bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytochrome c: A Thermodynamic Study of Relationships among Oxidation State, Ion‐Binding and Structural Parameters
Authors: Margalit Rimona , SCHEJTER Abel
Published: March 2005

The specific binding of anions to oxidized horse‐heart cytochrome c has been studied by two methods: measuring the effect of ion concentration on the redox potential, and directly by gel nitration. The anions investigated were: chloride, phosphate, ADP and sulfanilate. The binding constants estimated are in the range of 1–10 mM−1, and the number of sites per protein molecule are 1 to 2. The specificity of anion binding to the oxidized protein is demonstrated.
Reduced Rate of β‐Globin mRNA Translation in β‐Thalassemia
Authors: Conconi Francesco , DEL SENNO Laura , Borgatti Luigi
Published: March 2005

The synthesis of α and β‐globin have been determined in normal and thalassemic erythroid cells, incubated at temperatures decreasing from 37 to 10°C.

Synthesis of β‐globin by thalassemic reticulocytes decreased with temperature clearly less than α and β‐globin synthesis by normal erythroid cells, and also less than α‐globin synthesis by thalassemic reticulocytes. Consequently the excess of α‐globin synthesis, typical of β‐thalassemia, disappeared when cells where incubated at temperatures of approximately 10°C.

The normalization of β‐globin synthesis, obtained in our experimental conditions, clearly suggests that the defect of β‐globin synthesis, characteristic of β‐thalassemia, does not result from a reduction in amount of β‐globin messenger, but rather from a reduction of the rate of β‐globin mBNA translation.
Immunochemical Properties of Procollagen from Dermatosparactic Calves
Authors: Timpl Rupert , WICK Georg , Furthmayr Heinz , LAPIÈRE Charles M. , Kühn Klaus
Published: March 2005

After immunization of rabbits with dermatosparactic calf‐skin collagen a predominantly specific response to this kind of collagen and only a weak cross‐reaction with soluble skin collagen from normal calves was observed. Antibodies exclusively reacting with dermatosparactic collagen were obtained upon immunoadsorption. They displayed strong reactions with the constituent pα1‐ and pα2‐chain although both chains might not be equivalent in their antigenic structure. The serologic activity was entirely recovered on a characteristic additional peptide (molecular weight 20000) obtained from pα1‐chain after collagenase digestion. Reduction and alkylation of this peptide, but not alkylation alone, completely destroyed the antigenic activity which, on the other hand, was quite stable towards the action of various proteases. Antigenic determinants specific for the collagen α1‐chain of normal calf skin could also be demonstrated in the pα1‐chain except the one located in the N‐terminal pentapeptide sequence.

In indirect immunofluorescence tests antibodies specific for dermatosparatic collagen were shown to react with collagen fibers of skin and the interstitial connective tissue of kidney and muscle of normal calves. No staining of glomerular basement membranes was observed. Strong cross‐reaction with human but not with rat tissues was obtained. These results suggest that the extended collagen molecules found in dermatosparactic calves are normal tissue constituents which in all probability are identical to the biosynthetic precursor, procollagen.
Regulation of Pyruvate Metabolism in Rat‐Liver Mitochondria by K+ and Pi
Authors: Schaefer Phillip C. , Veneziale Carlo M.
Published: March 2005

The regulation of pyruvate metabolism in isolated rat‐liver mitochondria was studied in six media of varying ionic composition. In media containing low concentrations of Pi (<10 mM), 12 to 62% of the pyruvate was metabolized to citrate and malate; in media having high Pi concentrations (> 40 mM), pyruvate was metabolized exclusively to acetoacetate. Similarly, low K+ concentrations (9 mM) stimulated formation of citrate and malate from pyruvate when the Pi concentration was low but not when it was high. Inclusion of malate, as a source of oxaloacetate, in media which did not permit pyruvate alone to be metabolized to citrate and malate, eliminated accumulation of acetoacetate and caused pyruvate to be metabolized to citrate exclusively. Thus, it is possible that high Pi concentrations or high K+ concentrations inhibited pyruvate carboxylase but not citrate synthase. The dietary state (fasted or fed) of the rat prior to death affected the absolute or relative activities of these two path ways of pyruvate metabolism.
Electron‐Transport Chains of Escherichia coli
Authors: Haddock Bruce A. , Schairer Hans U.
Published: March 2005

The properties of a 5‐aminolaevulinic acid synthetase‐deficient mutant of Escherichia coli K12Ymel grown aerobically or anaerobically in the presence or absence of 5‐aminolaevulinic acid are described. In the absence of 5‐aminolaevulinic acid no detectable cytochromes are produced, respiration rates are lowered, and the concentration of menaquinone in the cell is increased. The absence of 5‐aminolaevulinic acid from the growth medium has no apparent effect on the Mg2+ dependent ATPase activity or on the concentrations of ubiquinone and flavoprotein(s) in the cell.

Two methods for the reconstitution of respiration by cells grown aerobically or anaerobically in the absence of 5‐aminolaevulinic acid are described. With intact cells, reconstitution is achieved in 3 h at 37 °C and is dependent on the presence of 5‐aminolaevulinic acid and a fermentable carbon source. With cell‐free extracts, respiration can be reconstituted by the addition of haematin in 15–20 min at 37 °C provided that ATP is also present.

It is concluded that the reconstitution of respiration by both methods is achieved in the absence of protein synthesis, indicating that the apoproteins of cytochromes, involved in electron transport with oxygen as terminal electron acceptor, are synthesized and incorporated into the membrane in the absence of haem synthesis.

From an examination of the cytochromes produced during the reconstitution experiments, it is suggested that two membrane‐bound electron transport systems using oxygen as terminal electron acceptor can co‐exist in E. coli. The first involving cytochrome b556 with cytochrome o as terminal oxidase is the system normally present under aerobic growth conditions, and the second is induced under conditions when the concentrations of NADH and ADP in the cell are likely to be high and involves cytochrome b558 with cytochrome a2 as the terminal oxidase.
Sex Dependency of Steroid Retention in Rat‐Liver Slices Incubated with Testosterone
Authors: Ghraf Rüdiger , Lax E. Rodney , Hoff H.‐Georg , Schriefers Herbert
Published: March 2005

The hepatic retention of steroids which occurs during the incubation of [4‐14C]testosterone with rat liver slices is demonstrated to be a sex‐specific phenomenon. After an incubation time of 30 min, slices from female animals can be shown to have retained significantly more [4‐14C] steroid than slices from male animals. The tissue/medium distribution ratio is 13 for the males and 30 for the females.

Δ4‐5α‐Hydrogenase activity of the liver tissue can be regarded as the driving force for the retention of steroids derived from testosterone turnover. Namely, the steroid retention is positively correlated with the testosterone hydrogenation rate. The Δ4‐hydrogenating activity of the female exceeds that of the male by a factor of 1.73; this is the same factor by which the hepatic retention of the female exceeds that of the male.

In contrast, there is an inverse correlation between the production rate of hydroxylated C19O2‐metabolites and the extent of retention. When compared to the female animal, slices from the male possess a higher hydroxylating activity accompanied by a much lower retention.

The majority of metabolites isolated from tissue have a retention which is proportional to their production rate; however, the proportionality factor varies from metabolite to metabolite. Thus, for each individual metabolite, the slice is able to maintain a specific concentration gradient against the medium. For these metabolites the retention is sex‐specific only in so far as their production rate is sex‐specific.

The retention of testosterone and androstenedione is independent of their production rate. They are found in the incubation medium of both sexes, but only in the tissue of males.
Multiple Molecular Forms of Pyruvate Kinase from Mucor rouxii
Authors: Friedenthal Myriam , Roselino Eduardo , Passeron Susana
Published: March 2005

Pyruvate kinase from Mucor rouxii constitutes a family of three isoenzymes which can be separated in DEAE‐cellulose columns. These have been named type I, II and III. They also have different electrophoretic mobility in polyacrylamide gels. Pyruvate kinases type I, II and III were partially purified by ion‐exchange chromatography. Antisera were prepared against types I and III. The antigenic behaviour of the three isoenzymes supported the hypothesis on the hybrid nature of pyruvate kinase type II and confirmed the identity of pyruvate kinase from yeast‐like cells and type I from mycelium.

The filamentous form contains the three types, none of them being constitutive and the proportion of each depends on the concentration and on the nature of the carbohydrate in the growth medium. Under gluconeogenic conditions of growth type III predominantes while in any condition in which aerobic glycosis is favoured type I is the dominant form.

Aerobic or anaerobic yeast‐like cells which display a fermentative mode of hexose utilization contain exclusively the type I isoenzyme.

Addition of glucose or xylose to a 17‐h‐old cultures devoid of carbohydrates leads to the reappearance of pyruvate kinase I and II activities. Experiments with cycloheximide and actinomycin D indicate that this process requires protein synthesis.

Results of preincubation of mixtures of extracts under several conditions indicate that the system of three pyruvate kinases does not represent proteolytic degradation of a single enzyme or interconvertible forms of the same enzyme.
Compartmentation of Orthophosphate and Adenine Nucleotides in Human Red Cells
Authors: Till Uwe , Köhler Wolfram , Ruschke Irene , Köhler Agathe , Löusche Wolfgang
Published: March 2005

Human red cells were incubated in plasma under physiological conditions and labelled with 32Pi. After separating the cells by Sephadex columns, the specific radioactivities of cellular Pi, [β‐32P]ADP, [β‐32P]ATP, [γ‐32P]ATP and 2,3‐diphosphoglycerate were estimated. From the third to the eighth hour after labelling, the specific radioactivities of cellular Pi and adenine nucleotides stayed at 20% of the specific radioactivity of plasma Pi without any tendency to equilibrate with the precursor. The specific radioactivity of 2,3‐diphosphoglycerate rose linearly with time and surpassed that of cell Pi and adenine nucleotides 5 h after labelling. A compartmentation of cellular Pi, ADP and ATP is suggested. It was confirmed experimentally and by means of computer simulation. Of the intracellular Pi, ADP and ATP, about 64% do not participate in tracer exchange, probably because of being bound to hemoglobin.
Isolation and Properties of Abrin: a Toxic Protein Inhibiting Protein Synthesis
Authors: Olsnes Sjur , Pihl Alexander
Published: March 2005

A new procedure is reported for the purification of the toxic protein abrin and for the isolation of its two constituent peptide chains.

Abrin was extracted from Semen jequiriti and purified by chromatography on a DEAE‐cellulose column and on a Sepharose‐4B column. The purified toxin behaved as a single protein on polyacrylamide‐gel electrophoresis. The LD50 dose in mice was about 70 ng indicating that the preparation was about 6‐times as toxic as the most active abrin previously described.

After reduction of the toxin with 2‐mercaptoethanol in the presence of galactose the two constituent peptide chains of abrin were isolated by chromatography on a DEAE‐cellulose column.

The smaller peptide, denoted the A‐chain, inhibited strongly protein synthesis in a cell‐free system from rabbit reticulocytes, whereas the larger peptide, the B‐chain, lacked this ability.

Human erythrocytes pretreated with intact abrin or with B‐chain were agglutinated by an antiserum against abrin, whereas erythrocytes pretreated with A‐chain were not agglutinated under these conditions.

The results indicate that the toxic action of abrin is associated with the A‐chain and that the B‐chain functions as a “carrier” moiety necessary for binding of the toxin to the cell surface.
Killing of 32P‐Labelled Single‐Stranded‐RNA Bacteriophage MS2
Authors: Lupker Jan H. , Glickman Barry W. , Rörsch Arthur , Bosch Leendert
Published: March 2005

The killing of highly 32P‐labelled bacteriophage MS2 by transmutation was studied. The efficiency of killing found was 0.60 ± 0.08. This value is significantly lower than the value expected for a single‐stranded bacteriophage. These results can be interpreted by assuming either that the MS2 RNA is replicated with the breaks caused by radiophosphorus decay, or that the breaks are repaired before replication.
Intermolecular Cross‐Links of Collagen
Authors: Zimmermann Bodo K. , Timpl Rupert , Küuhn Klaus
Published: March 2005

The present communication provides experimental proof for involvement in intermolecular cross‐links of the nonhelical area at the carboxy‐terminal end of the α1‐chain of collagen. Compared to soluble collagen, the content of α1‐CB6, the carboxy‐terminal peptide of the α1‐chain, was significantly low in cyanogen bromide digests of highly cross‐linked insoluble collagen from calf achilles tendon collagen. Approximately 50% of the α1‐CB6 could be identified by serological techniques within a peptide fraction of molecular weight approximately 50000. In this fraction, α1‐CB6 was thought to be cross‐linked by intermolecular bonds to certain cyanogen bromide peptides, i.e.α1‐CB7, α1‐CB8 and α2‐CB4. These intermolecular bonds were cleaved by brief pepsin treatment such that free α1‐CB6 was liberated by subsequent cyanogen bromide digestion. On the basis of the amino acid sequence of the α1‐chain presently known and the arrangement of the collagen within the fibrils, putative cross‐link regions were identified within the helical area which may cross‐link with lysine‐derived aldehydes present in the nonhelical amino‐ and carboxy‐terminal areas of adjacent molecules.
A Study of the Template Properties of Chromatin for DNA Polymerase I and of the Effects of Ionising Radiation
Authors: Itzhaki Ruth F. , Saffhill R.
Published: March 2005

The template activity of rat thymus chromatin for Escherichia coli DNA polymerase I has been found to amount to only 6% of that of pure DNA. Assays have been carried out on chromatin in 0.05 M NaCl, on a chromatin · polylysine complex, on the soluble and insoluble fractions of chromatin in the assay system and on DNA prepared from chromatin. The results suggest that the low activity is due only to a small extent to the insolubility of the chromatin in the assay system. Instead, it is proposed that initiation is restricted to certain “accessible’ DNA zones in chromatin.

After γ‐irradiation of the chromatin, the template activity increases with increasing dose up to a certain level and then decreases. It is therefore proposed that both active and inactive binding sites for DNA polymerase are formed on irradiation. Assays on a chromatin · polylysine complex indicate that the new initiation sites occur in the “accessible” zones.
Détermination d'un indice d'affinité des stéroïdes pour les protéines sériques, fondée sur une méthode de mesure de la liaison à l'équilibre
Authors: Goertz Guy R. , Guérin Marie A. , Crépy Odette C. , Longchampt Jacques E. , Jayle Max F.
Published: March 2005

A method has been developped to compare the competition between various unlabelled C19 and C18 steroids and [3H]testosterone for binding, during pregnancy, to human serum proteins. This method is based on equilibrium dialysis, according to the technic of Pearlman and Crépy, using Sephadex G‐25 in a batchwise fashion. Bound and unbound steroid may be measured without disturbing equilibrium and, in this way, the different rates of dissociation of complexes do not interfere with the measurement.

The partition coefficients K' of unlabelled and unbound steroid between the Sephadex‐external and internal volumes have also been determined.

Ten steroids have been studied by this method. Displacement of radioactive testosterone bound to serum proteins by increasing amounts of unlabelled steroids is measured. In order to get a linear relationship between the two parameters, graphs are plotted according to the method of Rodbard and Rees Midgley. Logit is plotted against the logarithm of unbound steroid in the external volume (Sue), where B = 100 × Sb*/Sb0*, Sb* being the still‐bound [3H]testosterone after addition of unlabelled steroid and Sb0* the total bound [3H] testosterone without addition of unlabelled steroid.

In order to compare the relative affinities of the steroids studied, a “displacement index” is determined. It is expressed as the ratio of unlabelled testosterone (ng) to the test substance (ng) required to decrease the bound [3H]testosterone to 50% of its initial value. The results thus obtained are similar (with the exception of 5‐androstenediol) to those described by Murphy, especially for 19 nor‐5‐androstenediol, which is the only 19 nor‐steroid showing a very high affinity for pregnancy human serum proteins. The displacement index, using testosterone as reference steroid, is higher for dihydrotestosterone, 5α‐androstanediol, 4‐androstenediol and 5‐androstenediol and lower for dehydroepiandrosterone, 19 nor‐testosterone, estradiol and 16‐hydroxy‐C19 compounds.
The Effect of Trimethoprim on RNA Synthesis in Relaxed and Stringent Escherichia coli Strains
Authors: Kaplan Ruth , Silman Nitza
Published: March 2005

Trimethoprim inhibits growth, protein synthesis, and RNA synthesis in both stringent and relaxed cells grown in unsupplemented minimal medium. A preferential inhibition of RNA synthesis in rel+ cells is encountered when these cells are grown in media supplemented with purines. Trimethoprim decreases the quantity of purine nucleoside triphosphates in the acid‐soluble pool of both relaxed and stringent cells grown in minimal medium. When trimethoprim is added to cells grown in the presence of guanine, it decreases the purine nucleoside triphosphate pool in the stringent cell only.
Phenol Hydroxylase from Yeast
Authors: Neujahr Halina Y. , Gaal Andràs
Published: March 2005

An enzyme, able to carry out an NADPH‐dependent hydroxylation of monocyclic phenols, was purified 20–30‐fold from Trichosporon cutaneum grown on phenol or resorcinol as a major carbon source. The purified enzyme was homogeneous upon analytical disc electrophoresis.

The enzyme is a bright‐yellow protein with an absorption spectrum typical of flavoproteins. Its molecular weight is 148000, and its estimated FAD content is approximately 1 mole per mole enzyme. The purified enzyme has essentially the same broad substrate specificity as crude preparations. In addition to phenol it also hydroxylates the three isomeric diphenols and a number of phenol derivatives to their corresponding o‐diols.

K
m values for the three substrates of the holoenzyme are all of the order 10 μM : Km (phenol) = 18 μM, Km(NADPH) = 71 μM. The absorption spectrum of the holoenzyme is modified in the presence of phenol.

FAD could be resolved from the purified enzyme by preparative disc electrophoresis. Reassembly of the holoenzyme required SH‐groups.

The enzyme activity is unaffected by chelators of iron and copper but it is inhibited by heavy metals. The inhibition by p‐chloromercuribenzoate is readily reversed by dithiothreitol.

Among oxidizing agents, hydrogen peroxide and peroxidase depressed the enzyme activity, whereas catalase was without effect. Among reducing agents ascorbate depressed enzyme activity. Sodium dithionite and sodium borohydride bleached the enzyme with concomitant loss of activity. After reduction with dithionite, the enzyme was rapidly re‐oxidized, re‐gaining its original activity. After reduction with borohydride re‐oxidation was very slow. However, the enzyme could be re‐activated by incubation with FAD.

The enzyme is very sensitive to inorganic salts, nitrogen bases and detergents. Chloride is quite deleterious whereas phosphate seems to stabilize the enzyme.
Molecular‐Weight Analysis of Helix pomatiaα‐Hemocyanin in Guanidine Hydrochloride, Urea and Sodium Dodecylsulfate
Authors: Brouwer Marius , Kuiper Harry A.
Published: March 2005

The molecular weight of the polypeptide chain(s) of Helix pomatia hemocyanin has been studied after denaturation and reduction of the protein in 6 M guanidine hydrochloride, 8 M urea and 1% sodium dodecylsulfate. The sedimentation equilibrium technique, which is a method for the measurement of mass directly, gives a value for the molecular weight of approximately 265000. Since methods for molecular weight determinations depending upon random coil behavior of polypeptide chains, such as gel chromatography in 6 M guanidine‐HCl, measurement of the intrinsic viscosity and s0 value in 6 M guanidine‐HCl, give the same value for the molecular weight as sedimentation equilibrium, we may conclude that 6 M guanidine‐HCl denatures hemocyanin to a randomly coiled state. Since hemocyanin of Helix pomatia binds one oxygen molecule per 50000 mol. wt, this polypeptide chain contains 5 or 6 active sites.
Animal DNA‐Dependent RNA Polymerases
Authors: Meilhac Madeleine , Chambon Pierre
Published: March 2005

The rifamycin derivative, AF/O13, was used to investigate the possible specificity of the sites where RNA synthesis is initiated on calf thymus DNA by the A1 and B animal RNA polymerases. It was found that the sites, which confer resistance to the inhibitor upon calf thymus AI, calf thymus B and Escherichia coli holoenzyme RNA polymerases are different; on the other hand, no difference is noticed either between calf thymus BI and BII enzymes, or between calf thymus and rat liver B enzymes. Studies of incorporation of nucleoside [γ‐32P]triphosphates indicate that there is a very limited number of DNA sites which can confer resistance to AF/013 on the calf thymus B enzymes. Calculations led to a figure of about 50000 sites per haploid genome. Studies ofthe ap‐ pearance of AF/013 resistance with various templates suggest that calf thymus enzyme AI, like E. coli core RNA polymerase, initiates RNA synthesis mainly a t nicks, ends or on denatured DNA regions, whereas the B enzymes initiate on double‐stranded sites.
Peptide Inhibitors and Activators of Carboxypeptidase B
Authors: Zisapel Nava , Kurn‐abramowitz Nurith , Sokolovsky Mordechai
Published: March 2005

A number of peptides containing d‐residues at the penultimate or C‐terminal position were tested for their ability to affect the carboxypeptidase‐B‐catalysed hydrolysis of basic and non‐basic peptides and esters. The effect of these modifiers, i.e. activation or inhibition is dependent on the nature of the modifier. The factors that determine the activity of the modifiers are: (a) the length of the peptide; (b) the basic or non‐basic nature of the C‐terminal and/or the penultimate residue and the optical configuration of these residues; (c) the substrate tested. The wide variations observed in the mode of activation and/or inhibition and the lack of correspondence between the Ki values is compatible with previous data on smaller inhibitors and larger peptide substrates, that the peptide and ester substrates of carboxypeptidase B occupy different loci on the enzyme.
The Selective Extraction of Histone Fractions from Deoxyribonucleoprotein
Authors: Bolund Lars A. , Johns Ernest W.
Published: March 2005

Three methods are described for quantitative depletion of calf thymus and chicken erythrocyte chromatin of various histone fractions. All methods involve dissociation of the histone fractions in question from chromatin in the presence of the ion‐exchange resin AG50W X 2 which, having strongly bound the free histones in solution, is easily removed from the chromatin. The depleted chromatins do not appear to be denatured.

Method 1. In 0.45–0.65 M NaCl, 0.05 M sodium phosphate buffer pH 7.0, histone F1 is quantitatively and selectively removed from calf thymus chromatin. In 0.55–0.65 M NaCl, 0.05 M sodium phosphate buffer pH 7.0 and a limiting amount of ion‐exchange resin, histone F2C is quantitatively and selectively removed from chicken erythrocyte chromatin. Histone F1 can be quantitatively and selectively removed from chicken erythrocyte chromatin by repeated extraction with 0.5 M NaCl pH 7. By further depletion with resin in 0.55–0.65 M NaCl, 0.05 M sodium phosphate buffer pH 7.0, chicken erythrocyte chromatin can be quantitatively and selectively depleted in both histones F1 and F2C.

Method 2. In 0.25–0.45M NaCl, 0.05 M sodium phosphate buffer, 4 M urea pH 7.0, histones F1, F2B and F2A2 are quantitatively and selectively removed from calf thymus chromatin.

Method 3. In 0.35 M NaCl, 30% ethanol, 4 M urea pH 7.0, an almost complete removal of histones F2A1 and F2A2 from calf thymus chromatin is achieved. In 0.35 M NaCl, 300% ethanol, 5–6 M urea pH 7.0, histone F3 is removed as well.
Spectrometric Investigation of Thaumatin I and II, Two Sweet‐Tasting Proteins from Thaumatococcus daniellii Benth
Authors: Korver Onno , van Gorkom Maarten , van der Wel Henrik
Published: March 2005

The sweet‐tasting proteins, thaumatin I and II, isolated from the fruits of Thaumatococcus daniellii Benth, both lose their sweetness on heating. Circular dichroism and proton magnetic resonance measurements indicate that the proteins undergo reversible conformational changes as the temperature is increased. Then at a certain temperature, which varies with pH, irreversible heat denaturation occurs. At pH 5.0, the native conformation is most stable and the conformational change is reversible up to 75 °C even on prolonged heating.

We concluded from the spectroscopic data that the proteins are flexible molecules and that tyrosine residues and at least one disulphide chromophore are involved in the conformational change. The results justify the assumption that the irreversible heat‐induced conformational transition is responsible for the loss of sweetness of the proteins.
A Comparative Study of Pig and Sheep‐Brain Glutamine Synthetases: Tryptic Peptides and Thiol Groups
Authors: Rajagopal Rao Desiraju , Beyreuther Konrad , Jaenicke Lothar
Published: March 2005

A comparative study of the tryptic peptides of glutamine synthetase from pig and sheep brain has been made. Individual peptide maps of S‐[14C]carboxamidomethylated enzymes as well as mixed peptide maps of S‐[14C]carboxymethyl and S‐[3H]carboxymethyl enzymes reveal close similarities of the enzyme. The S‐carboxyamidomethylated tryptic peptides have been separated by column chromatography. The amino acid composition of several of these peptides as well as their N‐terminal groups have been determined. Some of the tryptic peptides from both sources have very similar composition and properties. The major peptides obtained by cyanogen bromide cleavage of both the enzymes have identical N‐terminal groups.

The various thiol groups of both the pig and sheep brain glutamine synthetase have similar reactivity towards iodoacetic acid and iodoacetamide. The [14C]alkylated peptides from both sources have very similar properties. The pig brain enzyme is more sensitive to inhibition by N‐ethylmaleimide than by iodoacetamide. The inhibition can be prevented by ATP and Mg2+. Some thiol group or groups are located close to the active centre and are involved in the formation of active conformation rather than binding of ATP.
Activation of Human Plasminogen by an Insoluble Derivative of Urokinase
Authors: Wiman Björn , WallÉN Per
Published: March 2005

Activation of plasminogen was performed by perfusing a plasminogen solution through a small column loaded with insolubilized urokinase. The effluent was immediately collected in an excess of plasmin inhibitor (p‐nitrophenyl‐p‐guanidino‐benzoate). This provides a practically instantaneous inhibition of the plasmin formed and autocatalytic degradation is thus reduced to a minimum. Different degrees of activation are obtained by changing the perfusion rate.

Dodecylsulfate‐polyacrylamide electrophoresis, amino acid and NH2‐terminal amino acid analyses show that the activation of human plasminogen involves cleavage of at least two peptide bonds, the first event being the release of a peptide of 64 amino acid residues from the NH2‐terminal end. An intermediate inactive product with a molecular weight of about 86000 and methionine as NH2‐terminal amino acid is formed. Cleavage at the second site results in formation of a molecule with two chains connected by disulfide bridges. One chain which has a molecular weight of about 63000 is very sensitive to proteolytic degradation in its NH2‐terminal part. However, there is evidence that methionine is the true NH2‐terminal amino acid of this fragment. The other chain has a molecular weight of about 25000 and has valine as NH2‐terminal amino acid.
Mitochondrial Translation Products before and after Integration into the Mitochondrial Membrane in Neurospora crassa
Authors: Michel Rainer , Neupert Walter
Published: March 2005

Nascent translation products on mitochondrial ribosomes were selectively labeled in vivo in the presence of cycloheximide with radioactive leucine. They were isolated together with the ribosomes.

The labeled polypeptides show a high tendency to aggregate and can only be kept in solution in the presence of detergents such as dodecylsulfate. Also, mitochondrial ribosomes carrying nascent peptide chains easily form aggregates.

The polypeptides adhering to mitochondrial monomeric ribosomes differ from those adhering to polymeric ribosomes. Gel electrophoresis in the presence if dodecylsulfate shows for the peptidyl transfer RNA products at the monomer, an apparent molecular weight of 27000. After removing the transfer RNA, an apparent molecular weight of less than 10000 is registered. The peptides adhering to mitochondrial polymeric ribosomes display a broad range of apparent molecular weights. In contrast, translation products associated with cytoplasmic monomeric and polymeric ribosomes all show quite disperse molecular weights.

Using gel‐chromatographic analysis no difference in the elution characteristics between translation products associated with mitochondrial monomeric and polymeric ribosomes was found. In both cases apparent molecular weights of about 11000 were obtained.

A kinetic study of the appearance of mitochondrial translation products in the mitochondrial membrane was carried out. A conversion process of products with lower apparent molecular weights to those with higher apparent molecular weights is observed. This suggests that mitochondrial ribosomes form polypeptides which are modified during or after integration into the membrane.

The hypothesis is discussed that mitochondria possess their own system of transcription and translation, because the hydrophobic nature of the translation products makes it necessary that they are formed inside the inner mitochondrial membrane, into which they are integrated.
The Reaction of Hexokinase with Equilibrated d‐Glucose
Authors: Wurster Bernd , Hess Benno
Published: March 2005

In the hexokinase reaction with equilibrated d‐glucose (36.2%, α‐d‐glucopyranose and 63.8%β‐d‐glucopyranose) at saturating concentrations of ATP‐Mg, the ratio of phosphorylation of α‐d‐glucose and β‐d‐glucose was investigated.

It is shown that, knowing the Michaelis constants Km,(α), Km,(β), and the maximum velocities V(α), V(β), of hexokinase with α‐d‐glucose and β‐d‐glucose respectively, one can calculate the ratio of phosphorylation of α‐d‐glucose and β‐d‐glucose starting from the equilibrium mixture of d‐glucose.

In addition, the ratio of generation of α‐and β‐d‐glucopyranose‐6‐phosphate in the hexokinase reaction was determined from steady‐state analysis of the hexokinase/glucose‐6‐phosphate dehydrogenase system with equilibrated d‐glucose. The experimentally obtained and the calculated values were in agreement.
On the Regulation of the Activity of Immobilized Enzymes
Authors: Gestrelius Stina , Mattiasson Bo , Mosbach Klaus
Published: March 2005

Glucose oxidase, hexokinase, trypsin and urease were entrapped, either separately or together, within polyacrylamide particles, using a bead polymerization technique. The pH optima, for the immobilized enzymes in 5 mM buffer, were displaced compared to those of the enzymes free in solution.

Thus the pH optimum for trypsin activity towards benzoyl‐l‐arginine ethyl ester shifted 1.3 pH unit (to pH 9.6) and the pH optimum for glucose oxidase towards glucose shifted 0.3 pH units to the alkaline side (to pH 6.9). Reversely, urease activity towards urea leading to consumption of protons shifted its optimum 0.4 unit to the acidic side (to pH 5.8).

The effect on glucose oxidase activity of pH changes caused by activity of coentrapped trypsin or urease was studied. It was found that glucose oxidase activity in the alkaline region was stimulated during simultaneous trypsin activity, whereas at pH values below that of the optimum for glucose oxidase inhibition occurred. A reversed effect was measured during urease activity. At pH 8.6, for example, glucose oxidase activity was increased by a factor of three to 75% of its activity found at pH optimum due to trypsin activity. On the other hand, at pH 6.0, i.e. below the optimum, it increased by a factor of two to about 80% due to urease activity. Both simultaneous trypsin or urease activity gave distorted “two‐peak” pH‐activity profiles of glucose oxidase.

The effect of protons produced by trypsin on the simultaneous activities of glucose oxidase and hexokinase, both competing for the same substrate, glucose, was studied with a gel containing all three enzymes. It was found that the amount of glucose converted per minute by each enzyme reaction was effected. In one case, at pH 8.5 in the absence of trypsin activity, about one seventh of the glucose consumption was due to hexokinase activity. On addition of benzoyl‐l‐arginine ethyl ester, hexokinase activity decreased almost to zero, while glucose oxidase activity was stimulated, resulting in practically all conversion being due to the latter.
Isolation and Characterization of Cytoplasmic l‐Glycerol‐3‐Phosphate Dehydrogenase from Rabbit‐Renal‐Adipose Tissue and Its Comparison with the Skeletal‐Muscle Enzyme
Authors: Warkentin Donald L. , Fondy Thomas P.
Published: March 2005

Cytoplasmic NAD‐linked l‐glycerol‐3‐phosphate dehydrogenase was isolated from rabbit renal adipose tissue in a preparation that was apparently homogeneous by preparative isoelectric focusing and polyacrylamide‐disc‐gel electrophoresis. The adipose tissue and skeletal muscle enzymes were indistinguishable by disc‐gel electrophoresis, immunological comparisons, apparent Km, values for substrates and coenzymes, isoelectric point in preparative isoelectric‐focusing runs, and apparent molecular weight and Stokes radius on polyacrylamide‐gel‐filtration columns. The ultraviolet spectrum of the adipose tissue enzyme was identical to that of the muscle enzyme including the broad maximum at 275 nm indicative of the non‐protein component characteristic of the muscle enzyme. Crude homogenates of rabbit adipose tissue and muscle electro‐focused in polyacrylamide gels and stained for glycerol‐3‐P dehydrogenase activity showed four major zones present in slightly different proportions in the two homogenates, but fully superimposable in runs on mixed tissue homogenates.

Comparison of amino acid composition of the adipose tissue enzyme with analyses of the muscle done in five different laboratories showed that the adipose tissue enzyme cannot be distinguished from the muscle enzyme by this criterion. Similar comparison of the adipose tissue and muscle enzymes with the liver enzyme suggests that the liver enzyme is not identical to the muscle‐adipose form of the enzyme. Possible similarity in physiological role for the muscle‐adipose form of glycerol‐3‐P dehydrogenase in these two tissues and a rationale for their absence in rapidly growing cancer cells is suggested.
Studies on the Bacteriophage MS2
Authors: Haegeman Guy , Fiers Walter
Published: March 2005

After partial hydrolysis with T1‐RNAase of 32P‐labelled MS2 RNA and electrophoretic separation on different types of polyacrylamide gels, it is possible to isolate a fragment containing the nucleotide sequence G‐U‐U‐U‐Gp in combination with A‐U‐Gp. The complete primary structure of the 73‐nucleotide fragment is reported here. In addition to the usual techniques of sequence determination, the characterization involved identification of terminal oligonucleotides by exo‐nuclease treatment. The sequence is of interest as it shows a striking similarity to some ribosomal binding sites.
2‐Amino‐2‐deoxyhexuronic Acid: a Constituent of Shigella sonnei Phase I Lipopolysaccharide
Authors: Romanowska Elżbieta , Reinhold Vernon
Published: March 2005

2‐Amino‐2‐deoxyhexuronic acid was found to constitute the main sugar component of the specific part of Shigella sonnei phase I lipopolysaccharide. The sugar was isolated from the lipopolysaccharide by acid hydrolysis and purified on a Dowex 50×8 column. From the chemical reactivity, gas chromatography‐mass spectrometry, paper chromatography, high‐voltage electrophoresis and deamination experiments it has been concluded that the isolated compound is a 2‐amino‐2‐deoxyhexuronic acid.
Enzymatic Degradation of O‐Antigenic Lipopolysaccharides by Coliphage Ω8
Authors: Reske Konrad , Wallenfels Barbara , Jann Klaus
Published: March 2005

Incubation of the lipopolysaccharide from Escherichia coli O8: K27–: H– with coliphage Ω8 resulted in enzymatic degradation of the polysaccharide moiety. Investigation of the water‐soluble products showed that the polysaccharide was split into the trisaccharide repeating unit (α‐mannosyl‐1,2‐α‐mannosyl‐1,2‐mannose), its dimer, trimer and tetramer. As in the polysaccharide the trisaccharides are α‐1,3‐linked, the action of coliphage Ω8 may be described as that of an endo‐α‐1,3‐mannosidase.

Coliphage Ω8 also splits the lipopolysaccharides of E. coli O93 and Klebsiella O5 in the same fashion. These lipopolysaccharides are also receptor of coliphage Ω8; coliphage Ω8 is adsorbed on the respective bacteria and the lipopolysaccharides inhibit the infection of E. coli O8 with coliphage Ω8. In addition, all lipopolysaccharides susceptible to the enzyme action of coliphages Ω8 cross‐react serologically with that of E. coliΩ8. Two polysaccharides from E. coli strains O9 and O100 which do not cross react with O8 material are not receptors of coliphage Ω8 and are not degraded by it.
Association‐Dissociation Behaviour and Hydrodynamic Properties of Apoferritin Monomer and Dimer
Authors: BjÖRK Ingemar
Published: March 2005

The association behaviour of a homogeneous preparation of apoferritin has been investigated. Contrary to studies by other authors on less pure material, no evidence for a reversible association of the protein was detected in sedimentation equilibrium, light scattering or sedimentation velocity experiments. Correspondingly, no dissociation of an isolated dimer of apoferritin was observed. Hydrodynamic parameters, such as frictional ratio and intrinsic viscosity, indicate that both apoferritin monomer and dimer have a somewhat larger degree of hydration or a slightly more extended or irregular shape than most typical globular proteins. The solvent, presumably trapped inside the protein shell, cannot alone account for this behaviour.
Studies on the Mechanism of the Oxygen‐Induced Synthesis of Cytochrome Oxidase in Yeast
Authors: Schmitt‐Verhulst Anne‐Marie , Bex Françoise , Sels André A.
Published: March 2005

At concentrations where benzimidazole inhibited partially total protein synthesis in yeast, the drug also inhibited protein synthesis mediated by cytoplasmic ribosomes and by isolated mitochondria in vitro.

Except for cytochrome oxidase, the effects of benzimidazole on the anabolism of hemoproteins were also partial and unspecific; the induction by oxygen of the particulate cytochromes b and c1, the synthesis of which is determined by the obligate co‐operation of both protein‐synthesizing systems, was not affected more than the induction of catalase, an extramitochondrial hemo‐protein, which is synthesized exclusively in cytoplasm. The drastic inhibition of cytochrome oxidase was the consequence of a specific blocking of cytochrome (a, a3) production.

The delayed adaptation to oxygen, following the exclusion of benzimidazole, was characterized by its exceptionally high rate and correlated with the simultaneous increase of hemo‐protein (a, a3) content and the development of cytochrome oxidase activity; the latter activity ruled respiration rate. The process was largely unsensitive to the concerted actions of cycloheximide and chloramphenicol; under identical conditions, the synthesis of other hemoproteins was immediately stopped.

The reconstitution of cytochrome oxidase in vivo at the expense of the double set of cytoplasmic and mitochondrial adaptation products, which were simultaneously accumulated, was established by reference to the specific effects of cycloheximide and chloramphenicol. As the prevailing effect of benzimidazole was operationally to switch off the assembly of cytochrome (a, a3), it is inferred that the drug will afford new experimental approaches in further studies of cytochrome oxidase anabolism.
The Mechanism of Action of Methionyl‐tRNA Synthetase from Escherichia coli
Authors: Blanquet Sylvain , Iwatsubo Motohiro , Waller Jean‐Pierre
Published: March 2005

Titration of native methionyl‐tRNA synthetase from Escherichia coli with tRNAfMet and tRNAmMet leads to 37% and 38% quenching of the enzyme fluorescence respectively. The quenching curves for the two tRNAs are not additive. Analysis of the binding data indicates the presence of a single site for tRNA per mole enzyme, with a three‐fold higher affinity for tRNAmMet. The tRNA binding parameters do not vary significantly in the presence of a saturating concentration of methionine. On the other hand, titration of the enzyme · methioninyl‐adenylate complex (two moles of the ligand bound/mole enzyme) with tRNAfMet and tRNAmMet, leads to an extensive decrease of fluorescence, from an initial level of 134% to 40% and 35%, respectively, indicative of a major structural or conformational change of the enzyme. Analysis of the quenching curves reveals two distinct binding processes, corresponding to occupation of two tRNA sites on the enzyme.

Requirements for optimal interaction between enzyme and tRNA were investigated. Magnesium ions are essential for complex formation. Maximal interaction is attained at 1 mM and 4 mM MgCl2, respectively, for tRNAmMet and tRNAfMet. However, close to 40 mM MgCl2 are required to reach equivalence of the two tRNA sites which are available in the presence of methioninyl adenylate. Both spermine and spermidine are able to simulate the role of magnesium in the binding of tRNAMet to the free enzyme, but not in the occupation of the second tRNA site on the enzyme · methioninyl‐adenylate complex.

Addition of KCl results in a decrease of the fluorescence quenching produced by tRNA and in a lowering of the affinity for tRNA, both in the presence of MgCl2 and spermidine. Essentially similar quenching levels and affinities are obtained when titration experiments are performed in imidazole‐HCl or Tris‐HCl buffer in the pH range of 6.3 to 8.7. In contrast, in potassium phosphate buffer which inhibits the enzyme‐tRNA interaction, raising the pH leads to a progressive decrease of the fluorescence quenching caused by tRNA, with concomitant lowering of its affinity for the enzyme.

For comparative purposes the interaction of tRNAMet with trypsin‐modified methionyl‐tRNA synthetase was investigated. Analysis of the binding data indicates the presence of a single tRNA site per mole enzyme, even in the presence of saturating amounts of methioninyl adenylate (one mole of this ligand bound per mole enzyme). Furthermore, the magnesium ion concentration required to reach maximal quenching is the same (i.e. 4 mM) in the presence or absence of methionyl adenylate.
Pre‐Ribosomal RNA Synthesis in Isolated Rat‐Liver Nucleoli
Authors: Grummt Ingrid , Lindigkeit Ruth
Published: March 2005

Isolated rat liver nucleoli represent a simple system to study transcription and processing of ribosomal RNA in vitro. These organelles retain their ability to synthesize ribosomal precursor RNA in vitro as measured by the incorporation of nucleotides into RNA. The incorporation of labelled nucleotides is not affected by α‐amanitin or rifampicin AF‐013. Low concentrations of ammonium sulfate (60 mM) stimulate the nucleolar synthetic activity considerably. The (A + U)/(G + C) ratio of nucleolar RNA synthesized in vitro is 0.5–0.55 and resembles that of ribosomal precursor RNA. Discrete peaks of high‐molecular‐weight RNA, widely distributed as to size, appear in analytical sucrose gradients. Molecules with sedimentation coefficients up to about 80 S have been obtained a surprising result in view of the fact that, in general, only 45‐S precursors are found in vivo. The amount of rapidly sedimenting RNA increases with prolonged incubation. It is uncertain whether or not the presence of ribosomal RNA precursor molecules with sedimentation coefficients greater than 45‐S reflects a normal transcription mechanism in the living cell which remains undetected due to rapid processing. It is also possible that isolated nucleoli may have lost some specific factors which in vivo cause the RNA polymerase to terminate the RNA chains at the end of the 45‐S transcription unit.
Microbial Assimilation of Methanol
Authors: Sahm Hermann , Wagner Fritz
Published: March 2005

An NAD‐dependent alcohol dehydrogenase has been isolated from the yeast Candida boidinii grown on methanol. A study of the properties of the enzyme has shown that it is very similar to the alcohol: NAD oxidoreductase from baker's yeast. The alcohol dehydrogenase from Candida boidinii does not catalyze the oxidation of methanol and is constitutively formed.

A mutant, 48, has been isolated which is unable to grow on methanol as sole carbon and energy source. This strain lacks the methanol‐oxidizing enzyme but not NAD‐dependent alcohol dehydrogenase.

Cells grown on glucose or ethanol do not contain the methanol‐oxidizing enzyme. During adaptation from glucose to methanol, enzyme activity appears before growth on methanol begins.

The methanol‐oxidizing enzyme has been partially purified. It catalyzes the oxidation of methanol to formaldehyde and hydrogen peroxide, and is independent of the addition of a hydrogen acceptor.

The prosthetic group of this enzyme is FAD. The molecular weight was calculated to be 600000, one subunit has a molecular weight of 74000.

The optima of pH and temperature for enzyme activity are 7.5–9.5 and 30°C, respectively. It is unstable in acidic pH.

The enzyme is not specific for methanol. It also oxidizes lower primary alcohols. The Km value for methanol is 2.0 mM and that for ethanol 4.5 mM.
Subcellular Location of Superoxide Dismutase in Spinach Leaves and Preparation and Properties of Crystalline Spinach Superoxide Dismutase
Authors: Asada Kozi , Urano Mieko , Takahashi Masa‐aki
Published: March 2005

Localization of superoxide dismutase in spinach chloroplasts was demonstrated by differential and sucrose‐density centrifugations. About half to two thirds of the enzyme in chloroplasts was released by osmotic shock. In peroxisomes, no superoxide dismutase was detected. Disc electrophoresis of chloroplast and supernatant fractions revealed a single band of superoxide dismutase activity corresponding to the purified enzyme. Superoxide dismutase was purified from spinach leaves and prepared in a crystalline state. s020, w was 2.75 S and the molecular weight was approximately 32000 daltons. The enzyme was composed of two subunits of equal size and contained 2 atoms of copper and 2 atoms of zinc per molecule. In addition, visible and ultraviolet spectra, electron paramagnetic resonance spectrum and amino acid composition were similar to these properties of the enzyme isolated from mammalian tissues, Neurospora, green pea and yeast. A possible function of superoxide dismutase in chloroplasts is discussed.