bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Genetic Control of Molybdoflavoproteins in Aspergillus nidulans
Authors: Scazzocchio Claudio , Holl Frederick Brian , Foguelman Ada Isabel 
Published: March 2005

This paper describes the isolation and properties of a class of mutants (apl Ac), constitutive for the enzyme xanthine dehydrogenase. These mutants are superinducible and semidominant. Four independently isolated strains carry constitutive mutations which are allelic.Strains carrying the apl Ac mutation are, as the wild type, repressible by ammonia, and at variance with the wild type repressible by nitrate. The apl Ac mutation suppress mutations at the hx A locus that result in loss of xanthine dehydrogenase, and the ua Y locus that result in non‐inducibility for both xanthine dehydrogenase and urate oxidase, but only for xanthine dehydrogenase. Immunological evidence shows that the apl Ac mutation results in the production of a xanthine dehydrogenase (II) different from the one nom rally induced in the wild type by uric acid (I). Genetic evidence shows that whereas the hx A locus codes for a product specific for xanthine dehydrogenase I the hx B locus codes for a product necessary for both xanthine dehydrogenases I and II and that the cnx loci code for a cofactor present in xanthine dehydrogenase I and II and nitrate reductase. A model is presented by which xanthine dehydrogenase I and II are formed by a core comprising the polypeptide coded by the hx B locus and the cofactor specified by the cnx loci plus another peptide, coded in the case of xanthine dehydrogenase I by the hx A locus, carrying the substrate binding site and specific for each enzyme. The synthesis of the cnx cofactor could be constitutive, whereas the synthesis of the hx B peptide is under two independent systems of control and each of the specific components under a specific system of control. It is proposed that control systems for both xanthine dehydrogenase I and II are of the positive type. In the Appendix the induction of xanthine dehydrogenase II in wild type and mutant strains is described. The isolation and preliminary characterisation of mutants lacking xanthine dehydrogenase II but not xanthine dehydrogenase I is reported. Some of the mutations are allelic with apl Ac mutanta, while another one identifies a new locus coding possibly for the substrate binding site of xanthine dehydrogenase II.
Structure of Globin mRNA and mRNA‐Protein Particles
Authors: Dubochet Jacques , Morel Carlos , Lebleu Bernard , Herzberg Max 
Published: March 2005

The main advantage of dark‐field electron microscopy is to enhance considerably the contrast, of thin biological specimens. It is thus possible to decrease the amount of stain necessary for visualizing macromolecules. Finer details are visible and stain artefacts are reduced. The method has been used for visualizing mRNA and mRNA‐protein particles from rabbit reticulocytes and from duck erythroblasts. Two preparation methods involving no proteins have been used for the specimen preparation: spreading with glycerol and adhesion on positively charged film.Both species of mRNA and mRNA‐protein particles are linear structures about 170 and 220‐nm long, respectively. On the mRNA‐protein particles the proteins are mainly concentrated in 4 to 7 points along the molecule, but we cannot decide if these proteins are selectively bound on secondary structures of the mRNA.
Studies of Glutamate Dehydrogenase
Authors: Koberstein Rudolf , Sund Horst 
Published: March 2005

The detailed investigation of the binding of NAD(P)H to beef liver glutamate dehydrogenase in the presence of the effectors ADP and GTP shows that each oligomer (composed of six identical polypeptide chains) has a total of 18 nucleotide binding sites. They can be subdivided into three classes. A first set of six binding sites, the active sites, is occupied by NAD(P)H with nearly the same affinity for both coenzymes, and accompanied by negative cooperativity between the binding sites within the oligomer. The second set of six binding sites, the nonactive sites, binds the coenzyme less tightly than the first set and ADP competes with NAD(P)H for these sites. The third set of six binding sites, the GTP sites, is responsible for the GTP binding.ADP abolishes the negative cooperativity of the NAD(P)H binding to the active sites and causes a weaker binding of both reduced coenzymes to these sites (KE · ADP, NAD(P)H= 70 μM for NADH and 64 μM for NADPH). Also the reduced coenzymes weaken the binding of ADP to the enzyme. The dissociation constant for the enzyme · ADP complex is found to be 17 μM in the presence and 3 μM in the absence of NAD(P)H. The results are interpreted on the basis of free energy conservation in internal equilibria. The coupling energy ΔFc was calculated to be about 1 kcal/mol.In contrast to ADP/NAD(P)H, GTP and NAD(P)H enhance each other's binding to glutamate dehydrogenase causing positive cooperativity which is stronger in the case of NADH than of NADPH. The Hill coefficient is found to be 2.1 for NADH and 1.8 for NADPH. The nonactive sites are occupied by NADH in the presence of GTP with a dissociation constant of 23 μM indicating a three‐fold enhancement of the affinity of the enzyme for NADH in the presence of GTP. This is not the case with NADPH, where the dissociation constant is about 600 μM. This value is similar to the dissociation constant of the binary complex.The influence of glutamate on the enzyme · NAD(P)H binding is similar to the effect obtained in the presence of GTP; glutamate and GTP, however, are most likely bound to Merent sites. Glutamate induces strong positive cooperativity of three of the active sites within the oligomer characterized by a Hill coefficient of 2.4 for both reduced coenzymes. The extrapolations yield dissociation constants of 2–3 μM for both reduced coenzymes. The nonactive sites are occupied with dissociation constants of 57 μM for NADH and 700 μM for NADPH. These data indicate that glutamate has only a small effect on the binding of the reduced coenzyme to the nonactive site.
Kinetics of the Interaction between Ceruloplasmin and Reducing Substrates
Authors: Gunnarsson Per‐Olov , Nylén Ulf , Pettersson Gösta 
Published: March 2005

Decolourization of the 610‐nm chromophore of ceruloplasmin by ascorbate is due to a ratedetermining second‐order interaction between enzyme and substrate. Apparent rate constants for this process appear to be independent of the oxidation state of the 340‐nm chromophore of the protein, and decrease with pH over the range pH 5–7.Second‐order rate constants for the reduction of ceruloplasmin by various substrates are linearly correlated to the reciprocal value of the corresponding Km‐values, and agree in magnitude with the steady‐state kinetic quotient V/Km. This means that differences in the rate of oxidation of various substrates can be mainly attributed to differences in the rate of reduction of the 610‐nm chromophore and correlated to differences in the ionization potential of the reducing substrates.
X‐Ray Small‐Angle‐Scattering Investigation of a Giant Respiratory Protein: Hemoglobin Tubifex tubifex
Authors: Stöckel Peter , Mayer Adalbert , Keller Rainer 
Published: March 2005

The quaternary structure of hemoglobin of the worm species Tubifex tubifex was investigated by X‐ray small‐angle scattering. The desmeared scattering functions were observed to be similar for oxygenated and deoxygenated species having true radii of gyration of 11.0 and 11.2nm respectively. Comparison of experimental and theoretical scattering curves gives information on the overall shape of the macromolecule in solution. What is found in this way allows the postulation of structural models of uniform electron density which are equivalent in scattering to the macromolecule studied.The experimental scattering curves were compared with scattering curves calculated for various models. The overall shape of hemoglobin Tubifex can be approximated by a hollow cylinder of outer diameter D= 26.0 nm, inner diameter d = 0.3 D and height h = 0.6 D. The scattering functions calculated for hexagonal arrangements of 12 identical spheres of radius 4.5 nm in two superposed 6‐numbered rings, correspond to the experimental scattering curves we have obtained, as well as to electron microscopic observations on hemoglobins from some Annelida.However, the calculated scattering curves of a model of 108 identical spheres arranged in 12 nonamers proposed by Yamagishi and his colleagues in 1966 provided the best simultaneous fit for the radius of gyration and for the sizes and positions of the first and second subsidiary minima and maxima. A model of outer diameter 27.3 nm, inner diameter 9.1 nm, sphere diameter 3.4 nm for the oxygenated species and 3.45 nm for the deoxygenated species and total heights of 16.2 and 16.65 nm respectively is equivalent in scattering to the quaternary structures of the hemoglobin Tubifex molecules.
Proteinase K from Tritirachium album Limber
Authors: EBELING Wolfgang , HENNRICH Norbert , KLOCKOW Michael , METZ Harald , ORTH Hans Dieter , LANG Hermann 
Published: March 2005

The fungus Tritirachium album Limber was grown by submerged fermentation, investigating the conditions for maximal secretion of proteases into the medium. Enzyme secretion starts when the stationary phase of growth is reached, and when the culture medium is depleted of glucose and amino acids, suggesting a catabolite repression of the enzyme.The main proteolytic enzyme was named proteinase with respect to its keratin hydrolyzing activity. It was isolated from the culture medium and purified by crystallization and chromatography on DEAE‐Sephadex and Sephadex G‐75. Homogeneity was ascertained by disc gel electrophoresis and by isoelectric focusing. The molecular weight of proteinase K was determined by gel filtration to be 18500 ± 500, the isoelectrice point was pH 8.9. The enzyme was shown to be a serine protease by inactivation with diisopropyl phosphofluoridate and phenylmethylsulfonyl fluoride. It displayed strong proteolytic activity on denatured but also on native proteins as demonstrated by the rapid inactivation of bovine ribonuclease. The pH‐optimum was in the range between pH 7.5–12.0. A specificity of the enzyme for peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids was observed.
On the Determination of the Transmembrane pH Difference in Bacterial Chromatophores using 9‐Aminoacridine
Authors: CASADIO Rita , BACCARINI‐MELANDRI Assunta , MELANDRI Bruno Andrea 
Published: March 2005

The extent of hydrogen ion gradient across membranes from photosynthetic bacteria has been determined measuring the energy‐dependent quenching of fluorescence of 9‐aminoacridine in different experimental conditions.Examinations of the absorption or emission spectra of 9‐aminoacridine in aqueous solution reveal an ideal behaviour of this amine up to millimolar concentrations.The decrease in fluorescence observed in the presence of energized membranes corresponds to a general quenching in all regions of the absorption or emission spectra and is related to the amount of dye and of vesicles present in the assay according to the model of distribution of an ideal amine.The onset of a pH difference across the membrane is stimulated by the presence in the assay of lipophilic anions or of K+ and valinomycin; in these conditions a parallel decrease of membrane potential, as measured by carotenoid band shift, has been observed. The total protonmotive force evaluated by a combination of these two techniques however remains relatively constant during time and in almost all conditions tested.
Correlation between the Level of Vitamin‐B12‐Dependent Methionine Synthetase and Intracellular Concentration of Vitamin B12 in Some Bacteria
Authors: OHMORI Hitoshi , NAKATANI Kazuo , SHIMIZU Shoichi , FUKUI Saburo 
Published: March 2005

This paper presents several lines of evidence indicating that the synthesis of vitamin‐B12‐dependent 5‐methyltetrahydrofolate‐homocysteine transmethylase (“methionine synthetase”) is closely correlated to the intracellular level of vitamin B12 in manners different between bacteria which do and do not produce vitamin B12.1. The intracellular level of vitamin B12 in Rhodospirillum rubrum was reduced to one fifth that of the normal cells when the organism was cultivated in a medium lacking cobalt. In accord with the decrease in vitamin B12 production, the specific activity of methionine synthetase was lowered to about 50%. It was of much interest that the enzyme was always present as the holoenzyme and the apoenzyme could not be detected even in the vitamin‐B12‐deficient cells containing a markedly decreased level of the holoenzyme. When the vitamin‐B12‐deficient cells were incubated in a cyano‐cobalamin‐supplemented medium, the specific activity of methionine synthetase began to increase after 2 h lag time and reached the maximum in about 6 h. This enhancement of the specific activity was suppressed if an appropriate inhibitor of protein synthesis was added to the medium. These facts strongly suggest that synthesis of protein de novo is essential for the enhancement of the specific activity by incubation with cyanocobalamin. Similar results were obtained in other vitamin B12 producers such as Streptomyces olivaceus and Corynebacterium simplex.2. Chromatium D which is known to form cobalt‐free corrinoids but not produce complete vitamin B12, was found to possess methionine synthetase as apoenzyme. When the vitamin‐B12‐free cells were incubated in the presence of cyanocobalamin, all the apoenzyme was readily converted to the holoenzyme within 2 h. As the formation of the holoenzyme proceeded, the level of the total enzyme (holo + apo) increased to a certain degree. The enhancement, however, was not affected by the inhibitors of protein synthesis, indicating that the increment could not have resulted from de novo synthesis of protein. A similar phenomenon was observed in Escherichia coli which does not produce vitamin B12 but possesses apoprotein of methionine synthetase.It is suggested that vitamin B12 synthesis is closely correlated to that of methionine synthetase in bacteria producing the vitamin, such as Rhodospirillum rubrum, Streptomyces olivaceus and Corynebacterium simplex. On the contrary, the apoprotein of the enzyme is synthesized independently of vitamin B12 in bacteria not producing the vitamin, such as Chromatium D and Escherichia coli.
Interactions of a Purified Non‐Histone Chromosomal Protein with DNA and Histone
Authors: SHOOTER Kenneth V. , GOODWIN Graham H. , JOHNS Ernest W. 
Published: March 2005

1. The interactions of a purified calf thymus chromosomal non‐histone protein (designated protein HMG1) with bacteriophage T7 DNA and calf thymus DNA have been investigated by sedimentation analysis in the ultracentrifuge. The results obtained show that (a) the non‐histone protein HMG1 binds to DNA in an ionic‐strength‐dependent manner, (b) the DNA can bind up to approximately four to five times its weight of protein HMG1, the protein distributing itself evenly along the DNA chains, and (c) the interaction is a rapid reversible equilibrium. These results are interpreted as indicating that protein HMG1 binds to DNA through ionic bonding between the basic amino acids of the protein and the phosphate groups of the DNA.2. Equilibrium sedimentation studies were carried out on mixtures of protein HMG1 and histone F1. The results demonstrate that protein HMG1 combines with histone F1.3. The molecular weight of protein HMG1 has been determined by equilibrium sedimentation and a mean value of 26500 was obtained.
Comparative Kinetic Properties of α, β and Ψ Forms of Trypsin
Authors: FOUCAULT Georges , SEYDOUX François , YON Jeannine 
Published: March 2005

The kinetics of ester substrate hydrolysis catalysed by the α, β, and Ψ forms of trypsin have been compared. An affinity chromatography method for Ψ‐trypsin purification is described. At optimum pH, with esters as substrates, there is no significant difference between the activity of the α and β forms; β‐trypsin, however, is about 40% more active than α‐trypsin on benzoyl‐l‐arginine amide.At acidic pH, (∼ 3.6), β‐trypsin is more reactive toward p‐nitrophenyl‐p′‐guanidino benzoate HCl than α‐trypsin, and differential active‐site titrations of the α and β forms can be carried out in mixtures of both of them. Hydrolysis of specific esters by Ψ‐trypsin is very slow compared to native trypsin but it is not completely aspecific. Carbobenzoxy‐l‐lysine benzyl ester is a relatively good substrate of this enzyme form; kcat values for Ψ‐trypsin are very sensitive to the substrate leaving group, in contrast to that which is observed with native trypsin. Furthermore, experiments involving nucleophilic competition indicate that the acylation step becomes rate‐limiting for the hydrolysis of cationic ester substrates by Ψ‐trypsin.It is concluded that in tryptic catalysis enzyme acylation is a more specific step than substrate binding or enzyme deacylation. The acylation of the enzyme is greatly perturbed both by substrate modifications and by structural alterations of the native enzyme molecule. The role of the essential active‐site carboxylic group (Asp‐177) is discussed. It is proposed that its function in tryptic catalysis is to decrease the activation free energy for enzyme acylation instead of stabilizing the initial enzyme‐substrate complex.
Turnover of Malate‐Dehydrogenase Isozymes in Rabbit Liver and Heart
Authors: DÖLKEN Gottfried , LEISNER Elmi , PETTE Dirk 
Published: March 2005

1. The cytosolic and mitochondrial isozymes of malate dehydrogenase were purified from rabbit heart. Antisera against the two isoenzymes were obtained from sheep. The antiserum against mitochondrial malate dehydrogenase showed no cross‐reaction with the cytosolic isozyme and vice versa.2. Apparent turnover rates of the two isozymes were determined in rabbit liver and heart by means of single pulse labelling with [14C]leucine and isolation of the enzymes labelled in vivo by the immunochemical method. The apparent half‐lives of the cytosolic and mitochondrial isozyme are 8.6 and 21.9 h in liver and 21.6 and 33.9 h in heart. These values are shorter than the apparent half‐lives of total soluble protein of liver and heart.3. Turnover of mitochondrial malate dehydrogenase in rabbit liver was also calculated from the kinetics of enzyme accumulation and regression during a starvation‐refeeding cycle. The half‐life during starvation is 15.4 h and in the refeeding period 19.6 h. The approximately two‐fold increase in mitochondrial malate dehydrogenase content of liver during starvation appears thus to be brought about by an increased rate of synthesis.
Involvement of the Superoxide Anion Radical in the Autoxidation of Pyrogallol and a Convenient Assay for Superoxide Dismutase
Authors: MARKLUND Stefan , MARKLUND Gudrun 
Published: March 2005

The autoxidation of pyrogallol was investigated in the presence of EDTA in the pH range 7.9–10.6.The rate of autoxidation increases with increasing pH. At pH 7.9 the reaction is inhibited to 99% by superoxide dismutase, indicating an almost total dependence on the participation of the superoxide anion radical, O2·−, in the reaction. Up to pH 9.1 the reaction is still inhibited to over 90% by superoxide dismutase, but at higher alkalinity, O2·− ‐independent mechanisms rapidly become dominant.Catalase has no effect on the autoxidation but decreases the oxygen consumption by half, showing that H2O2 is the stable product of oxygen and that H2O2 is not involved in the autoxidation mechanism.A simple and rapid method for the assay of superoxide dismutase is described, based on the ability of the enzyme to inhibit the autoxidation of pyrogallol.A plausible explanation is given for the non‐competitive part of the inhibition of catechol O‐methyltransferase brought about by pyrogallol.
The Role of Acetyl‐CoA in the Reaction Pathway of Pig‐Liver Pyruvate Carboxylase
Authors: WARREN Graham B. , TIPTON Keith F. 
Published: March 2005

1. The kinetics of acetyl‐CoA activation of pig liver pyruvate carboxylase have been studied at varying concentrations of the other substrates.2. The activation of the enzyme by Mg2+ becomes increasingly co‐operative when the concentration of acetyl‐CoA is decreased.3. Free Mg2+ does not affect the co‐operative interaction between acetyl‐CoA and the enzyme.4. The binding of acetyl‐CoA and free Mg2+ to the enzyme appears to precede the binding of the other substrates.5. Possible models that would account for the observed allosteric effects are discussed.
Influence of Iron Deficiency on the Cytochromes of Rhodopseudomonas spheroides
Authors: AGALIDIS Ileana , JAUNEAU Edouard , REISS‐HUSSON Françoise 
Published: March 2005

Rhodopseudomonas spheroides Y cells were grown photosynthetically in a denned medium supplemented with either a limiting amount of iron or with an excess of iron. Soluble and bound cytochromes were determined in both cases.The composition of the soluble cytochromes was not altered qualitatively by iron deficiency. In both culture conditions, a number of cytochromes were isolated by chromatography and purified to various extents: cytochromes c‐550 (c2 type), c′, c‐551, c‐552, and b‐559. In normal cells cytochrome c2, and to a lesser extent cytochrome c′ predominated whereas the amounts of both were greatly lowered in iron deficiency.Bound cytochromes were detected by difference spectroscopy of particles after chemically or light‐induced oxidoreductions. When iron was not limiting, cells were also grown in presence of diphenylamine, which inhibited carotenoid synthesis and facilitated detection of cytochromes. Particles from those cells, as well as particles from uninhibited cells contained cytochrome c‐551 (bound form of cytochrome c2), cytochrome b‐560, and a minor component, possibly b‐type. All reacted to illumination. In iron‐deficient cells, the bound heme b was almost completely suppressed; difference spectra suggested the presence only of cytochrome c2 and c′.
Preparation of Immunoglobulin Light‐Chain mRNA from Microsomes without the Use of Detergent
Authors: HARRISON Timothy M. , BROWNLEE George G. , MILSTEIN Cesar 
Published: March 2005

A technique which releases ribosomes from membranes without the use of detergent described in the preceding paper is shown to allow the separation of two distinct pools of poly(A)‐containing RNA in microsomes. Thus, using preparations from mouse myeloma tissue‐culture cells, essentially 100% release of immunoglobulin mRNA occurs under conditions in which only 50–60% of the total microsomal poly(A)‐containing RNA is released. The RNA which is not released is principally mitochondrial ribosomal RNA and the method thus provides a good preparative procedure for this fraction.Messenger ribonucleoprotein particles involved in immunoglobulin biosynthesis have been prepared from the released material. The heavy‐chain mRNA‐containing particle sediments in a neutral sucrose gradient at about 22 S, and that containing the light‐chain mRNA at about 18 S. The ribosome‐release technique has been used for the rapid preparation of light‐chain mRNA in good yield and at over 60% purity after a single sucrose gradient centrifugation. Further purification has been achieved by polyacrylamide gel electrophoresis. Using [32P]RNA, the contaminants can be estimated directly at the chemical level using a fingerprint assay.