and Ib are further heterogeneous isoelectrically, and both together may contain at least six separable isoelectric species. As judged by analysis of their cyanogen bromide fragments, la and Ib are almost identical concerning their primary structure; the difference (charge only or size and charge) was located in a part of the protein that does not correspond to the C‐terminal or N‐terminal regions.Components la and Ib thus represent essentially the same polypeptide and they may arise by a modification process in vitro or the existence of two almost identical genes.
Synthesis of a Cleavable Protein‐Crosslinking Reagent for the Investigation of Ribosome Structure
Authors: PERETZ Hava , ELSON David 
Published: March 2005

This communication describes a simple method for synthesizing cleavable bifunctional imido esters of different chain lengths. These reagents. which form covalent crosslinks between lysine residues of proteins, contain a disulfide bond which is cleaved under mild conditions by reducing agents such as 2‐mercaptoethanol. The reagents are synthesized via the dithiobisnitrile which is prepared in high yield by reacting the appropriate omega‐activated nitrile with sodium polysulfide and is then converted quantitatively to the diimidate. Three such reagents were prepared: dimethyl 3.3′‐dithiobispropionimidate, dimethyl 4,4′‐dithiobisbutyrimidate, and dimethyl 6‐6′‐dithiobiscaproimidate. The first was synthesized from acrylonitrile, and the others from the appropriate omega hromonitriles.Experiments with the bispropionimidate and a test protein. pancreatic ribonuclease, have shown the reagent to be effective in producing multimeric crosslinked complexes, from which monomeric proteins can be recovered after treatment with 2‐mercaptoethanol. The reagents are suitable for studies of ribosomal structure.
Lysis of Yeast Cell Walls
Authors: ROMBOUTS Frank M. , PHAFF Herman J. 
Published: March 2005

Bacillus circulans WL‐12, when grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, produced five β‐glucanases. Two β‐(1→3)‐glucanases (I and II), which are lytic to yeast cell walls, were isolated from the culture liquid by batch adsorption on yeast glucan, and separated by chromatography on hydroxylapatite. Lytic β‐(1→3)‐glucanase I was further purified by carboxymethylcellulose chromatography. The specific activity of lytic β‐(1→3)‐glucanase I on laminarin was 4.1 U per mg of protein. The enzyme moved as a single protein with a molecular weight of 40000 during sodium dodecylsulfate electrophoresis in slab gels. It was specific for the β‐(l→3)‐glucosidic bond but the enzyme did not hydrolyze laminaribiose. Hydrolysis of laminarin went through a series of oligosaccharides, and laminaribiose and glucose accumulated till the end of the reaction. A small amount of gentiobiose was also produced from laminarin. Products from yeast cell walls and yeast glucan included laminaripentaose, laminaritriose, laminaribiose, glucose, and gentiobiose, but no laminaritetraose was detected. This glucanase has an optimum pH of 5.5. Laminarin hydrolysis followed Michaelis‐Menten kinetics. A Km of 0.105 mg of laminarin per ml and a V of 5.6 micro‐equivalents of glucose released/min per mg enzyme protein were calculated. The enzyme required no metal ions. The lytic β‐(1→3)‐glucanase I is a powerfully lytic enzyme, completely solubilizing the glucan of yeast cell walls. Synergism with lytic β‐(1→3)‐glucanase could be observed during yeast glucan hydrolysis.Lytic β‐(1→3)‐glucanase II could be further purified by carboxymethyl‐cellulose and diethylamino‐ethyl‐agarose chromatography. At this stage lytic β‐(1→3)‐glucanase II was still contaminated with some non‐lytic β‐(1→3)‐glucanase that could not be removed by any further chromatographic steps. The enzyme has an optimum pH of 6.5 to 7; its properties were not studied further.
Multiple Forms of Cyclohexanone Oxygenase from Nocardia globerula CL1
Authors: NORRIS David B. , TRUDGILL Peter W. 
Published: March 2005

The cyclohexanone 1,2‐monooxygenase of Nocardia globerula CL1 exists as two electrophoretically distinct forms. These are present in crude cell extracts and are not artifacts of enzyme purification or electrophoresis.They have been separated in mg amounts by preparative polyacrylamide gel electrophoresis and shown to have essentially identical kinetic, spectral and physical characteristics. They do differ in pH‐activity profile and temperature stability.Whether or not they are conformational isoenzymes or arise by gene duplication and divergent evolution has not been established. Cyclohexanone oxygenase constitutes 8% of the soluble protein of induced cells. This high level would correlate well with the presence of duplicate genes. It is proposed that the presence of a large amount of cyclohexanone oxygenase may confer an ecological advantage on the organism.
Purification and Properties of a Methanol‐Oxidizing Enzyme in Pseudomonas C
Authors: GOLDBERG Israel 
Published: March 2005

A methanol‐oxidizing enzyme has been purified from Pseudomonas C, grown on methanol as a sole source for carbon and energy. The purification procedure involved ammonium sulphate precipitation, ion‐exchange chromatography and gel filtration and resulted in a yield of 35.4 %.Enzyme activity can be coupled to phenazine methosulfate and requires the presence of ammonium ions in the assay mixtures.The enzyme possesses a broad specificity for primary alcohols. Formaldehyde is also oxidized by the purified enzyme. The Km value for methanol is 15 μM. The optimum pH for the oxidation of both methanol and formaldehyde is about 10.4.The enzyme has a molecular weight of about 128000 and consists of two subunits each having a molecular weight of 60000.
Ornithine Carbamoyltransferase from Escherichia coli W
Authors: LEGRAIN Christiane , STALON Victor 
Published: March 2005

Ornithine carbamoyltransferase from Escherichia coli W was purified to homogeneity. The enzyme has a molecular weight of 105000. It is composed of three apparently identical subunits with molecular weights of 35000. The mechanism of the ornithine carbamoyltransferase enzyme system from E. coli W was investigated kinetically by using the approach of product inhibition and dead‐end inhibition of both forward and reverse reactions. On the basis of the kinetic data and binding studies it appears that the mechanism of the reaction involves a compulsory sequence of substrate binding to the enzyme, in which carbamoylphosphate is the first substrate to bind to the enzyme and phosphate the last product to be released. The same studies also indicate that the mechanism involves dead‐end complexes.The reaction mechanism appears consistent with that proposed by Theorell and Chance. Values have been determined for the Michaelis and dissociation constants involved in the combination of each reactant with the enzyme. Comparison of the values for the kinetic constants which are common to both forward and reverse reaction have shown that they are always of a comparable magnitude.
Etude du lipopolysaccharide de Escherichia coli K12 CR 34
Authors: BENEDETTO Jean‐Pierre , BRUNETEAU Maud , MICHEL Georges 
Published: March 2005

Study of the lipopolysaccharide from Escherichia coli K12 CR34The structure of the lipopolysaccharide from Escherichia coli K12, strain CR34 has been investigated. The lipopolysaccharide contains d‐galactose, d‐glucose, d‐glucosamine, l‐glycero‐dmannoheptose, 2‐keto‐3‐deoxyoctonate and lipid A. The core region does not contain d‐glucosamine but contains galactose, glucose, and heptose in the molar ratios 1: 3:6. Methylations were performed an the lipopolysaccharide and an the degraded polysaccharide obtained after acetic acid hydrolysis of the lipopolysaccharide. It was found that galactose is in terminal position partly in the form of galactopyranose, partly in the form of galactofuranose. A part of the heptose was found as unsubstituted heptofuranose. A mole of glucose was 1 → 2 linked and another mole of glucose was 1 → 6 linked. Periodate oxidation of the lipopolysaccharide followed by borohydride reduction eliminates galactose and only a part of glucose and of heptose. A mole of heptose gave mannose and thus it is unsubstituted in C6 and C7. One mole of glucose and one mole of heptose were not degraded by periodate oxidation.On mild acid hydrolysis of the polysaccharide a mole of glucose phosphate was identified.After Smith degradation of the lipopolysaccharide a derivative of a trisaccharide (glucose, heptose, mannose) was obtained indicating that glucose phosphate is linked to heptose in the lipopolysaccharide.On the basis of these findings the partial structure of the E. coli K12 CR34 core polysaccharide is proposed. Some chains of the core must be incomplete an account of the high amount of heptose.
Multiple Forms of Cytochrome P‐450 in Rat‐Liver Microsomes
Authors: GUSTAFSSON Jan‐Åke. , INGELMAN‐SUNDBERG Magnus 
Published: March 2005

Cytochrome P‐450 from male and female rat liver microsomes has been solubilized with sodium deoxycholate, precipitated with ammonium sulphate and separated in the presence of deoxycholate into ten different fractions on a DEAE‐cellulose column eluted with a stepwise gradient of KCI. Each fraction was characterized with respect to its ability to catalyze different hydroxylation reactions of free and disulphurylated 5α‐androstane‐3α,17β‐diol, using a reconstituted system.All hydroxylases active on 5α‐androstane‐3α,17β‐diol (i.e. the 2α‐, 2β‐, 7α‐, 7β‐, 12β‐, 15α‐, 16α and 18‐hydroxylases) and 5α‐androstane‐3α,17β‐diol 3,17‐disulphate (i.e. the 15β‐hydroxylase) were solubilized with cholate. However, the 2β‐ and 18‐hydroxylases were partially inactivated when cholate was added to intact microsomes and these were also the only hydroxylase, activities that could not be detected in reconstitution experiments with lipid, NADPH‐cytochrome P‐450 reductase and the cytochrome P‐450 fractions recovered after DEAE‐cellulose chromatography.Using microsomal preparations from male rat liver it was possible to obtain a partial separation of–the 2α‐ and‐ 7α‐hydroxylases from the 12β‐, 15α‐ and 16α‐hydroxylases and also from the 7β‐hydroxylase. With preparations from female rat livers the 15β‐hydroxylase was well separated from the 2α‐ and 7α‐hydroxylases.The specific activities of the partially separated 2α‐, 7α‐, 7β‐, 12β‐, 15α‐ and 16α‐hydroxylases calculated per nmol of cytochrome P‐450 was 9–34% of the original activities in sonicated microsomes. There was an absolute requirement of the cytochrome P‐450 fractions for all hydroxylase activities except for the 2α‐hydroxylase activity. The requirement for NADPH cytochrome P‐450 reductase was not absolute for any of the hydroxylase activities and no lipid dependency was observed.Based on their behavior during solubilization and purification, elution pattern of the DEAE‐cellulose column and different modes of regulation, the various hydroxylases studied can be divided into different groups. It is suggested that one form of cytochrome P‐450 catalyzes the hydroxylation of 5α‐androstane‐3α,17β‐diol in the 2β‐ and 18‐positions, another form the 12β‐, 15α‐ and 16α‐hydroxylations of the same substrate and a third form the 15β‐hydroxylation of 5α‐androstane‐3α,17β‐diol 3,17‐disulphate.It is concluded that rat liver microsomes contain multiple forms of cytochrome P‐450 active on steroid hormones. It is suggested that the specificity of these forms is determined by the nature of the substrate binding site and by the distance between the binding and catalytic sites on the enzyme
On the Mechanism of Malonyl‐CoA‐Independent Fatty‐Acid Synthesis
Authors: HINSCH Wilhelm , KLAGES Christine , SEUBERT Werner 
Published: March 2005

NADPH‐specific mitochondrial enoyl‐CoA reductase can be assayed by a sensitive radioactive test, employing tritium‐labelled NADPH, synthesized in a prefixed reacton from d‐[1‐3H]glucose via the hexokinase and glucose‐6‐phosphate dehydrogenase reactions.
Liver, kidney cortex, heart muscle, skeletal muscle, brown adipose tissue, brain cortex, and aortic intimal tissue are investigated concerning chain lengths specificity of the chain elongation and the enoyl‐CoA reductase. Medium‐chain acyl‐CoA compounds prove to be the best primers for the chain, elongation. Enoyl‐CoA reductases still show large incorporation rates with hexadecenoyl‐CoA.
The differences in the chain lengths specificity of the chain elongation and enoyl‐CoA reductase can be explained, by the inhibitory effect of long‐chain acyl‐CoA derivatives on the 3‐hydroxyacyl‐CoA dehydrogenase.
The nucleotide specificity in the different tissues reveals two types of chain elongation: In addition to liver and kidney cortex, mitochondria of brown adipose tissue need NADH + NADPH for optimal chain elongation, whereas heart muscle, skeletal muscle and aortic intimal mitochondria only need NADH.
Different physiological roles are proposed for the two types. The “heart type” may he of importance in the conservation of reducing equivalents or acetate units in the anaerobic state the “liver type” may play a role in the transfer of hydrogen from NADPH to the respirator chain. In addition, the mitochondria(chain elongation may serve as bypass of the first part of the respiratory chain
A Presumptive Subunit of Elastic Fiber Microfibrils Secreted by Arterial Smooth‐muscle Cells in Culture
Authors: MUIR Larry W. , BORNSTEIN Paul , ROSS Russell 
Published: March 2005

Monkey smooth muscle cells, maintained for five weeks in confluent cultures, accumulated a substantial extracellular matrix. Elastic fibers possessing the characteristic amorphous elastin component and 11 rim diameter microfibrils could be isolated from this matrix by extraction with 5 M guanidine. Further extraction under reducing conditions selectively removed the microfibrillar component. Get electrophoresis of this extract in the presence of sodium dodecylsulfate revealed a single protein band with an apparent molecular weight of 270000. The amino acid composition of this presumptive subunit of the microfibrillar protein was rich in acidic and hydrophilic amino acids and distinctly different from that of collagen or elastin.When smooth muscle cell cultures were incubated with labeled cystine, the predominant labeled protein in the culture medium also had a molecular weight of 270000. This labeled protein comigrated with a major protein band and could be partially purified by DEAE‐cellulose ion‐exchange chromatography and gel filtration on agarose. It shared many of the properties of the presumptive microfibrillar protein.