bluuebunny/crossref_metadata_2025 huggingface dataset:
Biohydrogenation of Sterols by Eubacterium ATCC 21,408—Nova Species
Authors: Eyssen Hendrik J. , Parmentier Guido G. , Compernolle Frans C. , de Pauw Guido , Piessens‐Denef Maria
Published: March 2005

Biohydrogenation of Δ5‐steroids was studied in vitro by incubating various steroids with a pure culture of Eubacterium 21,408 in a brain‐thioglycollate medium under anaerobic conditions.

Eubacterium 21,408, a strictly anaerobic bacterium, was isolated from rat cecal contents. It differed from any previously described Eubacterium species by its requirements for a Δ5‐3β‐hydroxy steroid.

Eubacterium 21,408 reduced the 5,6‐double bond of cholesterol, campesterol, β‐sitosterol and stigmasterol, exclusively yielding the corresponding 5β‐saturated derivatives. Similarly, the 4,5‐double bond of allocholesterol was reduced to yield coprostanol. Neither the 7,8‐double bond in 7‐dehydrocholesterol or in lathosterol, nor the 22,23‐double bond in the side chain of stigmasterol were reduced.

In the absence of a 3‐hydroxyl function (e.g. in cholest‐5‐ene), when the 3‐hydroxyl function was in the 3α‐position (e.g. in epicholesterol) or was substituted (e.g. in 3β‐chloro‐cholest‐5‐ene and in cholesteryl esters), no saturation of the 5,6‐double bond was observed.

When incubated with Eubacterium 21,408, the carbonyl group of 4‐cholesten‐3‐one, was reduced to the corresponding 3β‐hydroxyl group along with saturation of the 4,5‐double bond. The bacterium also reduced the 3‐oxo group of cholestanone and coprostanone to a 3β‐hydroxyl group.
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.
Isolation and Properties of Isoleucyl‐tRNA Synthetase from Escherichia coli MRE 600
Authors: DurekoviĆ Andrija , Flossdorf Josef , Kula Maria‐Regina
Published: March 2005

A method is described for the isolation of isoleucyl‐tRNA synthetase from Escherichia coli MRE 600 involving gel filtration, ion‐exchange chromatography on DEAE‐Sephadex and chromatography on hydroxyapatite. The enzyme is prepared in high yield (60%) and is homogeneous as judged by equilibrium sedimentation, disc electrophoresis and electrophoresis in dodecylsulfate. The protein has a molecular weight of 102000 and a sedimentation coefficient sw, 20 of 5.37. It appears to be a single chain. The amino‐acid composition has been determined.
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.
Microbial Transformation of Cholesterol into Coprostanol
Authors: Björkhem Ingemar , Wrange Örjan , Gustafsson Jan‐Åke
Published: March 2005

The conversion of 4‐cholesten‐3‐one into 5β‐cholestan‐3‐one (coprostanone) by microorganisms in caecal contents from rats has been studied. The enzyme activity was present in the soluble part of a homogenate obtained by freeze‐pressing of caecal contents of rats. The major part of the activity could be precipitated between 25 and 50% saturation with ammonium sulphate. The 3‐oxo‐Δ4‐steroid 5β‐reductase activity required NADH as cofactor and was inhibited by p‐chloromercuribenzoate. The enzyme preparation also catalyzed the reduction of the Δ4‐double bond in progesterone and testosterone but did not catalyze reduction of the Δ5‐double bond in cholesterol, pregnenolone or dehydroepiandrosterone. The rate of reduction of the Δ4‐double bond in progesterone was more than twice as fast as the rate of reduction of the Δ4‐double bond in 4‐cholesten‐3‐one. The mechanism of reduction of Δ4‐double bonds in 3‐oxo‐Δ4‐steroids by the microbial 3‐oxo‐Δ4‐steroid 5β‐reductase was found to involve transfer of hydrogen from the 4B‐position of NADH to the 5β‐position of the steroid.
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.
Studies on Temperature‐Sensitive Mutants Affecting the Assimilatory Nitrate Reductase of Aspergillus nidulans
Authors: MACDONALD Donald W. , COVE David J.
Published: March 2005

In the ascomycete fungus, Aspergillus nidulans, mutations in the niaD gene and in five unlinked cnx genes lead to the loss of assimilatory nitrate reductase, and the inability to grow on nitrate as sole nitrogen source. Mutants have been isolated in the niaD gene and in three of the five cnx genes which are temperature‐sensitive for growth on nitrate and have nitrate reductase activity when grown at the permissive temperature. The half life of nitrate reductase in the wild‐type and in these temperature‐sensitive strains has been measured. Temperature‐sensitive mutants in the cnxE and cnxF genee havs a nitrate reductase with the same half life as in wild‐type strains. Temperature‐sensitive mutants in the niaD and cnxH genes have a nitrate reductase with a shorter half life than the wild‐type enzyme. These results can be interpreted as indicating that the products of the niaD and cnxH genes are structurally involved in the nitrate reductase molecule, whereas the products of the cnxE and cnxF genes are involved in the formation of a cofactor required for nitrate reductase activity.
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.
Transfer of Monosaccharide onto Exogenous Acceptors by Mitochondrial Glycosyl Transferases
Authors: MYERS Marjorie W. , BOSMANN H. Bruce
Published: March 2005

Mitochondrial glycosyl transferase have been studied by the transfer of labeled monosaccharide from nucleotide monosaccharide onto exogenous acceptor glycoproteins prepared by serial removal of carbohydrate from collagen, fetuin and immunoglobulin G. Triton X‐100 extracts of isolated mitochondria were used as enzyme source. Collagen minus glucose served as the best acceptor for the transfer of glucose from UDP‐glucose; immunoglobulin G minus sialic acid, galactose, N‐acetyl‐glucosamine and mannose served as the best acceptor for mannose from GDP‐mannose. The reaction has been studied for divalent cation requirement (glucosyl transfer is optimal with Mg2+; mannosyl transfer is optimal with Mn2+), temperature optima (30 °C) and pH optima (6.8). The data reveal some similarities and some differences between cytoplasmic and mitochondrial glycosyl transferases.
Adenine Nucleosides in Solution: Stabilisation of the anti‐Conformation by C‐5′ Substituents
Authors: FOLLMANN Hartmut , GREMELS Georg
Published: March 2005

Proton magnetic resonance spectra of 30 adenine nucleosides have been obtained in dilute (0.001 to 0.05 M) aqueous or dimethyl sulfoxide solutions and have been analysed with respect to structure‐dependent variations of the purine proton chemical shifts. In 2H2O the resonance signal of H‐8 shows a marked dependence upon the nature of substituents linked to C‐4′ of adenine ribofuranoside derivatives and upon the presence of a 2′: 3′‐O‐isopropylidene group while H‐2 is relatively unaffected. Therefore the chemical shift difference, δH‐8–δH‐2 =Δδ, may serve as an indicator of intramolecular ribose‐base interact ons. Δδ varies with the chemical nature of C‐4′ or C‐5′ substituents in the order: ammonium, sulfonium (0 ppm) < hydrogen < hydroxyl ∼ amino < thio < halogen < phosphate, carboxylate (0.4 ppm), and decreases with increasing chain length of these substituents. These results suggest the existence of electrostatic and dipolar repulsion or, in the majority of cases, attractive forces between the C‐5′ and C8‐H regions of adenine nucleosides which have been previously demonstrated for 5′‐nucleotides only. They imply a strong preference of most adenosine derivatives with unsubstituted 2′ and 3′‐hydroxyl groups for the anti‐conformation range and the gauche, gauche C‐5′ conformation. In dimethyl sulfoxide, basically the same situation exists but different substituents exhibit smaller individual Δδ variations than in water, indicating different solvation shells. In this solvent two additional conformation‐stabilizing contributions can be recognized, viz. an interaction of the bridge oxygen (O‐4′) of ribose with adenine, and a hydrogen bond between H‐8 and a 5′‐carboxylate anion. This energetic stabilisation of the anti and g,g‐conformers by most types of adenosine C‐5′ substituents supports the “rigidity” concept of nucleotides, extending it to the nucleoside level. The only exception found is 5′‐nitromethyladenosine in which ultraviolet, circular dichroism and magnetic resonance spectra suggest rotation of the adenine base to a “non‐classical” conformation due to base‐chain π‐electron interaction. The anti‐conformation of the adenosine derivatives used in this study can in some cases be correlated with their enzymatic deamination by adenosine and AMP aminohydrolase.
A Suppressor of Nonsense Mutations in Bacillus subtilis
Authors: CAMACHO Ana , CARRASCOSA Jose L. , VIÑUELA Eladio , SALAS Margarita , MORENO Felipe
Published: March 2005

The suppressor‐sensitive mutants of phage φ29, susB47 and susN212, do not induce the synthesis of proteins NP1 and II, respectively, when they infect Bacillus subtilis su−. A new polypeptide of molecular weight 75000, smaller than that of protein NP1, is produced in susB47‐infected cells. An analysis of the methionine‐containing tryptic peptides of protein NP1 and the 75000‐molecular‐weight polypeptide showed that both proteins are related. When mutants susB47 and susN212 infect B. subtilissu+3, a small amount of proteins NP1 and II, respectively, is synthesized, the level of the suppression being about 10%. The results indicate that the B. subtilis su+3 strain is a suppressor of nonsense mutations.
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.
Rifamycin Biosynthesis: Further Studies on Origin of the Ansa Chain and Chromophore
Authors: KARLSSON Anita , SARTORI Giuseppe , WHITE Richard J.
Published: March 2005

The metabolic origin of the ansa chain and chromophore of rifamycin S has been investigated by studying the incorporation of radioactive and 13C‐enriched precursors. A simplified degradation scheme has been utilised to cleave chemically the radioactive antibiotic into constituent chromophore and ansa chain. Rifamycin S labelled with [3‐14C]propionate and [Me‐3H]methylmalonate have the same pattern of isotope distribution. Unlabelled propionate efficiently dilutes the incorporation of [Me‐3H]methylmalonate but the reverse is not true. Although the results of [2‐14C]malonate and [2‐14C]acetate incorporation were inconclusive owing to randomisation, the proton‐decoupled carbon magnetic resonance spectrum of rifamycin S biogenetically enriched with [2‐13C]malonate revealed a specific incorporation into C‐5 and C‐18. We propose that the polyketide chain is formed by a linear condensation, in the appropriate sequence, of eight methylmalonates and two malonates with the subsequent esterification of the C‐25 hydroxyl with acetate. Two precursors were found preferentially to label the chromophore: [3,4‐14C2]glucose and [1‐14C] glycerate. Out of the seven carbons in the chromophore not deriving from acetate or propionate only C‐1 and C‐10 appeared significantly enriched by [1‐13C] glucose. This labelling pattern, coupled with the 14C incorporation data, suggests that the chromophore may derive from an intermediate of the common pathway of aromatic biosynthesis, before the level of shikimate.
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.
The Mechanism of Water‐Proton Relaxation in Enzyme · Paramagnetic‐Ion Complexes
Authors: JONES Robert , DWEK Raymond A. , WALKER Ian O.
Published: March 2005

The water proton‐relaxation times, T1 and T2, have been measured in solutions of Mn(II) and ATP in the presence and absence of rabbit muscle phosphofructokinase over a wide range of temperatures and nuclear frequencies, using pulsed nuclear magnetic resonance techniques. The equilibrium between the enzyme and the Mn · ATP complex has also been studied as a function of temperature, and the relaxation rates in the bound complex determined. An analysis of the relaxation rates in both free and enzyme‐bound Mn · ATP sites according to established theories of paramagnetic relaxation has been carried out by means of graphical and computing methods, and yielded estimates of the Mn(II) water co‐ordination number (q) and the relevant correlation times with their frequency and temperature dependence. Relaxation in the ternary enzyme complex is dipolar in nature and the correlation time (τc) is dominated by the water residence time (τM) or the Mn electron spin relaxation time (τs) according to frequency. The values obtained for both τc and τs are longer than earlier estimates. In the binary Mn · ATP complex the dipolar interactions are dominated by rotation (τR) but in addition there is a large scalar interaction governed by both τM and τs. The significance of the parameters in the structure of the ternary enzyme complex is discussed, together with the implications of the revised parameters τc and τs for earlier mapping studies of the Mn · ATP sites and the spin‐labelled enzyme. The limitations of the analysis have been stressed throughout.
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.
Purification and Characterization of Macromolecular Inhibitors of Proteinase A from Yeast
Authors: SAHEKI Takeyori , MATSUDA Yoshiko , HOLZER Helmut
Published: March 2005

Inhibitors for yeast proteinase A have been purified from boiled extracts from baker's yeast. Upon SP‐Sephadex C‐25 column chromatography, four different peaks of proteinase A inhibitory activity appear. The inhibitors IA2 and IA3 have been further purified by QAE‐Sephadex chromatography and preparative disc electrophoresis. Both inhibitors are homogeneous in dodecylsulfate‐gel electrophoresis. From the electrophoretic mobilities in dodecylsulfate‐polyacrylamide gels, a molecular weight of 6100 ± 200 was estimated for both inhibitors. In the absence of dodecylsulfate, both inhibitors showed one predominant band and a second faint, diffuse band. As a result of Sephadex G‐75 gel filtration, a molecular weight of 23000 ± 2000 was obtained for both inhibitors, indicating an aggregation of the monomers to a tetramer. Upon electrofocusing in acrylamide gels the isoelectric points of the inhibitors were found to be 5.7 for IA2 and 6.3 for IA3. Both inhibitors inhibited proteinase A. Neither proteinases B and C from yeast nor pepsin were inhibited. Both inhibitors were inactivated after incubation with purified yeast proteinase B, but not with the yeast proteinases A or C. The inhibition of proteinase A at high inhibitor/proteinase ratios was more pronounced at pH 5 and 7 as compared to pH 3. An autocatalytic activation of a mixture of inhibited proteinases A, B and C by addition or liberation of traces of active proteinase A or B is discussed.
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.
Renaturation of Escherichia coli Tryptophanase after Exposure to 8 M Urea
Authors: LONDON Jacqueline , SKRZYNIA Cécile , GOLDBERG Michel E.
Published: March 2005

The renaturation of Escherichia coli K 12 tryptophanase after treatment with 8 M urea has been studied. It is shown that, during the renaturation process, an inactive as well as an active form of the protein can be obtained. The influence on the relative amounts of these two forms of factors such as temperature, the presence of the coenzyme, the protein concentration and the conditions for removal of urea has been investigated. The results obtained indicate that the inactive form is characterized by incorrect quaternary interactions occurring specifically between tryptophanase protomers. These findings are discussed in the general frame of the mechanism of protein folding, and are taken as an evidence for the existence of nucleation centers in the folding of tryptophanase.
Human Leukemic Cells: RNA‐Directed DNA Polymerase
Authors: DESAI Laxman S. , SHORT Diane L. , FRIEDMAN Orrie M. , FOLEY George E.
Published: March 2005

The enzyme(s) RNA‐dependent DNA polymerase (reverse transcriptase) has been isolated from human lymphocytic cell lines which have been derived from patients with leukemia, infectious mononucleosis, and from normal healthy donors. The reverse transcriptase was purified through high‐speed glycerol gradients, and by DEAE‐cellulose and phosphocellulose column chromatography. The enzyme isolated from the cultured human lymphocytes had identical chromatographic patterns. The enzyme properties were studied by utilizing synthetic templates. These synthetic templates are known to discriminate between reverse transcriptase activity and that from other cellular polymerases. The activity of reverse transcriptase from human leukemic cells was much greater than that of the enzyme isolated from cells of normal or infectious mononucleosis origin. The reverse transcriptase isolated from human leukemic cells preferred oligo(dT) · poly(dA) over templates oligo(dT) · poly(dA) and oligo(dG) · poly(rC), wherease templates oligo(dT) · poly(dA) and oligo(dG) · poly(rC) were found to be active with the enzyme isolated from normal cells. The detection of the enzyme(s) reverse transcriptase in well‐established cell lines of normal and malignant origin and its possible relation to cellular metabolism is discussed.
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.
Control of Haemoglobin Synthesis
Authors: HUNTER Anthony R.
Published: March 2005

The distribution of ribosomes along the mRNAs for α and β chains was measured in cells which had achieved a steady state of protein synthesis after incubation with haem and iron or with the iron‐chelating agent 2,2′‐dipyridyl. The distribution was found to be uniform under both conditions for the α and β chain mRNAs, despite the dramatic breakdown of polysomes observed in the presence of dipyridyl. Since a uniform distribution of ribosomes along an mRNA indicates the absence of rate‐limiting steps in chain elongation or chain termination, we conclude that the slowing of protein synthesis during iron deprivation is not caused by the introduction of rate‐limiting steps into these processes. In the presence of dipyridyl the total numbers of ribosomes synthesising α and β chains were reduced to roughly the same extent indicating that iron starvation inhibits the synthesis of both α and β chains. In order to confirm that the rate chain elongation was not altered during iron starvation, we attempted to compare the rates of translation of the two mRNAs in the presence and absence of iron. Some of the difficulties in measuring the translation times for the α and β chain mRNAs under conditions of iron deprivation are discussed. Under conditions where a partial effect of iron deprivation was evident, we showed that the rate of translation of both α and β chain mRNAs was essentially unchanged. Both sets of findings support the hypothesis that, in vivo, the availability of haem controls the process of chain initiation.
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.
A Study on the Biosynthesis of Protein A in Staphylococcus aureus
Authors: MOVITZ Jan
Published: March 2005

Protein A is a cell wall constituent of Staphylococcus aureus. After being synthesized on the ribosomes, the protein A molecules are linked to the peptidoglycan. This is a very rapid reaction which is completed within approximately 1 min; it is suggested that the molecules leave the ribosomes and are then directly incorporated into the peptidoglycan without passing a soluble cytoplasmic stage.

The synthesis of protein A de novo and peptidoglycan take place independently of each other. Also, the incorporation of protein A into the cell wall proceeds when the synthesis of peptidoglycan has been completely inhibited by vancomycin, i.e. the synthesis of peptidoglycan is not essential for the incorporation of protein A. It is therefore concluded that protein A can be linked to pre‐existing peptidoglycan chains.
Heterogeneity of Erythrocyte Catalase II
Authors: AEBI Hugo , WYSS Sonja R. , SCHERZ Bernhard , SKVARIL František
Published: March 2005

Normal human erythrocyte catalase, when isolated by the method of Mörikofer et al. [Eur. J. Biochem. 11 (1969) 49–57], is heterogenous in two respects: (a) there are alternative molecular forms; (b) besides the active tetramer, enzyme dissociation products (dimer, monomer) are present.

By means of exclusion chromatography on Sephadex G‐150 it was shown that catalase preparations contain dimer and monomer in small amounts (∼ 4%). Upon storage, the yield in subunits can be considerably enhanced (up to 15%).

Residual catalase activity in the blood of an individual homozygous for Swiss Type Acatalasemia (1–2% of normal level) consists of a catalase variant, which is unstable, but of approximately normal specific activity. In this mutant, the tendency of dissociation into subunits is greatly enhanced. Upon purification, little tetramer but appreciable amounts of the inactive dimer are obtained.

The antigenic properties of these molecular species were investigated using anti‐catalase and anti‐catalase‐subunit immunoglobulins G. There is complete antigenic identity between the normal and the variant catalase; the same is true for the normal and variant dimer. Different antigenic properties of dimer and monomer, however, offer evidence for the existence of hidden determinants in the subunits.
Human Glutathione Reductase: Purification of the Crystalline Enzyme from Erythorocytes
Authors: WORTHINGTON David J. , ROSEMEYER Michael A.
Published: March 2005

Glutathione reductase from human erythrocytes has been purified 40 000‐fold in 10 steps with an overall yield of 36%.

The procedure included bulk separations on DEAE‐Sephadex and CM‐Sephadex, gel‐filtration on Sephadex G‐200, and salt fractionation with ammonium sulphate. The purified enzyme was crystallised from aqueous solution.

The final preparation had a specific activity of 240 units/mg. The purification procedure was completed in 4 weeks, and gave 40 mg enzyme from 35 1 blood. The schedule for glutathione reductase allowed the concurrent isolation of glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase.

In solutions of thiols, the glutathione reductase preparation was homogeneous, as indicated by gel‐filtration, ultracentrifugation and electrophoresis. In the absence of thiols, the enzyme showed a tendency to form aggregates. Some of the physical properties of the enzyme are discussed in relation to previous difficulties encountered in its isolation.
Binding of Salicylate in the Adenosine‐Binding Pocket of Dehydrogenases
Authors: EINARSSON Roland , EKLUND Hans , ZEPPEZAUER Eila , BOIWE Torne , BRÄNDÉN Carl‐Ivar
Published: March 2005

Binding of salicylate derivatives to liver alcohol dehydrogenase has been studied by crystallographic and spectrofluorometric methods. We have found that salicylate binds in the hydrophobic adenosine‐binding pocket which has been shown to be structurally similar in all investigated dehydrogenases. The binding of salicylate in this pocket strongly resembles adenosine binding in spite of their very different structures. The position of the phenyl ring partly overlaps the position of adenine; the hydroxyl group of salicylate forms a hydrogen bond to one of the carboxyl oxygen atoms of Asp‐223. This residue has been shown to be invariant in several dehydrogenases. It forms a hydrogen bond to one of the hydroxyls of the adenosine ribose which is important for NAD‐binding. We have also shown that 8‐anilino‐1‐naphthalene sulfonate competes with salicylate for this binding site. A simple fluorometric method based on this competition is proposed for rapid screening of salicylate binding to other enzymes, such as kinases which have coenzyme binding domains similar to those found in dehydrogenases.
Isolation and Characterization of Oligosaccharides from Canine Submaxillary Mucin
Authors: LOMBART Christian G. , WINZLER Richard J.
Published: March 2005

Pure mucin isolated from canine submaxillary glands was treated with alkaline borohydride and products of degradation were fractionated by a procedure that included ion‐exchange chromatography, gel filtration, high‐voltage electrophoresis and paper chromatography.

Seven acidic, reduced oligosaccharides were isolated being divided into two distinct types: one, containing sialic acid and no glucosamine or sulfate (type A) and the other containing glucosamine and sulfate but no sialic acid (type B).

The most complete oligosaccharide of type A designated component (I) was a tetrasaccharide α‐l‐fucopyranosyl‐(1 → 2)‐d‐galactopyranosyl‐[N‐acetylneuraminyl‐(2 → 6)]‐2‐acetamido‐2‐deoxy‐d‐galactitol. The most complex oligosaccharide of type B designated component (V) has a hexasaccharide with the following structure: α‐l‐fucopyranosyl‐(1 → 2)‐β‐d‐galactopyranosyl‐(1 → 3, 4, or 6)‐β‐2‐acetamido‐2‐deoxy‐d‐glucopyranosyl 3 or 4 sulfate‐(1 → 6)‐α‐l‐fucopyranosyl‐(1 → 2)]‐β‐d‐galactopyranosyl‐(1 → 3) 2‐acetamido‐2‐deoxy‐d‐galactitol. All the other acidic isolated oligosaccharides were derived from these two main components.

No oligosaccharide containing both sialic acid and glucosamine sulfate could be demonstrated. Based on these structural features, a possible biosynthetic pathway leading to the two different oligosaccharide chains is discussed.

Variations in ratios of sugars observed in canine submaxillary mucin and especially the reciprocal relationship between sialic acid and fucose noted earlier (1962) by Dische, Pallavicini, Kavasaki, Smirnow, Cizek and Chien may reflect variations in the proportions of these two types of oligosaccharides.