bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Lysines in Liver Alcohol Dehydrogenase
Authors: Mckinley‐McKee John S. , Morris David L. 
Published: March 2005

Liver alcohol dehydrogenase is reversibly inactivated by pyridoxal 5′‐phosphate. When the enzyme is saturated with pyridoxal‐P the residual activity is about 20%. The enzyme can be completely inactivated by successive covalent labelling with pyridoxal‐P, using sodium borohydride reduction. A maximum of about 11 pyridoxal‐P groups per subunit were covalently incorporated into the enzyme.The binding to the enzyme of several known inhibitors was investigated by studying their effect on the reversible inactivation of the enzyme by pyridoxal‐P at pH 8.0. The effect of these ligands on the activation of the enzyme by methyl picolinimidate was also studied. AMP protected competitively against inactivation by pyridoxal‐P and also protected strongly against activation by methyl picolinimidate. After picolinimidylation of the enzyme the binding of AMP at pH 10 was 8‐fold weaker.AMP and NADH both protected one lysine residue per subunit from reaction with pyridoxal‐P. It is concluded that there is one lysine residue at the active centre of the enzyme and that this lysine plays an important part in binding the coenzyme by attracting the phosphate group of the AMP moiety of the coenzyme. Coenzyme binding to the picolinimidylated enzyme is weakened by steric interference between the picolinomidylated active‐centre lysine and the AMP moiety of the coenzyme. Since effects of neutral zinc‐binding ligands cannot be explained by steric or electrostatic effects, it is suggested that they produce conformational changes in the enzyme, perhaps by binding at a site other than the active‐centre zinc atom.
Kinetic Studies of the Interaction of Pigeon‐Liver NAD Kinase with Adenine Nucleotides and Divalent Cations
Authors: Apps David K. , Marsh Adrian 
Published: March 2005

NAD kinase is inhibited by free Mg2+, free ATP and by a number of other adenine nucleotides.
Inhibition by ATP, ADPMg−, ADP‐ribose2− and AMP2− is competitive with respect to both ATPMg2− and NAD+.
ATP appears to bind equally well to the free enzyme and to binary enzyme · substrate complexes.
The other adenine nucleotides studied have markedly less affinity for the free enzyme than for binary complexes.
Mg2+ probably inhibits by forming a complex with NAD+, which binds to the enzyme but is inactive in the reaction.
Low concentrations of Mg2+ reduce KmNAD+ in an unexplained manner.
ATPCa2− is a substrate for NAD kinase, but the rate‐limiting step of the reaction may be different when other metals activate the enzyme.
Comparison of Polypeptide‐Chain Structure of Four Mammalian Ceruloplasmins by Gel Filtration in Guanidine Hydrochloride Solutions
Authors: RydÉN Lars 
Published: March 2005

In a recent paper in this journal it was shown that human ceruloplasmin contains a single continuous polypeptide chain composed of about 1050 amino acid residues. Together with the carbohydrate this accounts for the molecular weight of the native protein, 132000. In contrast to this porcine ceruloplasmin has been reported to contain two subunits of molecular weight 70000–80000. This was the reason for performing a direct comparison of polypeptide‐chains from ceruloplasmin from different mammalian sera.Ceruloplasmin was prepared from sera of man, pig, horse and rabbit. The reduced and alkylated proteins were gel filtered on a calibrated column of 6% agarose in 6 M guanidine hydrochloride. The four proteins all contained a single main component eluting in the same position within errors of measurements. It is concluded that the four proteins investigated all contain a single polypeptide chain of very similar size.
Magnetic Resonance Studies on Manganese‐Activated Phosphofructokinase
Authors: Jones Robert , Dwek Raymond A. , Walker Ian O. 
Published: March 2005

Rabbit skeletal‐muscle phosphofructokinase exhibits similar kinetic behaviour and specific activity when manganese is used as the essential divalent cation in place of magnesium. At pH 7.5, the kinetics are Michaelis‐Menten with respect to fructose 6‐phosphate, ATP and manganous ion concentrations; the reaction, as a function of metal ion concentration, is inhibited competitively by excess free ATP and by calcium ions. Between pH 7.0 and 6.5, in the presence of high ATP concentrations, there is a sharp transition to sigmoid kinetics as indicated by an increase of the Hill interaction coefficient from 1 to 4.1. Preincubation of the enzyme with ligands has a marked effect on activity, particularly free divalent metals which cause dissociation and an almost total loss of activity.Studies on the rates of water‐proton longitudinal relaxation at pH 7.5 and 35 MHz indicate that, in the absence of nucleotides, the phosphofructokinase‐manganese system shows low relaxation enhancements (ɛb∼ 2). On titrating with ATP at fixed enzyme and manganese ion concentrations, the enhancement rises sharply, then falls again at very high ATP levels. An analysis of enhancement data at fixed ATP concentrations as a function of enzyme and metal ion concentrations, using a combination of graphical and computational methods, indicates that the protomer of 90000 molecular weight has 2 Mn‐ATP sites with dissociation constant 240 μM and an enhancement for the ternary complex ɛt, of 18. Free ATP binds competitively with a dissociation constant of 3 mM. Line‐broadening studies on the H‐2 and H‐8 resonances of the adenine ring in the high resolution spectrum of ATP reveal that the proton‐manganese distances in Mn‐ATP are almost unchanged in forming the ternary complex with enzyme. The kinetic and magnetic resonance data indicate that Mn‐ATP is the true substrate, which probably binds to the enzyme via a nucleotide bridge.
Effect of Sulfur Binding on Rhodanese Fluorescence
Authors: Finazzi Agrò Alessandro , Federici Giorgio , Giovagnoli Carlo , Cannella Carlo , Cavallini Doriano 
Published: March 2005

Intrinsic fluorescences of the sulfur‐transferring protein rhodanese (or thiosulfate: cyanide sulfurtransferase) and of artificially persulfurated bovine serum albumin were studied. Both are attributable mainly to tryptophan side‐chains. Addition of cyanide or sulfite to these proteins causes a 20–25% increase in fluorescence.
Both proteins exhibit a low intensity absorption band at about 330 nm which disappears on addition of cyanide or sulfite. This absorption can be restored in rhodanese, after the treatment with cyanide, by excess thiosulfate; in the same time the fluorescence decreases to that of sulfur‐containing enzyme.
A change in solvent viscosity, obtained by addition of glycerol, reduces the quenching effect in both proteins. On the basis of these data a long‐range energy‐transfer mechanism between the donor (tryptophan) and an acceptor is proposed. The acceptor involved seems to be one or more persulfide groups (R‐SSH) in both proteins.
The presence of a persulfide group into rhodanese fits very well with the current knowledge of the mechanism of this enzyme.
Effect of Ionic Strength, pH, Amines and Divalent Cations on the Lytic Activity of T4 Lysozyme
Authors: Jensen Harald B. , Kleppe Kjell 
Published: March 2005

The influence of ionic strength, pH, basic amines and divalent cations on the apparent lytic activity of T4 lysozyme on chloroform‐treated Escherichia coli B cells has been studied, and in some cases compared with the effect on hen egg‐white lysozyme.
The lytic activity of T4 lysozyme was found to be markedly dependent on ionic strength and pH. Maximum activity was observed at pH 7.3 at I= 0.07. Very low activity was observed above pH 8.5 whereas maximum activity of hen egg‐white lysozyme at I= 0.06 was obtained at pH 9.2.
Low concentrations of spermine, 40 μM, enhanced the lytic activity of T4 lysozyme 3 to 4‐fold. No activating effect was observed on hen egg‐white lysozyme.
Higher concentrations of spermine, above 100 μM inhibited lysis, presumably by stabilizing the protoplasts formed. The muramidase activity of T4 lysozyme was not inhibited, but rather stimulated also under these conditions.
Several other amines and Mg2+ and Ca2+ were also tested. They exhibited similar effects as spermine. In contrast to spermine, the inhibition observed at high concentrations of Mg2+ is presumed to be due to inhibition of the enzyme.
Studies on the Conformation of DNA‐Dependent RNA Polymerase in Solution by Small‐Angle X‐Ray Measurements
Authors: Pilz Ingrid , Kratky Otto , Rabussay Dietmar 
Published: March 2005

The size and shape of DNA‐dependent RNA polymerase from Escherichia coli K 12 were studied by small‐angle X‐ray measurements. The equilibrium between the monomeric and dimeric form of the polymerase was investigated in dependence on the ionic strength of the solution.Homodisperse solutions of the holo‐enzyme monomer and dimer and of the core enzyme monomer were investigated. For each particle the radius of gyration R, the molecular weight Mr and the volume V were determined; in detail the following values were obtained: RNA polymerase holo‐enzyme monomer (R= 6.2 nm, Mr= 4.9 × 105, V= 9.8 × 102 nm3), holo‐enzyme dimer (R= 9.5 nm, Mr= 9.95 × 105, V= 35 × 102 nm3), core enzyme (R= 6.0 nm, Mr= 3.8 × 105, V= 9.3 × 102 nm3).The hole‐enzyme dimer is equivalent in scattering to a flat hollow cylinder of the following dimensions: length 10 nm, outer diameter 25 nm, inner diameter 5 nm. The shape of the holo‐enzyme monomer is not equivalent in scattering to any body which can be obtained by dividing this cylinder into two halves. The shape of the monomer is similar to that of the core enzyme; both are elongated bodies, which show a cross‐section factor.
Octopine Dehydrogenase
Authors: Olomucki Anna , Huc Claude , Lefebure Francine , van Thoai Nguyen 
Published: March 2005

The amino acid composition of octopine dehydrogenase from muscles of Pecten maximus was determined. The calculate dpartial specific volume of 0.74 allowed us to evaluate the molecular weight of the enzyme as 38000.It is concluded that this enzyme consists of a single polypeptide chain on the basis of the molecular weight determined from equilibrium ultracentrifugation in 6 M guanidine hydrochloride and from electrophoresis on polyacrylamide gel containing sodium dodecylsulfate as well as on the basis of the number of peptides obtained by tryptic digestion. The presence of one essential –SH group per mole of enzyme and of one binding site for NADH are also consistent with the existence of a single active center.No NH2‐terminal amino acid can be detected by reaction with dansyl chloride and with phenyl‐isothiocyanate.
Esterase Activity of Chymotrypsin on Oxygen‐Substituted Tyrosine Substrates
Authors: Kundu Nakuleswar , Roy Sujata , Maenza Frederick 
Published: March 2005

The replacement of the phenolic proton of tyrosine by alkyl, aryl or acyl groups completely abolishes the chymotryptic hydrolysis of tyrosine esters. Similarly, chymotrypsin fails to hydrolyze the tyrosyl bonds of dinitrophenyladrenocorticotropin.
Several model substrates have been synthesized and characterized. The action of chymotrypsin was followed potentiometrically in buffer containing some alcohol. The hydrolysis of the analogous compounds unsubstituted at the phenolic group was normal.
We conclude that inhibition is due, not to an inductive effect, but to a blocking effect which is markedly affected by replacement of the phenolic proton, even with a group as small as a methyl group.
Metabolism of Guanosine Tetraphosphate in Escherichia coli
Authors: Lund Elsebet , Kjeldgaard Niels Ole 
Published: March 2005

In rel+ strains of Escherichia coli guanosine 2′(3′)‐diphosphate‐5′‐diphosphate (ppGpp) was found to accumulate (a) during amino acid starvation of an auxotrophic strain, (b) during heating to the non‐permissive temperature of a valSts strain, (c) during heating of a wild‐type strain to temperatures above 46°C and (d) during treatment with trimethoprim or hydroxylamine. A rapid decay of the accumulated ppGpp was observed after addition of chloramphenicol, tetracycline, fusidic acid, erythromycin, puromycin or colicin E3 to susceptible cultures. All of these antibiotics give a rapid and complete inhibition of protein synthesis. Streptomycin showed a delayed inhibitory action on protein synthesis and had little effect on the level of accumulated ppGpp. The addition of rifampicin also results in a delayed decay of ppGpp.These results indicate that the synthesis of ppGpp is independent of RNA synthesis, but suggest that a coupling exists between ppGpp formation and an idling reaction of one of the initiation steps of protein synthesis.
Properties of the Sarcoplasmic ATPase Reconstituted by Oleate and Lysolecithin after Lipid Depletion
Authors: The R. , Hasselbach W. 
Published: March 2005

The ATPase activity of the sarcoplasmic membranes and the phosphoryl transfer reaction from ATP to the membranes depend on the composition of their lipid phase.
Lipid depletion leads to a drastic (∼ 60%) reduction of phosphoprotein formation. Oleate raises and lysolecithin decreases phosphoprotein formation. In absence of calcium and/or magnesium neither the native nor the delipidated vesicular preparations are phosphorylated.
The ATPase activity of lipid‐depleted vesicular preparations reactivated by oleate or lysolecithin and that of the preparation containing the lipid hydrolysis products after phospholipase A treatment reaches half‐maximal activation at concentrations of ionized calcium between 0.2 and 0.3 μM. The maximal ATP‐splitting rates, however, differ considerably.
Half‐maximum activation by Mg · ATP for the oleate and lysolecithin‐reactivated ATPase is reached at 10 μM and 100 μM, respectively. The corresponding Hill coefficients are 0.5 and 0.6.
For the oleate‐reactivated ATPase an activation energy of about 35 kcal/mol has been calculated which is similar to that of the extra ATPase of native vesicles. The activation energy of the lysolecithin‐reactivated ATPase is about 8 kcal lower.
The ATPase activity of the reconstituted vesicular preparations shows an identical steep decline at pH values >7. An irreversible damage of the vesicular preparations at higher values could be excluded.
The properties of the reconstituted sarcoplasmic ATPase are discussed in relation to the sarcoplasmic calcium transport.
Isolation of a High‐Molecular‐Weight Hybrid between Ribosomal RNA and DNA
Authors: Spadari Silvio , Falaschi Arturo , Mazza Giorgio 
Published: March 2005

The genes for rRNA of Bacillus subtilis have been purified as high molecular weight DNA: RNA hybrids with a DNA/RNA ratio of 1. Two different methods were used. (1) High molecular weight denatured DNA (Mr= 2.2 × 107) was hybridized with rRNA and the excess of single‐stranded DNA was digested with Neurospora crassa endonuclease and Escherichia coli exonuclease I. The hybrid was then purified on Sepharose 4B and two subsequent CsCl gradients. This is a marked improvement on the previously published procedure of Sgaramella, Spadari and Falaschi (1968) insofar as it gives high molecular weight products (8 × 105 for the DNA moiety of the hybrid). (2) A simplified non‐enzymic procedure that used relatively short single‐stranded DNA molecules (Mr= 4 × 106) and several subsequent CsCl gradients led to a hybrid with a DNA: RNA ratio of 1.1 and a molecular weight higher than the previous procedure (1.7 × 106 for the DNA moiety). The DNA of the isolated hybrid, after removal of the RNA moiety, rehybridizes completely with rRNA.After hybridization of rRNA with DNA having a single‐stranded molecular weight of 2.2 × 107, the hybrid bands mainly at a density corresponding to that of denatured DNA; hybrid with a density corresponding to that of 1:1 DNA/RNA ratio can be obtained only if the DNA has been reduced by enzymic digestion or shearing to a single‐stranded molecular weight close to 2 × 106; this indicates that between each set of 16‐S + 23‐S rRNA genes, there must be regions of DNA non‐complementary to rRNA; these regions have a single‐stranded molecular weight that can be estimated as greater than 1.8 × 107.When the isolated DNA is treated with N. crassa endonuclease, in conditions that cause 95% hydrolysis of denatured DNA and 5% digestion of native DNA, it is digested only up to 50%. This suggests the presence of self‐annealing sequences in the rRNA genes, in agreement with the secondary structure of rRNA.
An Induced Mitochondrial DNA Polymerase from Tetrahymena
Authors: Westergaard Ole , Lindberg Birte 
Published: March 2005

The activity of mitochondrial DNA polymerase from Tetrahymena pyriformis can be induced up to 50‐fold by several kinds of DNA damage (irradiation, thymine starvation etc.). The induced mitochondrial enzyme has been purified more than 5900‐fold based on whole treated cells. The purification procedure involved the following steps: (a) isolation of mitochondria from cells with induced enzyme; (b) extraction of the polymerase; (c) chromatography on two columns of phosphocellulose at different pH. Homogeneity studies of the purified polymerase indicate that the enzyme is more than 50% pure.The purified polymerase is able to use both native and heat‐denatured DNA as primer, although the optimal ion concentrations differ quite considerably with the primer. At low Mg2+ concentrations (5–12 mM) optimal conditions are observed for the reaction with heat‐denatured DNA, while the reaction with native DNA primer demonstrates optimum around 45 mM MgCl2. In the reaction with native DNA primer the high MgCl2 concentrations can be partially substituted by high KCl concentrations of same ionic strength. The reactions with the bihelical polymers poly[d(A‐T)] · poly[d(A‐T)] and poly[d(I‐C)] · poly[d(I‐C)] demonstrate optima similar to those found for the reaction with heat‐denatured DNA.The purified DNA polymerase has a temperature optimum around 28°C and heat‐inactivation experiments demonstrate that the enzyme is very labile at the high Mg2+ concentrations required for the reaction with native DNA.
Revised Structure of Aspartokinase I‐Homoserine Dehydrogenase I of Escherichia coli K12
Authors: Falcoz‐Kelly Françoise , Janin Joël , Saari John C. , Véron Michel , Truffa‐Bachi Paolo , Cohen Georges N. 
Published: March 2005

After revision, the molecular weight of the subunit of Escherichia coli K12 aspartokinase I‐homoserine dehydrogenase I is 86000 ± 4000 instead of 60000 as previously published. The enzyme is a tetramer, not an hexamer. The electrophoretic homogeneity of the subunits, the number of unique sequences around cysteinyl and tryptophanyl residues, the determination of the N‐terminal and C‐terminal sequences all point to the probable identity of the four subunits.After revision of the molecular absorption coefficient, a new set of binding data for various ligands of the enzyme is given.
Phylogeny of the Neurohypophysial Hormones
Authors: Acher Roger , Chauvet Jacqueline , Chauvet Marie‐Thérèse 
Published: March 2005

Two oxytocin‐like hormones have been isolated from a cartilaginous fish, the spiny dogfish Squalus acanthias, belonging to the order Pleurotremata. They have been characterized by amino acid analysis and partial sequence determination. The chemical data suggest that the oxytocin‐like hormones are [Val8]‐oxytocin (Valitocin) and [Asn4]‐oxytocin (Aspartocin). The biological properties of natural products are compatible with those previously published for the synthetic analogues of oxytocin. On the other hand arginine‐vasotocin, present in very small amount, has been pharmacologically recognized.In contrast to the other classes of vertebrates, the cartilaginous fishes show a particular neurophyophysial hormone heterogeneity since the three main groups, rays, sharks and chimaeras seem to have different oxytocin‐like hormones. If four species of rays have the same active principles, only one species of shark has so far been investigated and the distribution of the two new hormones remains to be studied. It is not known whether valitocin and aspartocin are present in a single gland of Squalus acanthias or each one in a particular strain.Cartilaginous fishes have arginine‐vasotocin as other nonmammalian vertebrates but the amount in the pituitary gland is comparatively much lower; the quantity per mg of pituitary powder is roughly one thousand times weaker in marine cartilaginous fishes than in bony fishes. This decrease might be explained by the existence of an uraemic regulation of osmotic pressure in the former instead of the current ionic regulation found in the latter.
Utilization of Methionine‐Accepting tRNA Species from Escherichia coli, Ascites‐Tumor Cells, and Yeast in Homologous and Heterologous Cell‐Free Systems
Authors: Drews Jürgen , Grasmuk Hans , Weil Rudolf 
Published: March 2005

The methionine‐accepting tRNA species from Escherichia coli, ascites tumor cells, and yeast were tested for their capacity to function in peptide‐chain elongation in homologous and heterologous cell‐free systems from these organisms comprising polysomes and elongation factors. The following results were obtained.

The utilization of Met‐tRNAfMet in chain elongation is not restricted to mammalian systems but can also be observed in a polysome system from E. coli with initiator tRNA from ascites cells and homologous Met‐tRNAfMet.
In polysome systems from E. coli and ascites tumor cells the magnesium concentrations optimal for the utilization of the homologous methionine‐accepting tRNAs in chain elongation were found to be higher than the Mg2+ ion concentrations at which the discrimination between homologous Met‐tRNAMet and Met‐tRNAfMet is maximal.
Of the three systems studied, the polysome system from yeast is characterized by the most stringent exclusion of any initiator tRNA from functioning in peptide‐chain‐elongation.
Initiator‐tRNA from yeast, while recognized by initiation factors from E. coli, is not utilized for chain elongation in any of the cell‐free polysome systems tested.

Additional results pertaining to the T factor‐induced attachment of all six Met‐tRNA species to ribosomes from E. coli, ascites cells and yeast were in complete agreement with these findings.Finally, the influence of a formylation or acetylation of the α‐amino group on the recognition of Met‐tRNA species from E. coli and ascites tumor cells by factors T1, Tu,s, and a crude initiation factor preparation from E. coli was studied. The presence of a free α‐amino group could be shown to be an important prerequisite for the interaction of aminoacyl‐tRNA and factors T1 or Tu,s. On the other hand, the acetylation of the α‐amino group of Met‐tRNAMet from E. coli appeared to introduce a critical structural element into this tRNA which rendered it suitable for partial recognition by bacterial initiation factors.
Studies on Alkaline Phosphatase
Authors: Byers David A. , Fernley H. Neville , Walker Peter G. 
Published: March 2005

The three common homozygous variants of human placental alkaline phosphatase, types F1, S1 and I1 were partly purified and the concentration of active sites determined using a stopped‐flow procedure. A comparison of the variant steady‐state activities indicated that any differences in catalytic‐centre activity are marginal, both at alkaline pH (with 4‐nitrophenyl phosphate) where phosphorylation is rate limiting and at acid pH (with 4‐methylumbelliferyl phosphate) where dephosphorylation is rate limiting.Though the I1 variant is significantly more heat labile, it is suggested that differences in activity on starch gel and in placental tissue are due to a lower rate of synthesis of the I1 subunit, approximately one half that of the F1 and S1 subunits.It is proposed that where kinetic identity between closely‐related phosphatases is observed, especially with regard to inhibition by l‐phenylalanine, then the catalytic activities are probably identical.