bluuebunny/crossref_metadata_2025 huggingface dataset:
Zur Wirkung von Liganden auf das Assoziations‐Dissoziations‐Gleichgewicht des Methämoglobins der Flußneunaugen (Lampetra fluviatilis L.)
Authors: Behlke Joachim , Scheler Werner
Published: March 2005

The dissociation‐association equilibrium of lamprey methemoglobin as a function of protein concentration, pH, and ligandation of the heme Fe3+ has been studied using sedimentation analysis.

The sedimentation constant s°20,w of MetHb in the pH range 4.5 < pH < 10 amounts to 1.94 S leading to a calculated molecular weight of 17000 which corresponds to the Hb protomer.

At pH 7.5 the MetHb · N3 complex is monomeric. Below pH 5.7 and at high protein concentration the dimer of 34000, s°20,w= 3.13 S, is formed, the dimerization being accompanied by an uptake of two protons per protomer: (a) 2 MetHb + 2 N3−+ 2 H+⇌ (H+· MetHb · N3)2. At high concentrations of protein and azide the association of MetHb · N3 becomes a function of pH only: (b) 2 MetHb · N3+ 2 H+⇌ (H+· MetHb · N3)2. The pK value of equilibrium (b) is found to be 6.7 pointing to a heme‐linked imidazole determining the equilibrium.

As compared to the azide complex, other ligands, such as F− > OCN− > CN− > SCN−, favour the dimerization of lamprey MetHb to a lesser degree.

Dimerization of the heme polypeptide chains in the MetHb‐ligand complexes and their association in DeoxyHb supposedly involves different aminoacids in the interface between the protomers.
Oxygen‐17 Hyperfine Splitting in the Electron Paramagnetic Resonance Spectrum of Enzymically Generated Superoxide
Authors: Bray Robert C. , Pick Frances M. , Samuel David
Published: March 2005

The electron paramagnetic resonance spectrum of 17O in O2− generated by reduction of O2 by two related systems has been observed using the rapid freezing method. The systems were: (a) during re‐oxidation of xanthine oxidase following reduction with dithionite and (b) during steady‐state oxidation of xanthine catalysed by xanthine oxidase. The spectrum was similar to that reported for O2− adsorbed on MgO. Both the 11‐line spectrum from 17O17O− and the six‐line spectrum from 17O16O− were detected. The results provide final confirmation that one‐electron reduction of oxygen can occur in biological systems.
Thermolysin: Kinetic Study with Oligopeptides
Authors: Morihara Kazuyuki , Tsuzuki Hiroshige
Published: March 2005

Thermolysin is a well‐known protease which exhibits its specificity against hydrophobic amino acid residues such as l‐leucine, l‐phenylalanine, etc. whose amino groups donate the susceptible peptide bonds (amino‐endopeptidase). The present study was undertaken to investigate the effects of neighboring residues surrounding the sensitive amino acid residues at the amino‐side in peptide substrates. For the purpose, a kinetic study was made using various synthetic oligopeptides such as Z‐A‐(Gly)n↑–Leu‐Ala or Z‐Gly–↑Leu‐(Gly)n‐B (A or B = various d‐ or l‐amino acid residues; n= 0, 1 and/or 2; the arrow shows the bond split) as substrates. Other kinetic or inhibition studies were also made. These studies indicated that the specificity is affected by at least three amino acid residues on the N‐terminal side and by two amino acid residues on C‐terminal side from the sensitive amino acid residue (at amino‐side) in peptide substrates. The effect of each of the five neighboring amino acid residues for appearance of the specificity was similar with that of the corresponding one which had been observed in a neutral protease of Bacillus subtilis, but that was not completely the same.
On the Conformation of Phenylalanine Specific Transfer RNA Studies on Size and Shape of the Molecule by X‐Ray Small Angle Scattering
Authors: Pilz Ingrid , Kratky Otto , Cramer Friedrich , VON DER Haar Friedrich , Schlimme Eckhard
Published: March 2005

The scattered X‐ray intensities from dilute solutions of tRNAphe (yeast) in 0.05 M Tris buffer at pH 7.5 were measured at 17°. The radius of gyration R (24.4Å), the molecular weight M (26100) and the volume V (41500 Å3) were determined.

A comparison of experimental with theoretical scattering curves of different bodies shows that the shape of the molecule cannot be well represented by a simple triaxial body. The calculated scattering curves which fit the measured scattering data best are for an elongated body with two different cross‐sections.

No significant change of X‐ray small angle scattering was observed on increasing the temperature from 17° to 40°. At higher temperature the radius of gyration is increased with increasing temperature and at 70° the molecule has a random coil conformation.
Biosynthèse du noyau naphtoquinonique des ménaquinones bactériennes
Authors: Guérin M. , Leduc Mireille M. , Azerad Robert G.
Published: March 2005

Incorporation of some 14C‐ or 3H‐labeled compounds in menaquinones of various Gram + or Gram – bacteria has been tried during bacterial growth or with resting cells of Mycobacterium phlei, Mycobacterium avium, Bacillus megaterium, Micrococcus lysodeikticus, Aerobacter aerogenes, and Proteus vulgaris. [14C]p‐Hydroxybenzaldehyde and [3H]3,4‐dihydroxybenzaldehyde are not incorporated into the menaquinones; [14C‐ring labeled]p‐hydroxybenzaldehyde is incorporated into ubiquinone‐8 of P. vulgaris. [U‐14C]shikimic acid (or [1,2‐14C2]shikimic acid) are good precursors of the naphthoquinone ring of M. phlei menaquinone. Only a slight incorporation of [1‐14C]α‐naphthol into menaquinones could be demonstrated, except with a mutant strain of A. aerogenes which incorporated also with a high yield [14C]2‐methyl‐1,4‐naphthoquinone and [14C]1,4‐naphthoquinone. A comparison of radioactivity dilutions of these precursors indicated that menadione is probably not a true intermediate in menaquinone biosynthesis.
Studies on Methionyl Transfer RNA Synthetase from Escherichia coli K12 Amino Acid Composition and Relation of Sulfhydryl Groups to Enzyme Activities
Authors: Lawrence Françoise Jeane
Published: March 2005

The purified methionyl‐tRNA synthetase isolated from Escherichia coli K 12 was shown to possess 24 cysteine residues, but no disulfide bridges from the amino acid composition and titration of the sulfhydryl groups with para‐mercuribenzoate and 5–5′‐dithio‐bis (2‐nitrobenzoic acid). All of these sulfhydryl groups were titratable with para‐mercuribenzoate, but in the presence of l‐methioninyl adenylate (a structural analogue of methionyl adenylate), eight of them were protected from reaction.

The enzyme catalyzes two reactions: (I) the conversion of methionine and ATP to methionyl adenylate enzyme complex, and (II) the transfer of the methionine from the complex to the tRNA.

Titration of the sulfhydryl groups with para‐mercuribenzoate in the presence of l‐methioninyl adenylate resulted in a loss of ability to transfer the methionine to tRNA (reaction II) while the ability to catalyse reaction I was essentially unaffected. This result demonstrated that: (a) there are different sites on the enzyme for the two reactions, and (b) different sulfhydryl groups in the enzyme are involved in reactions I and II.
Characterization of Protein Bound to Rapidly‐Labelled RNA in Polyribosomes from Rat Liver
Authors: Olsnes Sjur
Published: March 2005

A complex of rapidly labelled RNA and protein that can be liberated from polysomes by treatment with EDTA has been characterized in attempts to establish whether it is derived from the nuclear particles (“informofers”) containing RNA of DNA‐like nucleotide composition.

After short time of labelling with [3H]orotic acid, polysomes were isolated, either by a method involving Triton X‐100, or without any detergent (“free” polysomes). After treatment of polysomes with EDTA, the sedimentation rate of the rapidly‐labelled RNA was considerably higher than that expected for pure RNA. CsCl density gradient centrifugation demonstrated that the buoyant density of the labelled material had a value intermediate between that of pure RNA and protein. Treatment with sodium deoxycholate or with 0.5 M KCl, increased considerably the buoyant density of the labelled material, while that of the ribosomal sub‐units was only slightly affected. The results indicate that in polysomes the rapidly labelled RNA exists in a complex with protein that can be selectively removed by sodium deoxycholate or KCl.

The proteins released by treatment with deoxycholate or with KCl were characterized by polyacrylamide gel electrophoresis. One of these proteins behaved similarly to the protein‐component of the nuclear particle containing RNA of DNA‐like base composition. It is suggested that this protein follows the rapidly labelled RNA (presumably messenger RNA) during its transfer from the nucleus to the polysomes.
Monosubstituted 2,2‐Dimethyl‐3‐Formyl‐L‐Thiazolidine‐4‐Carbonyl‐Insulins
Authors: Lindsay David G. , Shall Sydney
Published: March 2005

The treatment of insulin in aqueous solution at pH 6.9 and 8.5, with the hydroxysuccinimide ester of 2,2‐dimethyl‐3‐formyl‐l‐thiazolidine‐4‐carboxylic acid leads to the incorporation of thiazolidine residues into the protein.

Examination by DEAE‐Sephadex chromatography of the products formed shows the presence of three products. The relative amount of products formed depends upon the reaction conditions. Characterisation of these products by end‐group analysis, cellulose acetate electrophoresis, amino acid analysis of the performic acid‐oxidised chains and treatment with TPCK‐treated trypsin suggests that they are monosubstituted derivatives formed by reaction at each of the free amino groups of insulin.

The effect of modification on the biological activity and crystallisation properties of the hormone is discussed as well as the application of the preparation of covalent heavy atom derivatives of proteins.
R 17 Coat Protein Interaction with Multi‐Stranded R 17 RNA
Authors: Knolle Peter , Hohn Thomas
Published: March 2005

Phage coat protein reacted with preparations of multi‐stranded RNA at high protein inputs to yield complexes containing phage‐like particles. RNA complexes containing predominantly double‐stranded RNA (replicative form, “RI”) were considerably less efficient in promoting particle formation than those rich in single strands (replicative intermediate, “RF”). Similarly, binding of coat protein at low input multiplicities was less efficient with “RF” than with “RI” preparations.
The So‐Called Non‐Histones from Acid‐Treated Calf Thymus Chromatin
Authors: Sonnenbichler Johann , Nobis Peter
Published: March 2005

When chromatin from calf thymus is treated with dilute hydrochloric acid under mild conditions variable proportions of histones become bound to the DNA, thus becoming insoluble and appearing as non‐histones. This is confirmed by model experiments in which artificial mixtures of pure histones and DNA were treated with acid.

The acid insoluble residual proteins formed are strongly bound (presumably covalently) to the DNA, but can be separated by density gradient centrifugation at pH 11.5. Some properties of these “non‐histones” are discussed. Their amino acid analyses are different from those of the histones. The differences, also recognizable in model experiments must originate from the mild acid treatment in presence of DNA and suggest modifications of amino acids. These “non‐histones” are considered to be masked histones, because after strong alkali treatment definite histone fractions can be recognized by electrophoreses.

The conclusion is drawn that mild acid treatment of chromatin artificially produces “non‐histones”.
Specificity in the Binding of Escherichia coli Ribosomes to Natural Messenger RNA
Authors: Revel Michel , Greenshpan Haim , Herzberg Max
Published: March 2005

Initiation factor C(F2) promotes 30 S ribosome binding to RNA templates. However, another initiator protein, factor B(F3), determines the specific recognition in messenger RNA of the initiation signal for translation. Template competition experiments demonstrate that this signal is not merely the codon AUG.
Rifampicin Resistance of RNA Polymerase in the Binary Complex with DNA
Authors: Sippel Albrecht E. , Hartmann Guido R.
Published: March 2005

Addition of rifampicin to a preincubated mixture of RNA polymerase and DNA does not lead to inhibition of the subsequent initiation of RNA chain synthesis. The formation of the enzymatically active binary complex between RNA polymerase and DNA, however, proved to be very sensitive to the action of the inhibitor. Once the complex has formed in the absence of substrate the enzyme is resistant to the antibiotic. The presence of sigma factor is not required for the formation of the resistant complex. In the presence of sigma, however, more resistant complex is formed between enzyme and DNA containing sigma specific initiation sites. The binary complex between RNA polymerase containing sigma factor and sigma specific DNA is more rapidly inactivated during prolonged incubation with rifampicin than a complex formed with unspecific DNA or in the absence of sigma. Rifampicin may therefore be used as a tool to discriminate sigma specific and unspecific active complexes of RNA polymerase and template.
Purification of Rat Liver Acetyl Coenzyme A Carboxylase and Immunochemical Studies on its Synthesis and Degradation
Authors: Nakanishi Shigetada , Numa Shosaku
Published: March 2005

Acetyl coenzyme A carboxylase from rat liver was isolated in homogeneous form as evidenced by sedimentation velocity experiments and Ouchterlony double diffusion analyses. The specific activity and biotin content of the purified enzyme preparation were similar to those of pure chicken liver enzyme isolated previously.

Antibody against rat liver acetyl‐CoA carboxylase was prepared by injecting rabbits with the purified enzyme. With the use of this antibody, the mechanisms were studied by which the level of liver acetyl‐CoA carboxylase activity was varied in fasted rats, in those subsequently refed a fat‐free diet and in diabetic rats.

Immunochemical analysis demonstrated that the wide variations in the level of acetyl‐CoA carboxylase activity in liver extracts derived from rats under the different dietary and hormonal conditions were accompanied by proportionate changes in the quantity of immunochemically reactive protein. This indicates that the changes in the activity level are determined by changing quantities of the carboxylase protein.

Isotopic leucine incorporation studies showed that the relative rate of synthesis of liver acetyl‐CoA carboxylase was decreased 1.9‐fold and 1.7‐fold by fasting and diabetes, respectively, whereas increased 4.0‐fold by fat‐free refeeding. The rate of degradation of the carboxylase, expressed as half‐life, was found to be essentially the same in normal, refed and diabetic rats, i.e. 59, 55 and 59 h respectively, and accelerated in fasted rats, i.e. 31 h. Thus, the increase or decrease in the carboxylase quantity in refed or diabetic animals, which are presumably in a steady state, can be ascribed to a rise or fall in the rate of enzyme synthesis, whereas the decrease in the carboxylase quantity in fasted animals, which are not in a steady state, is due both to diminished enzyme synthesis and to accelerated enzyme degradation. On the basis of these results, it is discussed that the control of enzyme quantity by changes in the rate of enzyme degradation may play an important role when the animal deviates from a steady state for adjustment to a new environment.
The Structure of the Porcine Pancreatic Secretory Trypsin Inhibitor I
Authors: Tschesche Harald , Wachter Elmar
Published: March 2005

Porcine pancreatic secretory trypsin inhibitor I (56 residues) was rendered sensitive to tryptic digestion by reduction and carboxymethylation. Nine tryptic fragments were formed by the action of trypsin. They were separated by taking advantage of the size fractionating and ion exchange properties of Bio Gel P‐2 in 0.01 N pyridine. The major tryptic peptides were purified on SE‐Sephadex C‐25.

The amino acid sequence of each peptide was determined by a special technique of Edman degradation using p‐bromophenyl‐isothiocyanate. This reagent facilitated the identification of the extracted p‐bromophenyl‐thiohydantoines by mass spectrometry. The characteristic fragment and molecular ions are emphasized by a significant double peak of a mass difference Δm/e= 2, which is caused by the isotope ratio of the p‐bromosubstituent. In general this technique permitted up to thirteen consecutive steps of degradation and the unequivocal positioning of the amides.

The complete amino acid sequence of the polypeptide trypsin inhibitor was determined.
Purification and Comparison of the N‐(5′‐Phosphoribosyl)anthranilic Acid Isomerase/Indole‐3‐glycerol Phosphate Synthetase of Tryptophan Biosynthesis from Three Species of Enterobacteriaceae
Authors: Mcquade John F. , Creighton Thomas E.
Published: March 2005

A measure of evolutionary divergence between three species of the enterobacteriaceae was made by studying the physical properties of a protein common to all three, N‐(5′‐phosphoribosyl)‐anthranilic acid isomerase indole‐3‐glycerol phosphate synthetase, a bifunctional enzyme of the tryptophan biosynthetic pathway. The protein was purified to near homogeneity from Aerobacter aerogenes, Escherichia coli, and Salmonella typhimurium.

The purified proteins were found to be very similar in their ultraviolet absorption spectra, fluorescence emission and excitation spectra, and molecular weights. Various determinations of the molecular weights varied from 4.4 to 5.1 × 104 daltons, and no significant differences were observed between the purified proteins and the enzymes in crude extracts. The electrophoretic behaviour of each of the three purified proteins on polyacrylamide gels with sodium dodecyl sulphate were similar and consistent with each enzyme consisting of a single polypeptide chain.

Peptide patterns of each of the proteins were obtained following digestion with trypsin plus chymotrypsin. Close homology in primary structure between the proteins was suggested by the observation that approximately half the peptides of any two proteins were identical. Comparison with similar data from a previous study on the α subunit of tryptophan synthetase suggests that there has been no great difference in the rate of divergence of the α subunit and phosphoribosylanthranilate isomerase‐indole‐glycerol‐phosphate synthetase in the species studied.
Induced Fit in Yeast Hexokinase
Authors: Fuente Gertrudis Dela , Lagunas Rosario , Sols Alberto
Published: March 2005

In the nucleoside triphosphatase activity that yeast hexokinase shows in the absence of acceptor substrate the Km values for MgATP and for MgITP, as well as the Ki for MgGTP, are markedly different from those in the phosphotransferase reaction. In the presence of lyxose or xylose, two non‐phosphorylable analogues of the sugar substrates, these Km or Ki values change to those corresponding to the hexokinase reaction. Besides this effect on the apparent affinities, lyxose and xylose can enhance by about 20 times the maximal rate of hydrolysis of the terminal phosphoryl group of the nucleoside triphosphates. In addition, xylose slowly induces a specific, apparently irreversible, inactivation of the enzyme, both as hexokinase and as nucleoside triphosphatase. The relative hydrolytic activities on ATP, ITP, and GTP resemble the relative efficiencies of these nucleotides as phosphate donors, as well as those in the inactivation of the enzyme in the presence of xylose. The presence of a phosphate group on carbon 6 of glucose does not prevent the sugar effects on the Km values of hexokinase for the nucleoside triphosphates, although it reduces their rates of hydrolysis. These effects indicate the occurrence of an “induced fit” by which certain sugars specifically modify the binding of the nucleoside triphosphate substrates and enhance the labilizing action of the enzyme on their terminal phosphoryl bond. Moreover, hexokinase seems to induce a peculiar configuration of the pyranose ring of the sugar substrate, probably a boat form. When bulky substituents prevent the latter to be formed, sugars, even if they are still able to bind to the enzyme, do not induce an increased labilization of the nucleoside triphosphates as glucose or lyxose do.
The Kinetics of Yeast Hexokinase in the Light of the Induced Fit Involved in the Binding of its Sugar Substrate
Authors: DelaFuente Gertrudis , Sols Alberto
Published: March 2005

The kinetics of the initial reaction rates of yeast hexokinase, both in the forward and in the backward direction, have been re‐examined in the light of recent findings concerning the properties of the ATPase activity of hexokinase and its modification by certain non‐phosphorylable analogues of the sugar substrates.

A single pattern has been found in the double reciprocal plots even if the concentrations of both the acceptor and the donor substrates are varied over a wide range, with ITP as well as with ATP as donor substrates. A similar pattern has been found for the backward reaction.

These results are interpreted as indicative that in the forward as well as in the backward reaction, the interconversion of the ternary complex of the enzyme with both substrates is the limiting step. This fact and the complex effect of glucose 6‐phosphate as an initially‐added product, prevents in principle the distinction between the possible kinetic mechanisms leading to the ternary complex. Nevertheless, taking into account the change in affinities for the nucleotide substrates specifically induced by certain sugars, it becomes possible to formulate a plausible hypothesis. It is postulated that binding of a sugar to an enzyme‐nucleotide complex results in an abortive ternary complex. Accordingly, formation of active ternary complexes in the hexokinase reaction requires the sequential addition of a nucleotide substrate to a binary complex of the enzyme with a sugar substrate.
Steroids in Germfree and Conventional Rats
Authors: Eriksson Håkan
Published: March 2005

Radio‐gas chromatography and gas chromatography‐mass spectrometry were used to study unconjugated metabolites of administered [4‐14C]corticosterone and [4‐14C]pregnenolone in faeces from germfree and conventional female rats. 3α‐Hydroxy‐5α,17α‐pregnan‐20‐one, 3α‐hydroxy‐5α,17β‐pregnan‐20‐one, 3β‐hydroxy‐5α,17β‐pregnan‐20‐one, 3β,15β‐dihydroxy‐5α‐pregnan‐20‐one, 3β,15α‐dihydroxy‐5α‐pregnan‐20‐one and 3α,16α‐dihydroxy‐5α‐pregnan‐20‐one were characterized as metabolites of pregnenolone whereas 3α,20β‐dihydroxy‐5α‐pregnan‐11‐one and 3β,20β‐dihydroxy‐5α‐pregnan‐11‐one were identified as metabolites of corticosterone in the conventional rats. Two other 3,15‐dihydroxypregnan‐20‐one isomers, tentatively identified as the 3α,5α,15α(and 15β) epimers, were also found. In addition 3β‐hydroxy‐5‐androsten‐17‐one, 5α‐androstane‐3β,17β‐diol, 5α‐androstane‐3α,7α,17β‐triol, 3α(and 3β),19‐dihydroxy‐5α‐pregnan‐20‐one and 5α‐pregnane‐3α(and 3β),20α‐diol were recovered in the free steroid fraction in faeces from conventional female rats. The amounts of these steroids were too small to permit a study of the degree of labelling.

Although some radioactivity was present in the free steroid fraction from the germfree animals no steroid could be identified in this fraction. The findings are discussed with particular reference to enzyme activities of the intestinal flora.
Excretion of Steroid Hormones in Adults
Authors: Eriksson Håkan , Gustafsson Jan‐Åke
Published: March 2005

The steroids in urine from a pregnant woman have been studied by gas chromatography‐mass spectrometry. The major part, 64%, of the steroids identified occurred in the glucuronide fraction, whereas the free, monosulphate and disulphate fractions contained 24%, 5% and 7%, respectively. The compounds had the following general structures: 3‐hydroxy‐17β‐pregnan‐20‐one, 3α‐hydroxy‐5β,17α‐pregnan‐20‐one, 3α‐hydroxy‐5α‐androstan‐17‐one, pregnane‐3,20‐diol, 3α,21‐dihydroxy‐5β‐pregnan‐20‐one, 3α,16α‐dihydroxy‐5β‐pregnan‐20‐one, 3,15β‐dihydroxy‐pregnan‐20‐one, pregnane‐3,16α,20‐triol, 5β‐pregnane‐3α,17α,20α‐triol, 3β,16α‐dihydroxy‐5‐androsten‐17‐one, 5‐pregnene‐3β,20α‐diol, 5α‐pregnane‐3,20α,21‐triol, 5‐androstene‐3β,16,17‐triol, oestriol, 15‐hydroxyoestriol and pregnane‐3,16,20,21‐tetrol. In the free and glucuronide fractions the C21O2 steroids mainly had a 5β configuration whereas about 90% of the mono‐ and disulphurylated C21O2 steroids had a 5α structure. The results are discussed in relation to what is known about faecal excretion of steroids during pregnancy.
Glycine Formation via Threonine and Serine Aldolase. Its Interrelation with the Pyruvate Formate Lyase Pathway of One‐carbon Unit Synthesis in Clostridium kluyveri
Authors: Jungermann Kurt A. , Schmidt Wilhelm , Kirchniawy F. Hans , Rupprecht Eckhard H. , Thauer Rudolf K.
Published: March 2005

14C‐labeling experiments with growing Clostridium kluyveri have shown that two pathways each are operative for glycine and one‐carbon unit synthesis: about two‐thirds of the cell's glycine is formed from threonine and one third from serine; serine simultaneously yields about a quarter of the cell's C1‐units, while the remaining three‐quarters are derived from CO2via the pyruvate carboxyl group.

The key enzymes, threonine aldolase and pyruvate formate lyase as well as serine aldolase interrelating glycine and C1‐unit synthesis, could be demonstrated in cell‐free lysates in activities sufficient to account for the anabolic requirements during growth. The substrate specificity, cofactor requirements and Km values indicate that threonine aldolase and serine aldolase are two separate, constitutive and pyridoxal phosphate dependent enzymes.
An Optical Rotatory Dispersion Study of 6‐Phosphogluconate Dehydrogenase
Authors: Rippa Mario , Pontremoli Sandro , Fasella Paolo
Published: March 2005

The optical rotatory dispersion of purifed 6‐phosphogluconate dehydrogenase in the 500 to 195 nm region was examined in the presence and absence of its coenzymes and substrates. Identical curves were obtained in all cases.

In particular, coenzyme binding does not cause an extrinsic Cotton effect. In this respect, 6‐phosphogluconate dehydrogenase, like several other dehydrogenases, differs from alcohol dehyudrogenase.

The ultraviolet portion of the optical rotatory dispersion curve suggest that 6‐phosphogluconate dehydrogenase may contain other structures besides α‐helix, β‐structure and random coil.
The Subunit Structure of Thiolase
Authors: Harris J. Ieuan , Gehring Ulrich
Published: March 2005

Thiolase from pig heart has been carboxymethylated in 6 M guanidine‐HCl, and the N‐terminal sequence of the carboxymethylated protein is shown to be uniquely Val‐Ser‐Lys. A trypsin digest has been examined by peptide mapping techniques and the number of peptides (42–46) detected is in good agreement with the total number of lysine and arginine residues calculated from the amino acid composition of the enzyme tetramer and assuming 4 identical chains per 170 000 g of protein. No evidence for the presence of more than one type of subunit was found when both the untreated and the carboxymethylated proteines were examined by polyacrylamide and starch gel electrophoresis in 6 M urea. From these results it is proposed that thiolase is composed of four very similar and probably identical protein chains each comprising approximately 400 amino acids including one catalytically active cysteine.
Action Patterns of Phosphorylase and Glycogen Synthetase on Glycogen
Authors: Parodi Armando J. , Mordoh José , Krisman Clara R. , Leloir Luis F.
Published: March 2005

The action patterns of liver and muscle glycogen synthetases and of muscle phosphorylase b on glycogen samples of different molecular weight and on β‐amylase limit dextrins were studied. For this purpose a method for measuring the number of newly added glucose residues that are at non‐reducing ends was developed.

It was found that glucose transfer to the non‐reducing ends of glycogen catalyzed by liver glycogen synthetase and muscle phosphorylase followed a Poisson distribution.

The number of outer chains in the glycogen molecules available to both enzymes appeared to be smaller than the actual number of outer chains in the polysaccharide. The number of such available chains diminished as the glycogen was heavier. For the same glycogen sample, the number of available chains to phosphorylase appeared to be equal or smaller than that to glycogen synthetase.

It was found that muscle phosphorylase transferred 1.2 to 1.4 glucose moieties successively per outer chain, independently of the molecular weight of the glycogen.

The number of glucose units added successively to the non‐reducing ends of glycogen by liver glycogen synthetase increased from 1.7 to 6.8 with the molecular weight of the polysaccharide.

Both phosphorylase and liver glycogen synthetase transferred more glucose units in a repetitive way to the same outer chain of the β‐amylase limit dextrin than to the same outer chain of the undegraded glycogen.

Muscle glycogen synthetase transferred a greater number of glucose units successively per outer chain of glycogen than the liver enzyme.
Purification of Animal Neurotoxins
Authors: Miranda François , Kupeyan Charles , Rochat Hervé , Rochat Catherine , Lissitzky Serge
Published: March 2005

The purification of eleven neurotoxins from the venom of three scorpions (Androctonus australis Hector, Buthus occitanus tunetanus and Leiurus quinquestriatus quinquestriatus) has been performed by Sephadex G‐50 filtration (with and without recycling) and equilibrium chromatography on Amberlite CG‐50, CM‐Sephadex C‐50 and DEAE‐Sephadex A‐50. All the chromatographic steps were carried out in ammonium acetate buffers. Purity of the neurotoxins was assessed by amino acid analysis and zone electrophoresis. All have a molecular weight of about 7000 daltons and do not contain methionine. As shown by amino acid composition and chain length scorpion neurotoxins show a high degree of homology.
Ultraviolet Difference Spectral Studies on Concanavalin A
Authors: Hassing Gordon S. , Goldstein Irwin J.
Published: March 2005

The interaction of concanavalin A, the phytohemagglutinin of the jack bean, with a variety of glycosides has been studied by the technique of ultraviolet difference spectroscopy. Whereas methyl α‐d‐gluco‐ and manno‐pyranoside gave rise to relatively low intensity difference spectra, p‐nitrophenyl α‐d‐mannopyranoside and α‐d‐glucopyranoside yielded large difference spectra upon interaction with concanavalin A. Using this technique as a measure of concanavalin A activity, it was demonstrated that the protein specifically binds low molecular weight carbohydrates at much lower pH values (e. g. pH 2.4) than previously believed. Although polysaccharides are also bound at these low pH values, they are not precipitated by concanavalin A. Molecular weight studies in acid media indicate that the protein does not dissociate and it is suggested that electrostatic repulsion of the protein molecules due to their high net positive charge prevents protein‐polysaccharide lattice formation and hence failure of the complex to precipitate.
On the Morphology of Phytohemagglutinin from Phaseolus vulgaris Seeds
Authors: Höglund Stefan , Dahlgren Karin
Published: March 2005

A purified sample of phytohemagglutinin molecules from Phaseolus vulgaris seeds was shown to be homogenous by analytical ultracentrifugation, electrophoresis and by electron microscopy. A test for the agglutination capacity of phytohemagglutinin a” in the contrast agent used for electron microscopy indicated that the contrast agent did not interact with the phytohemagglutinin a” molecule to destroy its biological activity.

The phytohemagglutinin a” molecules were distributed uniformly in the electron microscopic specimen, where they appeared as small light dots against the background. The general shape of the projection of the molecule was almost rectangular with the minimum mean dimensions: 77 Å X 43 Å. Often it was also observed that phytohemagglutinin a” seemed to be irregularly constructed, and that it might consist of two chain units, joined lengthwise. An approximation of the molecular weight of phytohemagglutinin a” from its size and partial specific volume gave a value just below 100000; this value is close to the molecular weight of the monomer of phytohemagglutinin a” of 80000 daltons which was obtained from the chemical composition.
Large Scale Purification of A‐Protein from Bacteriophage R17
Authors: Osborn Mary , Weiner Alan M. , Weber Klaus
Published: March 2005

A new method is described for maximizing the yield of A‐protein per liter of crude lysate. It involves a modified growth procedure for bacteriophage R17 which allows titers in excess of 1013 plaque forming units per ml, and a new procedure for purifying the A‐protein by acetic acid treatment of the whole phage.
Specific Staining for Electron Microscopy of Murein Sacculi of Bacterial Cell Walls
Authors: Formanek Helmut , Fobmanek Sibylle
Published: March 2005

The murein sacculi of the cell walls of Spirillum serpens consist of a two‐dimensional network of polysaccharide chains covalently connected with oligopeptides. Each oligopeptide in the murein sacculi is a stainable locus which reacts with one heavy atom compound by addition of a stoichiometric amount of the staining agent to a sacculi suspension.

The single free amino groups of the oligopeptides are specifically stained by stoichiometric amounts of the heavy atom compounds 3‐hydroxy‐mercuri‐4‐hydroxy‐5‐carboxy‐benzenesulfonic acid, anhydro‐3‐hydroxymercuri‐4‐toluene‐sulfonic acid, Zeise's salt, platinum blue and tri‐bromoacetaldehyde.

In a two‐step staining reaction sulfur compounds are bound to the free amino‐ or carboxyl groups of the oligopeptides by reaction with propylenesulfide, N‐acetyl‐DL‐homocysteine‐thio‐lactone and S‐acetylmercaptosuccinic anhydride. These sulfur atoms then react specifically with stoichiometric amounts of 2,4,6‐tris‐acetoxymercuri‐3‐acetaminotoluene. Stained and unstained murein sacculi are observed by high resolution electron microscopy. Unstained murein sacculi can hardly the recognized in bright field and do not expose any structural details. Sacculi specifically stained show distinct contrast and reveal a network pattern in which the stain is localized as discrete particles of about 5 Å diameter in periods of about 10 Å.
Immunological Properties of Amylose, Dextran and Polyvinylalcohol Conjugated with Polytyrosyl‐Peptides
Authors: Sorg Clemens , Rüde Erwin , Westphal Otto
Published: March 2005

Tyrosyl‐peptide side‐chains were linked to amylose, dextran and polyvinylalcohol in order to study their influence on the immunological properties of these polymers. For this purpose aminoethyl groups were introduced by reacting the polymers with ethylenimine. Side‐chains consisting on the average of six to eight tyrosine residues were bound to these amino groups by polymerization ofN‐carboxy‐L‐tyrosine‐anhydride. The polytyrosyl conjugates were found to be weakly immunogenic in rabbits with respect to the antibody response obtained upon primary and secondary injections. The antibodies formed were directed against the polytyrosyl side‐chains and in the case of polytyrosyl‐aminoethyl‐dextran also against the dextran backbone. These results indicate that the polytyrosyl side‐chains are of major importance in determining the immunological properties of these conjugates.
Chemical Characterization of Tetanus Toxin and Toxoid
Authors: Bizzini Bernard , Blass Judith , Turpin André , Raynaud Marcel
Published: March 2005

The amino acid analysis of tetanus toxin as purified from bacterial extracts or from culture filtrates of Plectridium tetani, was carried out. The results that have been obtained support the assumption of the identity of both types of toxin.

Tetanus toxoid amino acid composition was comparatively determined. Significant decreases in the amounts of some amino acids were found. These deficits are accounted for within the reactions with formaldehyde which lead to the formation of Mannich‐like derivatives.

The presence of 6 free SH‐groups on the molecule of toxin could be substantiated. Two of these groups are difficult to reveal. The existence of two disulfide bridges was established. These results are discussed, in particular, with regard to the expression of the toxicity.

The presence of only one N‐terminal amino acid residue could be revealed.
Neutral Steroids in Urine during Pregnancy
Authors: Jänne Olli A. , Vihko Reijo K.
Published: March 2005

Three 15‐hydroxylated C21 steroids, 3β,15α‐dihydroxy‐5α‐pregnan‐20‐one, 3β,15α‐dihydroxy‐5β‐pregnan‐20‐one and 5‐pregnene‐3β,15β,17α,20α‐tetrol, were identified and measured in late‐pregnancy urine by gas‐liquid chromatography and gas chromatography‐mass spectrometry. All these steroids were present in urine as monosulphates, as evidenced by their chromatographic properties on Sephadex LH‐20 chromatography and by their cleavage by solvolysis. Quantitatively, 5‐pregnene‐3β,15β,17β,20α‐tetrol was the most abundant compound among these 15‐hydroxylated C21 steroids in the urine pool studied.

The precursors and origin of the steroids identified are discussed.
The Partial Specific Volume of Chymotrypsinogen A in Aqueous Urea Solutions
Authors: ŠKerjanc Jože , Doleček Valter , Lapanje Savo
Published: March 2005

The partial specific volume of chymotrypsinogen A in water and aqueous urea solutions has been determined from density measurements. The partial specific volume of the protein first increases with urea concentration, reaches maximum, decreases, reaches minimum, and then increases again. An interpretation of this behavior has been attempted considering especially the binding of urea to the protein and imperfect atomic packing in the native protein. From dilatometric experiments the differences between the partial molar volumes of the protein in water and urea solutions of different concentrations, respectively, have been obtained. The agreement of the values of these differences with those calculated from the partial specific volumes is satisfactory. It can be seen that the volume changes reflect the interaction of urea with the protein which eventually results in complete unfolding, i. e., denaturation, in about 8 M urea. Subsequent papers will deal with further aspects of this denaturation.
Quantitative Correlation between the Distribution of Anions and the pH Difference across the Mitochondrial Membrane
Authors: Palmieri Ferdinando , Quagliariello Ernesto , Klingenberg Martin
Published: March 2005

The factors regulating the distribution of anions between the inner and outer mitochondrial phase have been investigated in the presence of respiratory inhibitors and oligomycin.

As the external pH is decreased, the ratio of the intra‐ to extramitochondrial concentration of added anions increases with acetate, malate and phosphate, and with citrate in the presence of malate. In contrast, the uptake of thiocyanate does not change significantly with the pH.

In the pH range used, the log of the anion distribution ratio is proportional to the pH difference across the mitochondrial membrane for acetate, malate and phosphate.

The anion distribution ratio is highly sensitive to pH at low external concentration and becomes largely independent of pH at high external concentration.

In double reciprocal plots of intra‐ against extramitochondrial concentration, it was found that the capacity (Am) for the uptake of phosphate, acetate, malate and succinate is independent of pH. The slopes (A1/2/Am) increase with pH due to the increase of the external concentration required for half maximal uptake.

The distribution ratio of phosphate and malate is proportional to the relative “free capacity” (α) at all the external pH tested. The dependence of the distribution ratio on the external pH is maximal for α= 1 and nil for α= 0.

The following relation holds for the dependence of the slope A1/2/Am on Δ pH: log Am/A1/2=a+bΔ pH. This relation was used to obtain the equation describing the dependence of the anion distribution on the Δ pH and on the capacity.

It is concluded that the distribution of anions, such as acetate, phosphate and Krebs cycle intermediates, at equilibrium can be explained essentially as a function of two parameters: (a) the pH difference across the mitochondrial membrane (b) the capacity of the mitochondria for anions.
The Amino Acid Sequence of Neurotoxin I of Androctonus australis Hector
Authors: Rochat Hervé , Rochat Catherine , Miranda François , Lissitzky Serge , Edman Pehr
Published: March 2005

The complete amino acid sequence of neurotoxin I of the North African scorpion Androctonus australis Hector collected in Algeria (area of Chellala) is described.

S‐Methylation of cysteine residues formed by reduction of disulphide bonds was found useful for their identification in sequence determination using the phenylisothiocyanate method.

From the N‐terminal sequence (38 residues) established in the protein sequenator, and from the sequence of chymotryptic peptides the complete sequence of the protein has been deduced. The molecule consists of a single polypeptide chain cross‐linked by four disulphide bridges. An isoneurotoxin (I′) has been found in the venom of A. australis collected in Tunisia (area of Tozeur). It differs from neurotoxin I in the replacement of the valine residue in position 17 by an isoleucine residue.
The Activation of Mutant β‐Galactosidase by Specific Antibodies
Authors: Melchers Fritz , Messer Walter
Published: March 2005

An immunosorbent was prepared of antiserum against purified wild type β‐galactosidase coupled to Sepharose. Extracts of wild type Escherichia coli K12 3300 cells and of eleven mutant cells producing proteins activatable by specific antibodies were adsorbed to this immunosorbent. The adsorbed proteins could be eluted from the immunosorbent by 8 M urea. By co‐electrophoresis in polyacrylamide gels containing 8 M urea, 0.01 M mercaptoethanol and 1% sodium dodecylsulfate in a buffer at pH 7.2 subunits of all the eleven [3]leucine‐labelled mutant proteins were found to have the same size (molecular weight 130000) within the accuracy of the method (± 15000) as subunits of [14C]leucine‐labelled wild type enzyme. After renaturation from urea solutions the molecular weights of the native forms of the eleven [3H]leucine labelled mutant proteins were compared to that of the 14C‐labelled native wild type enzyme by co‐sedimentation in sucrose gradients. The native proteins were found to be identical in size as well (molecular weights 520000—540000). Thus, activation of these eleven mutant proteins by specific antibody occurs at the four subunit structure level.

Such purification on immunosorbents represents a rapid, easy and generally applicable method to be used in systems with immunologically cross‐reacting mutant proteins, where the wild type protein can be obtained in sufficiently pure form.
Energy‐Dependent Quenching of Chlorophyll a Fluorescence in Isolated Chloroplasts
Authors: Wraight Colin A. , Crofts Antony R.
Published: March 2005

A light‐induced, energy dependent quenching of chlorophyll a fluorescence in the presence of 2,3,5,6‐tetramethyl‐p‐phenylene diamine (diamino‐durene) and 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea is described for isolated chloroplasts, and is related to the formation of the high‐energy state of photophosphorylation.

The quenching is distinguished from the normal type, operative through the redox state of the Photosystem II quencher, by its light‐intensity dependence, reversibility in the dark and sensitivity to uncoupling agents.

The sensitivity of the quenching to uncouplers and ionophorous antibiotics closely parallels that of photophosphorylation.

A similar quenching is described for other electron transport systems in the absence of dichlorophenyl‐dimethylurea, when it is distinguished from Q‐quenching on a time‐basis.

The pH‐optimum for the uncoupler‐reversible quenching is about pH 8.0 for all electron flow systems used. This apparent discrepancy with the pH optimum for XE‐formation is discussed.

Two other types of slow quenching seen during the Hill reaction with ferricyanide are described. One is found to be uncoupler enhanced and is related to acceleration of electron transport giving rise to a lower level of reduced Q in the steady state; the other is an irreversible loss of total fluorescence yield, which is most pronounced in coupled chloroplasts, and it is suggested that it is due to photochemical destruction of bulk pigment molecules via a side reaction of the excited state of chlorophyll.
Turnover Rates and Intracellular Pool Size Distribution of Citrate Cycle Intermediates in Normal, Diabetic and Fat‐Fed Rats Estimated by Computer Analysis from Specific Activity Decay Data of 14C‐Labeled Citrate Cycle Acids
Authors: Sauer Frank , Erfle James D. , Binns Michael R.
Published: March 2005

Specific radioactivity curves of citrate, malate, glutamate and aspartate were measured in vivo in liver from normal, fat‐fed and diabetic rats with [1‐14C]acetate as precursor at time intervals of 0.1 to 20 min. A mathematical model of 14 pools and 40 interconnecting flux rates been constructed and by computer analysis on the Univac 1108 flux rates and intra‐ and extra‐mitochondrial metabolite pool distribution calculated from the measured specific radioactivity curves. Flux rates and pool distributions were varied by using an automatic minimization routine to minimize the difference between observed and theoretical values until an acceptable fit of computer generated specific radioactivity time curves to experimentally observed specific radioactivities was obtained. Steady state kinetics were assumed, i.e. the model was fixed with no change in pool sizes over the measured time interval. The main conclusions were:

A state of non‐equilibration of isotope exists for the acetyl‐CoA and acetylcarnitine, citrate, malate, aspartate, glutamate and α‐ketoglutarate pools distributed between cytoplasmic and mitochondrial compartments.

Cytoplasmic pools of citrate, acetyl‐CoA and acetylcarnitine account for the slow rate of radioactivity decay observed for these metabolites in vivo.

Key flux rates were acetate entry into the citrate cycle of 0.36, 0.28 and 1.05 μmoles × min−1× g liver−1 in normal, fat‐fed and diabetic rats. Citrate turnover rates were 1.21, 0.37 and 1.94 μmoles × min−1× g liver−1 for normal, fat‐fed and diabetic rats. The acetate pool was 0.23 μmoles/g liver in normal, 0.44 μmoles/g liver in fat‐fed and 0.38 μmoles/g liver in diabetic rats. Metabolite pool distribution was greater in the extra‐ than intramitochondrial space: the shift was most marked in the diabetic rat where the intra‐ to extramitochondrial malate distribution was in the ratio of 1:21.5.
The Cleavage of Prolyl Peptides by Kidney Peptidases
Authors: Dehm Peter , Nordwig Arnold
Published: March 2005

Peptides of the X‐Pro‐Y type were found to be readily cleaved by crude swine kidney homo‐genates. Using Gly‐Pro‐Hyp as a substrate, a peptidase releasing glycine was isolated and partially purified from the microsomal fraction of the homogenate. The purification steps used included treatment of the kidney microsomes with n‐butanol, and chromatography of the protein fraction thus released on DEAE‐cellulose as well as gel filtration on Sephadex G‐200. The increase in specific activity was approximately 250‐fold.

The purified preparation consisted of two enzymatically active components exhibiting molecular weights of one million and two millions, respectively. There is evidence that these components are lipoproteins, i.e. membrane fragments of different size.

The activity of the enzyme was optimal at pH 8 and was greatly increased in the presence of manganese ions and albumin. Since the peptidase required a prolyl residue in the second position of the substrate, the designation “X‐prolyl‐aminopeptidase” is suggested. Although the enzyme acted on substrates of different chain length, the reaction rates were greatest with peptides of the structure Gly‐Pro‐Y. Therefore, a role of X‐prolyl‐aminopeptidase in the degradation of collagen appears plausible.