bluuebunny/crossref_metadata_2025 huggingface dataset:
UDP‐glucuronyltransferase in Perfused Rat Liver and in Microsomes
Authors: ABOU‐EL‐MAKAREM Mahmud M. , BOCK Karl Walter
Published: March 2005

In perfused rat liver and in fistula rats the formation of bilirubin conjugates was studied after labeling with [14C]bilirubin,5‐amino [14C]levulinic acid and [14C]hemin. The latter two compounds were used to study heme degradation to bilirubin from intrahepatic and extrahepatic sources, respectively. Bilirubin glucuronides were the major conjugates in fistula bile. In liver perfusion bile the proportion of non‐glucuronide conjugates was increased. After a high dose of hemin (2.5 gmol) bilirubin glucuronides were decreased compared with other bilirubin conjugates both in fistula bile and in liver perfusion bile. In addition green pigments were formed. These alterations were reversed in chronically hemin‐treated rats in which heme oxygenase had been induced.

The interference of UDP‐glucose and UDP‐glucuronic acid with bilirubin glucuronidation and glucosidation was studied in liver microsomes. UDP‐glucose did not affect bilirubin glucuronidation in native microsomes in which UDP‐glucuronyltransferase activity is constrained. When this constraint was released by various treatments altering membrane structure UDP‐glucose markedly inhibited bilirubin glucuronidation. However, under these conditions bilirubin glucosidation was unaffected by UDP‐glucuronic acid.

The results suggest that the release of the constraint of UDP‐glucuronyltransferase in vivo may lead to a decrease of the proportion of bilirubin glucuronides to other bilirubin conjugates in bile.
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.
Replication of Escherichia coli DNA in vitro: Inhibition by Oxolinic Acid
Authors: STAUDENBAUER Walter L.
Published: March 2005

Oxolinic acid, a quinolone antibacterial agent, inhibits reversibly the ATP‐dependent replicative DNA synthesis in permeable cell systems as well as in cellophane disk lysates. It is about 10‐fold more active than the structurally related nalidixic acid. Both drugs have no effect on the ATP‐independent DNA repair, but interfere to some extent with RNA synthesis in permeable cells. They appear to interact with the same target since spontaneous nalidixic‐acid‐resistant mutants of nalA phenotype are also resistant to oxolinic acid. Full sensitivity to oxolinic acid can be conferred to lysates from resistant cells by addition of extracts from sensitive cells.
Decreased Rate of Metabolism Induced by a Shift of the Double Bond in Prostaglandin F2α from the 5 to the 4 Position
Authors: GRÉEN Krister , SAMUELSSON Bengt , MAGERLEIN Barney J.
Published: March 2005

The synthesis of an isomer of prostaglandin F2α, 9α,11α,15(S)‐trihydroxyprosta‐4‐cis, l3‐trans‐dienoic acid is described. The metabolism of this compound in the rat has been investigated. The rate of degradation by β;‐oxidation was slowed down considerably. Thus 10–20% of the injected isomer was excreted in the urine unchanged indicating a longer half‐life in the circulation than for prostaglandin F2α. More over 2 % was excreted as C20 metabolites, 11–18 % as C18 metabolites and 8 – 15 % as C16 metabolites. This relative resistance to degradation by β‐oxidation is of considerable biochemical and pharmacological interest.
A Simple Method for Measuring Specific Radioactivities of Ribonucleoside Triphosphates using RNA Polymerase
Authors: MAXSON Robert E. , WU Roy S.
Published: March 2005

We describe a method for the rapid, one‐step determination of the specific radioactivity and pool size of ATP, UTP, CTP or GTP. Escherichia coli RNA polymerase and poly[d(A‐T)] or poly[d(G‐C)] are used to synthesize an alternating copolymer from a [3H]nucleoside triphosphate of unknown specific activity and a [14C]nucleoside triphosphate of known specific activity. The fact that [3H]nucleotide and [14C]nucleotide are incorporated into poly[r(A‐U)] or poly[r(G‐C)] in equilnolar amounts, coupled with a knowledge of the [14C]nucleotide specific activity, permits calculation of the [3H]nucleotide specific activity. The requirement for direct knowledge of the [14C]nucleotide specific activity may be bypassed by an isotope dilution procedure. The pool size of a nucleoside triphosphate can be estimated either from isotope dilution data or by determining the fraction of [3H]nucleotide polymerized, dividing the number of counts 3H/min in the polymer by this fraction and by the [3H]nucleotide specific activity. The method was successfully applied to acid extracts made from sea urchin embryos labeled with a [3H]RNA precursor.
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.
Membrane‐Reversible H+‐ATPase from Micrococcus lysodeikticus
Authors: MILEYKOVSKAYA Evgenia I. , TIKHONOVA Galina V. , KONDRASHIN Andrei A. , KOZLOV Igor A.
Published: March 2005

Treatment of phosphorylating fragments of bacterial membrane from Micrococcus lysodeikticus with trypsin leads to increased ATPase activity. As a result of this treatment, the membrane fragments acquire the ability to transform the ATP energy into transmembrane difference in potential. Dithiothreitol has a similar effect to that of trypsin on the membrane fragments from M. lysodeikticus. Dicyclohexylcarbodiimide inhibits ATPase of the membrane fragments of M. lysodeikticus, and also the ATPase‐reaction‐coupled generation of membrane potential. It has been suggested that the increased ATPase activity of membranes from M. lysodeikticus during treatment with trypsin and dithiothreitol is connected with the effect of these agents on the protein inhibitor of ATPase.
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.
The Use of Acetone Precipitation in the Isolation of Ribosomal Proteins
Authors: BARRITAULT Denis , EXPERT‐BEZANÇON Alain , GUÉRIN Marie‐France , HAYES Donal
Published: March 2005

Acetone precipitation is a rapid and efficient method for concentrating ribosomal proteins. This procedure also permits the elimination of sodium dodecylsulphate or urea from protein solutions containing these compounds. Acetone‐precipitated 30‐S ribosomal proteins can be used for the reconstitution of active 30‐S ribosomal subunits.
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 CR34

The 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
The Binding of Tritiated Elongation‐Factors 1 and 2 to Ribosomes from Krebs II Mouse Ascites‐Tumor Cells
Authors: NOLAN Robert D. , GRASMUK Hans , DREWS Jürgen.
Published: March 2005

The effect of a number of different antibiotics and toxins on the capacity of Krebs II mouse ascites ribosomes to bind 3H‐labelled elongation factors (EF‐1 and EF‐2) has been examined. It was found that abrin and ricin inhibit the binding of EF‐2, while diphtheria toxin, sparsomycin, streptovitacin A, and cycloheximide had essentially no effect on its binding. Of the other compounds examined, sparsomycin was unique in its capacity, under some circumstances, to significantly affect the binding of aminoacyl‐tRNA and EF‐1 to ribosomes. Fusidic acid appears to nonspecifically enhance the binding of both EF‐1 and EF‐2.
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.
Characterization of Cardiac Sarcoplasmic Reticulum ATP‐ADP Phosphate Exchange and Phosphorylation of the Calcium Transport Adenosine Triphosphatase
Authors: SUKO Josef , HASSELBACH Wilhelm
Published: March 2005

The terminal phosphate of (γ‐32P)ATP is rapidly incorporated into cardiac sarcoplasmic reticulum membranes (0.7–1.3 μmol/g protein) in the presence of calcium and magnesium. Cardiac sarcoplasmic reticulum membranes catalize an ATP‐ADP phosphate exchange in the presence of calcium and magnesium.

Half‐maximum activation of the phosphoprotein formation and ATP‐ADP phosphate exchange is reached at an ionized calcium concentration of about 0.3 μM. The Hill coefficients are 1.3.

Transphosphorylation and ATP‐ADP phosphate exchange require magnesium and are maximally activated at magnesium concentrations close to or equal to the ATP concentration.

The phosphoprotein level is reduced to about 45% at an ADP/ATP ratio of 0.1. The rate of calcium‐dependent ATP splitting declines, whilst the rate of the calcium‐dependent ATP‐ADP phosphate exchange increases when the ADP/ATP ratio is varied from 0.1 to 1. The sum of both, the rate of ATP splitting and the rate of ADP‐ATP phosphate exchange remains constant.

Phosphoprotein formation and ATP‐ADP phosphate exchange are not affected by azide, dinitrophenol, dicyclohexyl carbodiimide and oubain, whilst both activities are reduced by blockade of –SH groups localized on the outside of the sarcoplasmic reticulum membrane.

The isolated phosphoprotein is acid stable. The trichloroacetic acid denatured 32P‐labelled membrane complex is dephosphorylated by hydroxylamine, which might indicate that the phosphorylated protein is an acyl‐phosphate.

Polyacrylamide gel elctrophoresis (performed with phenol/acetic acid/water) of phosphorylated sarcoplasmic reticulum fractions demonstrates that the 32P‐incorporation occurs into a protein of about 100000 molecular weight.

It is suggested that the phosphoprotein represents a phosphorylated intermediate of the calcium‐dependent ATPase which formation occurs as an early step in the reaction sequence of calcium translocation by cardiac sarcoplasmic reticulum similar as in skeletal muscle.
The Structure of Kinetoplast DNA
Authors: KLEISEN Coen M. , WEISLOGEL Paul O. , FONCK Kees , BORST Piet
Published: March 2005

Degradation of highly purified kinetoplast DNA (kDNA) networks with restriction endonucleases yields ‘extra’ bands in agarose gels that are absent from digests of mini‐circles. Each of the five endonucleases tested, i.e. AluI, HapII, EcoRI, Hsu and HindII + III, yields a unique set of ‘extra’ bands. The ‘extra’ bands consist of linear DNA; they are not mini‐circle oligomers and their added molecular weights, calculated from mobility in gels, are around 2 × 107. Double digests with two restriction endonucleases yield a new set of ‘extra’ bands, showing that the ‘extra’ bands obtained with different enzymes are all derived from the same complex component of kDNA. In digests of 32P‐labelled kDNA an average of 2.3% of the radioactivity is recovered in the ‘extra’ bands.

Treatment of kDNA networks with the single‐strand‐specific S1 nuclease of Aspergillus oryzue preferentially releases a linear DNA with a molecular weight of 26 × 106, calculated from mobility in gels. We present evidence that the ‘extra’ bands obtained with restriction endonucleases are derived from this component.

DNA‐DNA renaturation analysis of fragmented kDNA shows the presence of a minor complex component with a complexity of about 3 × 107, making up less than 10% of the total kDNA.

From these results we conclude that 3–5% of the kDNA consists of a homogeneous class of maxi‐circles catenated in the mini‐circle network. The molecular weight of these maxi‐circles is about 26 × 106 and they contain a unique, non‐repetitive, non‐mini‐circle nucleotide sequence. This component is a prime candidate for the true mitochondrial DNA of trypanosomes.
Purification of Protease II from Escherichia coli by Affinity Chromatography and Separation of Two Enzyme Species from Cells Harvested at Late Log Phase
Authors: PACAUD Michèle.
Published: March 2005

N‐Acetyl‐d‐arginine linked to an agarose matrix has been used to purify protease II from Escherichia coli by affinity chromatography. The specific adsorption of protease II to this absorbent was achieved in 220 mM potassium phosphate buffer pH 7.6, and the enzyme was‐eluted with l‐arginine.

Enzyme preparations from cells harvested at late log phase have been resolved into two molecular species, which differ in specific activity, kinetic constants and carbohydrate content. Both species appeared homogeneous by electrophoresis in conventional buffers and also in the presence of sodium dodecyl sulfate. Only one enzyme species was obtained by the same procedure using bacteria harvested at the middle of exponential growth.
The Selective Blocking of the Polymerization Reaction of Striated Muscle Actin Leading to a Derivative Suitable for Crystallization
Authors: BENDER Norbert , FASOLD Hugo , KENMOKU Akitsugu , MIDDELHOFF Gert , VOLK Karl‐Ernst
Published: March 2005

The polymerization reaction of rabbit muscle actin was completely inhibited by reaction of one amino acid side chain per protein monomer with 5‐diazonium‐(1 H)[14C]tetrazole. A tryptic peptide fingerprint showed a single peptide labeled by the reagent. The peptide was isolated and the labeled amino acid identified by amino acid analysis as Tyr‐53. This side chain is not accessible to the reagent in F‐actin. The modification is compared to similar inhibitions by other reagents.
Lactose Synthetase
Authors: IVTT Raymond J. , ROSEMEYER Michael A.
Published: March 2005

Galactosyl transferase and α‐lactalbumin were concurrently, isolated from bovine milk.

The galactosyl transferase had an S20,W of 3.0 S and D20,W of 60 μm2/s. It exists as a monomer of 46000 molecular weight, as determined by sedimentation equilibrium and dodecylsulphatepolyacrylamide electrophoresis. Aggregation of the enzyme was promoted by N‐acetylglucosamine.

Sedimentation velocity experiments show an association between galactosyl transferase and α‐lactalbumin in the presence of N‐acetylglucosamine. In contrast, UDP‐galactose, UDP or lactose do not promote protein‐protein association.

A complex between galactosyl transferase and α‐lactalbumin was isolated by gel filtration in the presence of excess α‐lactalbumin and either N‐acetylglucosamine or glucose. The complex was stable over arrange of concentrations of these components.

The complex is a discrete homogeneous entity with a molecular weight of 60000, corresponding to one molecule of galactosyl transferase and one molecule of α‐lactalbumin.

The estimated association constants for the ternary complexes of the two proteins and either of the sugars, suggest that α‐lactalbumin enhances equally the binding of N‐acetylglucosamine or glucose to the galactosyl transferase.
The Enzyme Complex Citramalate Lyase from Clostridium tetanomorphum
Authors: BUCKEL Wolfgang , BOBI Angeliki
Published: March 2005

The enzyme citramalate lyase from Clostridium tetanomorphum is not stable in crude extracts. However, the inactive enzyme can be reactivated by incubation with dithioerythritol followed by acetylation with acetic anhydride. Reactivation was also obtained with acetate, ATP. MgCl2 and acetate :SH‐enzyme ligases (AMP) from C. tetanomorphum or Klebsiella aerogenes.

Incubation of the inactive enzyme with iodoacetate resulted in rapid loss of enzymic activity as determined by reactivation with acetic anhydride whereas the active enzyme was stable in the presence of iodoacetate. Using iodo[2‐l4C]acetate the sites of carboxymethylation and acetylation where identified as cysteamine residues of the enzyme. The results demonstrate that the active enzyme contains acetyl thiolester residues which play the central role in the catalytic mechanism.

Citramalate lyase was purified by a procedure almost identical to that already described for citrate lyase from K. aerogenes. The molecular weight of citramalate lyase is equal to that of citrate lyase (Mr= 5.2–5.8 × 105) as estimated by gel chromatography and sucrose gradient centrifugation. Polyacrylamide gel electrophoresis of citramalate lyase in sodium dodecylsulfate yielded three polypeptide chains (Mr: α 5.3–5.6 × 104; β 3.3–3.6 × 104; γ 1.0–1.2 × 104) in probably equal molar amounts. These data lead to a hexameric structure (α,β,γ)6 of the complete enzyme.

Pantothenate (5 mol/mol of enzyme) and the essential cysteamine residues were exclusively present in the γ‐chain, the acyl carrier protein of citramalate lyase. The acyl exchange and cleavage functions, probably catalysed by the α and β‐subunits, were measured with acyl‐CoA derivatives which were able to substitute for the natural acyl carrier.

The results demonstrate that citramalate lyase is an enzyme complex with structure and functions closely resembling those of citrate lyase. Although the similarity between citramalate lyase and citrate lyases from various organisms suggests a close evolutionary relationship, these occur in very different, unrelated bacteria. A parallel situation found in the distribution of the nitrogenase system among procaryotes is discussed
The Prosthetic Group of Citrate‐Lyase Acyl‐Carrier Protein
Authors: DIMROTH Peter
Published: March 2005

The acyl carrier protein of citrate lyase contains adenine, phosphate, sugar, cysteamine, β‐alanine and pantoic acid in a molar ratio of 1:2:2:1:1:1. Peptides containing these components in the same stoichiometric relationship were isolated after proteolytic digestion of acyl carrier protein. All components wefe.linked together in a single prosthetic group. This was released from the peptide by mild alkaline hydrolysis. Under these conditions a phosphodiester bond is cleaved which links the prosthetic group to a serine residue of the peptide.

Incubation of the prosthetic‐group‐containing peptide with phosphodiesterase I yielded 4′phosphopantetheine and adenylic acid. The 5′‐AMP was not free but was substituted by presumably an acidic sugar residue, which was released by mild acid hydrolysis yielding free 5′‐AMP.

It was concluded from these results that the prosthetic group of citrate Iyase acyl carrier protein consists of a substituted isomeric dephospho‐CoA. This is bound to the protein by the 5′‐phosphate group of adenylic acid. The 4′‐phosphopantetheine residue is bound by a phosphodiester linkage to the 2′ or 3′ position of ribose and the remaining hydroxyl group of ribose is substituted with presumably an acidic sugar residue. The structural similarities of this prothetic group and coenzyme A are discussed and related to the catalytic properties of citrate Iyase.
The Binding of Indole to the α‐Subunit and β2‐Subunit and to the α2β2‐Complex of Tryptophan Synthase from Escherichia coli
Authors: WEISCHET Wolfgang O. , KIRSCHNER Kasper
Published: March 2005

The binding of indole and indolepropanol phosphate, an analogue of the substrate indoleglycerol phosphate, to the individual α and β2‐subunits and to the α2β2‐complex of tryptophan synthase was studied by equilibrium dialysis. The use of [14C]indole and indolepropanol [32P]phosphate permitted simultaneous binding studies to be carried out. Competition between indole and indolepropanol phosphate in binding to a particular site was taken as evidence for that site being part of the active site of the α‐subunit.

The binding of indole to the active site of the α‐subunit is weak (Kd= 18 mM). A second distinct site binds indole more strongly (Kd= 1.5 mM) and interacts with the active site indirectly. It is therefore designated an effector site. Furthermore, the binding of indole and/or indolepropanol phosphate appears to stabilize different conformations of the α‐subunit. The β2‐subunit binds indole only weakly (Kd= 12 mM) to many (n = 10) sites per polypeptide chain. The α2β2‐complex retains one or two sites per αβ‐equivalent of relatively high affinity (Kd= 1.2 mM). The active sites of the component α and β‐subunits probably belong to the second class of many (n = 40) sites of low (Kd= 30 mM) affinity for indole. These findings support conclusions from the literature that both bi‐substrate reactions involving indole catalyzed by tryptophan synthase and its subunits must follow strictly ordered addition mechanisms with the respective other substrate adding first.
Peroxidase from Human Cervical Mucus
Authors: SHINDLER Julian S. , CHILDS Robert E. , BARDSLEY William G.
Published: March 2005

A peroxidase has been isolated from pooled specimens of human cervical mucus. Investigation of substrate and inhibitor specificity, pH optimum, chromatography on Sephadex G‐200, disc electrophoresis, ultraviolet absorption spectrum and the effects of temperature and ionie strength variation are described.

A study of the steady‐state kinetics is reported using hydrogen peroxide and 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) as substrates and a catalytic mechanism is proposed
The Mechanism of the Synthesis of Indoleglycerol Phosphate Catalyzed by Tryptophan Synthase from Escherichia coli
Authors: WEISCHET Wolf gang O. , KIRSCHNER Kasper
Published: March 2005

The mechanism of indoleglycerol phosphate synthesis from indole and d‐glyceraldehyde 3‐phosphate catalyzed by tryptophan synthase has been investigated by steady‐state kinetic techniques. The equilibrium constant and the progress curves were measured by use of the difference in absorbance between indole and indoleglycerol phosphate. Stopped‐flow measurements show that only the nonhydrated form of d‐glyceraldehyde 3‐phosphate serves as substrate. The product analogue indolepropanol phosphate was used as an inhibitor to discriminate between possible mechanisms.

The data agree well with an ordered addition mechanism with d‐glyceraldehyde 3‐phosphate adding first. Mechanisms involving random addition of substrates or ordered addition with indole adding first can he excluded because indolepropanol phosphate is a competitive inhibitor only towards glyceraldehyde 3‐phosphate. The high affinity of tryptophan synthase for indoleglycerol phosphate leads to product inhibition even at small extents of reaction. Glyceraldehyde 3‐phosphate combines with the enzyme with an apparent second‐order rate constant, which is not diffusion controlled and generates a site with. high affinity for indole
Steady‐State Kinetic Studies of the Synthesis of Indoleglycerol Phosphate Catalyzed by the α Subunit of Tryptophan Synthase from Escherichia coli
Authors: WEISCHET Wolfgang O. , KIRSCHNER Kasper
Published: March 2005

For the α subunit of tryptophan synthase and at constant concentration of d‐glyceraldehyde 3‐phosphate the saturation curves with respect to indole concentration are weakly sigmoidal. This phenomenon can be explained by interaction between indole bound to the effector site established previously and the active center of the monomeric α subunit. Kinetic studies of the inhibition of indoleglycerol phosphate synthesis by the analogue indolepropanol phosphate show that the inhibition is competitive with respect to d‐glyceraldehyde 3‐phosphate and non‐competitive with respect to indole. Mechanisms with random addition of substrates or ordered addition with indole binding first can therefore be excluded. A quantitative fit of the data has been obtained to an ordered addition mechanism with d‐glyceraldehyde 3‐phosphate binding first and with a distribution of the enzyme between two states differing in V, governed by the binding of indole to the efector site.

The kinetic constants obtained for the α subunit have been compared with those of the α2β2 complex of tryptophan synthase. Protein‐protein interaction of the α subunit with the β2 subunit (a)‐does not alter the catalytic mechanism of indoleglycerol phosphate synthesis, (b) suppresses the substrate activation by indole, and (c) changes the various equilibrium, rate and steady‐state constants in the sense of conveying higher substrate specificity and catalytic efficiency to the α‐subunit. The ocurrence of local and gross conformational changes in the tryptophan synthase system is discussed
A Comparison between the Common Type and a Rare Genetic Variant of Human Cupro‐Zinc Superoxide Dismutase
Authors: MARKLUND Stefan , BECKMAN Gunhild , STIGBRAND Torgny
Published: March 2005

Human cupro‐zinc superoxide dismutase is polymorphic in northern Sweden. The genetic variant type has a lower mobility at electrophoresis in alkaline buffer. The enzyme was isolated from erythrocytes from one of the rare homozygotes and its properties compared to those of the common type.

The isoelectric point of the variant was higher (4.85) than that of the common type (4.7). Small differences in amino acid composition were found but no definite amino acid substitutions could be pointed out. The molecular weights were equal as judged from electrophoreses in polyacrylamide gels in the presence of dodecylsulphate. The ultraviolet spectra were similar.

Parameters related to the active site of the enzyme were very similar; i.e. specific activity and sensitivity to inhibition by cyanide and by H2O2. These parameters, especially the latter two, differ widely between species. Both enzymes were stable for weeks at neutral pH at 37 °C, whereas the common type was significantly more stable at pH 4 and pH 11 and also at incubation in neutral buffer at 70 °C.

It appears that the active site of the variant is conserved whereas the stability of the enzyme is affected.