bluuebunny/crossref_metadata_2025 huggingface dataset:
A Nuclear‐Magnetic‐Resonance Study of the Globular Structure of the H1 Histone
Authors: CHAPMAN George E. , HARTMAN Peter G. , CARY Peter D. , BRADBURY E. Morton , LEE David R.
Published: March 2005

The globular structure of the H1 histone has been studied by 1H NMR, as an aid to elucidating its mode of action, and also as a model small protein molecule for investigating the capabilities of NMR for gaining information about the tertiary folding when the structure has not been determined by other techniques. Guanidylation of the lysine residues does not effect the capability of the peptide chain to fold into the native globular structure, suggesting that the lysine residues are on the surface of the structure. Partial assignment has been made of some of the ring‐current‐shifted resonances (i.e. resonances from methyl groups of apolar residues which are shifted by their proximity to the magnetic fields of aromatic rings) by a combination of spin‐decoupling experiments and theoretical ring‐current‐shift stimulations. Substitution of the tyrosine residue of histone H1 considerably weakens the globular structure, and observation of specific changes in the perturbed methyl region of the spectra on substitution indicates the aromatic residues giving rise to ring‐current perturbation of specific peaks in the spectrum. Interproton Overhauser effects are demonstrated, arising from throughspace dipole interaction between protons of the aromatic residues and methyl residues, the resonances of which are perturbed by close proximity to the aromatic rings. The results of these experiments confirm the assignments made from the results of the tyrosine modification experiments.
Effects of Steroid Hormones on the Level of Corticotropin Messenger RNA Activity in Cultured Mouse‐Pituitary‐Tumor Cells
Authors: NAKAMURA Masahiro , NAKANISHI Shigetada , SUEOKA Satoru , IMURA Hiroo , NUMA Shosaku
Published: March 2005

Studies have been made with the mouse pituitary tumor cell line AtT‐20 in culture to determine whether or not the suppression of pituitary corticotropin messenger RNA activity observed upon the administration of glucocorticoids to adrenalectomized rats is due to a direct action of these steroid hormones on the pituitary. The levels of corticotropin messenger RNA activity in AtT‐20 cells treated with various steroid hormones were measured with the use of the cell‐free protein‐synthesizing system derived from wheat germ. The addition of dexamethasone to culture medium reduced the level of corticotropin messenger RNA activity to 30–40% of that in untreated cells. Corticosterone and cortisol exhibited a suppressive effect to a lesser extent. In contrast, nonglucocorticoids such as testosterone and 17β‐estradiol were essentially ineffective. These results indicate that at least part of the glucocorticoid action is exerted directly on the pituitary to suppress corticotropin messenger RNA activity.
dd‐Carboxypeptidase Activity of Membrane Fragments of Mycobacterium smegmatis
Authors: EUN Hyone‐Myong , YAPO Alexandre , PETIT Jean‐François
Published: March 2005

A dd‐carboxypeptidase activity is present in membrane fragments of Mycobacterium smegmatis. Kinetic parameters of the enzymatic activity have been studied using UDP‐N‐glycolylmuramyl‐l‐alanyl‐γ‐d‐glutamyl‐meso‐2,2′‐diaminopimelyl‐d‐[14C]alanyl‐d‐[14C]alanine as substrate. The dd‐carboxypeptidase can be solubilized by Triton X‐100 and Genapol X‐100. It is inhibited by β‐lactam antibiotics although intact cells of M. smegmatis are insensitive to that kind of antibiotics. Inhibition by penicillin G is slowly reversible. By storage, at ‐ 20°C, kinetic parameters and sensitivity to penicillin G vary non‐concomittantly, suggesting a penicillin binding site different from the substrate binding site.
Purification and Some Properties of l‐Glutamate Decarboxylase from Human Brain
Authors: BLINDERMANN Jean‐Marc , MAITRE Michel , OSSOLA Lucienne , MANDEL Paul
Published: March 2005

Glutamate decarboxylase (EC 4.1.1.15) from human brain has been purified 8000‐fold with respect to the initial homogenate. The molecular weight of the native enzyme was found to be 140000 by electrophoresis on a polyacrylamide gradient gel slab. The presence of a single protein band (Mr 67000) on sodium dodecylsulphate/polyacrylamide gel and the existence of only one N‐terminal amino acid suggest that the enzyme consists of two similar if not identical polypeptide chains. The Km of the enzyme at the optimum pH of 6.8 is about 1.3 × 10−3 M for glutamate and 0.13 × 10−6 M for pyridoxal phosphate. The analysis of the effects of various inhibitors of mouse brain glutamate decarboxylase on the human enzyme confirms the strong competitive inhibition caused by 3‐mercaptopropionic acid (Ki= 2.7 × 10−6 M) while the Ki values for allylglycine and chloride ion are 1.8 × 10−2 M and 2.2 × 10−2 M, respectively.
Chorismate Mutase/Prephenate Dehydratase from Escherichia coli K12
Authors: GETHING Mary‐Jane , DAVIDSON Barrie E.
Published: March 2005

The binding of phenylalanine to the allosteric site of chorismate mutase/prephenate dehydratase has been studied by steady‐state dialysis.

Under most of the experimental conditions examined positive co‐operativity was observed for the binding of ligand up to 50% saturation and negative co‐operativity above 50% saturation.

In the presence of 0.4 M NaCl at pH 8.2 the co‐operativity was positive at all phenylalanine concentrations and the maximal stoichiometry of 1 mol of phenylalanine/mol of enzyme subunit was observed. It was concluded that there is a single phenylalanine‐binding site per subunit which is associated with the regulation of each of the mutase and dehydratase activities.

The effects of enzyme concentration, NaCl, temperature and pH on the binding of phenylalanine have been investigated. Neither tyrosine nor tryptophan bound to the allosteric site of the enzyme. Enzyme that was desensitized to inhibition by phenylalanine following modification of three sulphydryl groups with 5,5′‐dithio‐bis (2‐nitrobenzoic acid) did not bind phenylalanine.

The mechanism of co‐operativity, the binding of the enzyme to Sepharosyl‐phenylalanine and the physiological significance of the inhibition of the enzyme by phenylalanine are discussed in terms of the results obtained.
On the Significance of the Prosthetic Group Composition of Citrate Lyase
Authors: BAYER Ernst , EGGERER Hermann
Published: March 2005

Klebsiella areogenes contains two different acyl carrier proteins, one specific for citrate lyase, the other for fatty acid synthetase.

The acyl carrier protein of fatty acid synthetase from K. aerogenes was isolated and compared with the corresponding protein from Escherichia coli and with the acyl carrier protein of citrate lyase from K. aerogenes.

As judged from prosthetic group compositions as well as amino acid and fingerprint analyses, the acyl carrier proteins of the two fatty acid synthetases are nearly identical but different from that of citrate lyase from K. aerogenes.

Therefore, the different prosthetic groups alone cannot be responsible for the different specificities of the acyl carrier proteins of fatty acid synthetase and citrate lyase in K. aerogenes.

The prosthetic group of citrate lyase, phosphoribosyl dephospho‐CoA, apparently represents no incidental, phosphopantetheine‐replacing aberration. The requirement of citrate lyase for the CoA‐like prosthetic group may arise from the substrate requirement of both subunit enzymes of the enzyme complex.
The Amino‐Acid Sequence of Kangaroo Pancreatic Ribonuclease
Authors: GAASTRA Wim , WELLING Gjalt W. , BEINTEMA Jaap J.
Published: March 2005

Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N‐terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobic‐hydrophilic interchanges in the sequence 51–55, which forms part of an α‐helix in bovine ribonuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn‐Val‐Thr (62–64). The enzyme differs at about 35–40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red‐necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found.
Oxidation of Sulfhydryl Groups in Lactate Dehydrogenase under High Hydrostatic Pressure
Authors: SCHMID Gerhard , LÜDEMANN Hans‐Dietrich , JAENICKE Rainer
Published: March 2005

Lactate dehydrogenase from rabbit skeletal muscle in the presence of substrate exhibits irreversible deactivation at hydrostatic pressures beyond 1 kbar [Schmid. G., Lüdemann, H.‐D. & Jaenicke, R. (1975) Biophys. Chem. 3, 90–98]. In the absence of substrate and coenzyme the lability towards pressure is enhanced. The pH dependence of the effect and its inhibition by SH‐protecting agents suggest the oxidation of sulfhydryl groups to be involved in the mechanism of deactivation. Partial deactivation observed at pH 5.5–7.0 becomes complete in the range of the intrinsic pK of cysteine; addition of dithiothreitol and/or EDTA protects the enzyme from complete deactivation, and leads to the residual enzymatic activity observed at pH 7.0. Incubation of the enzyme with dithiothreitol after pressure deactivation at pH 8.5 causes partial reactivation.

From pressure‐dependent measurements of the kinetics of deactivation an activation volume of δV‡=−285 ± 30 cm3· mol−1 is calculated, which exceeds numerical data reported for typical reactions in organic chemistry. Therefore, the assumption can be made that the oxidation of sulfhydryl groups is connected with structural changes in the enzyme in the rate‐determining step of the deactivation. The proposed mechanism may contribute to the toxicity of oxygen towards bacteria under high hydrostatic pressure.
Antibodies against a Synthetic Peptidoglycan‐Precursor Pentapeptide Cross‐React with at least Two Distinct Populations of Uncross‐linked Soluble Peptidoglycan Secreted by Micrococcus luteus Cells
Authors: ZEIGER Allen R. , EATON Stephen M. , MIRELMAN David
Published: March 2005

Antibodies elicited by a synthetic immunogen, (Glu60 Ala40)n [Ala‐dGlu(Lys‐dAla‐dAla)]5.1, related to a major class of peptidoglycan precursors, were purified by an affinity column of Sepharose 4B, to which the hapten, Ala‐dGlu(Lys‐dAla‐dAla), was covalently attached and were then coupled to Sepharose 4B. Low‐molecular‐weight [14C]alanine‐labeled products secreted by Micrococcus luteus cells grown in the presence of penicillin G were applied to the antibody‐coupled gel. The bound material, which should be univalent to the antibody, was eluted completely by 0.02 mM α‐tert‐butyl‐oxycarbonyl‐lysyl‐d‐alanyl‐d‐alanine. High‐molecular‐weight uncross‐linked soluble peptidoglycan secreted by M. luteus cells grown in the presence of penicillin G was completely bound to the affinity column, regardless of whether the soluble peptidoglycan was labeled in its alanine moieties or in its glucosamine and muramic acid residues. A minor fraction of the bound soluble peptidoglycan was released with 0.02 mM tripeptide (‘paucivalent’ fraction). The remainder of the bound material was eluted with 2 mM tripeptide, indicating that a major portion of the high‐molecular‐weight material was ‘multivalent’.

When glycan‐labeled soluble peptidoglycan was stored in buffered saline at – 5°C for several months, a fraction of the labeled material was no longer bound to the affinity gel. Digestion of this non‐binding fraction, as well as the paucivalent and multivalent fractions with hen egg‐white lysozyme, was consistent with the affinity chromatographic data. These results indicate that an amidase present in these preparations cleaved different amounts of peptide from the soluble peptidoglycan preparations, producing strands which are polydisperse with respect to their peptide substitution.
Control of Enzymic Hydrolysis of Polyadenylate Segment of Messenger RNA: Role of Polyadenylate‐Associated Proteins
Authors: MÜLLER Werner E. G. , ARENDES Josef , ZAHN Rudolf K. , SCHRÖDER Heinz C.
Published: March 2005

The role of poly(A)‐associated proteins in the breakdown of poly(A) sequences in both mammalian polyribosomes and in isolated poly(A) · protein complexes has been studied on an enzymic level. Two nucleases (alkaline exoribonuclease and endoribonuclease IV; both isolated from eukaryotic tissue), which preferentially hydrolyze poly(A) sequences, have been applied to determine the susceptibility of poly(A) in dependence on the presence of poly(A) · protein(s).

Polysomes, isolated from L5178y mouse lymphoma cells, do not contain endogenous poly(A) nuclease activity.

The poly(A) segment in polysomes is hydrolyzed by the exoribonuclease, irrespective of the preincubation conditions used. Pretreatment of the polysomes with 1 M urea increases the rate of hydrolysis considerably. In contrast to susceptibility of poly(A) towards the exoribonuclease, the endoribonuclease IV does not hydrolyze poly(A) in mammalian polyribosomes under low salt conditions (140 mM KCl). However pretreatment of the polysomes with 1 M urea renders the poly(A) susceptible to endoribonuclease IV.

Similar results were obtained using poly(A) · protein complex, which has been isolated from mRNA · protein by ribonuclease digestion; two polypeptide species were found to be present in this complex, one with a molecular weight of 79000 and a second one with a molecular weight of 51000. While the poly(A) segment is hydrolyzed by exoribonuclease both after low and 1 M urea salt pretreatment, the homopolymer can be degraded by endoribonuclease IV only after a pretreatment with urea.

In control experiments it was established that under incubation conditions of 1 M urea a poly(A)‐associated protein is dissociated from the homoribopolymer. Therefore we conclude that the susceptibility of poly(A) to nucleolytic enzymes is determined by the interaction with a poly(A)‐associated protein.
Nucleotide Sequence of the Simian‐Virus‐40 DNA Region Coding for the Carboxyl‐Terminal Part of the T Antigen
Authors: VAN HEUVERSWYN Hugo , VAN DE VOORDE André , FIERS Walter
Published: March 2005

Fragment Hind‐B‐C2, the second largest DNA restriction fragment generated by digestion of fragment Hind‐B with the endo R ·EcoRII enzyme, represents 4.4% of the Simian virus 40 (SV40) genome. The complete nucleotide sequence (231 base pairs) of this fragment has been determined by application of the procedure of Maxam and Gilbert (1977). This region is believed to code for the carboxyl‐terminal part of the T antigen. On the basis of the termination code words in the strand with the same polarity as the early mRNA, two open reading frames for translation could be defined. Only one of these, however, extends through the adjacent Hind‐B‐Ci fragment and is therefore believed to dictate the primary structure of the T antigen. It terminates within Hind‐B with a TAA signal. The region, determined by the second open frame, is large enough to code for a protein of Mr about 11000.

The regions in SV40 DNA from which the non‐translated regions of the early and late mRNAs are transcribed overlap over nearly their entire length. Although this spacer region is very (dA + dT)‐rich, a secondary structure model can be proposed which is centered around map position 0.17. The RNA copy of the untranslated region also contains the sequence A‐A‐U‐A‐A‐A, common to all other eukaryotic mRNA molecules sequenced so far; it is present once on the late mRNA and twice on the early messenger RNA. Also a sequence of 6 or 7 successive thymidine residues is present in the early and late DNA strand respectively, 131 and 95 nucleotides beyond the polypeptide termination codons, and these may perhaps play a role in termination of transcription.

Of particular interest may be the carboxyl terminus of the T antigen; the terminal tryptic peptide is 10 residues long, six of which are proline.
Isolation and Chemical Characterization of the Enterobacterial Common Antigen
Authors: MÄNNEL Daniela , MAYER Hubert
Published: March 2005

The isolation of the soluble form of the Enterobacteriaceae common antigen from Salmonella montevideo was achieved by a combination of the phenol/water method and the phenol/chloroform/petroleum ether extraction procedure. A phenol‐soluble fraction highly enriched in the antigen was obtained which was further purified by DEAE‐cellulose chromatography. A fraction eluted with 0.9 M ammonium acetate/methanol was highly active in coating erythrocytes for passive hemagglutination with antisera specific for enterobacterial common antigen and was an active inhibitor of the hemagglutinating system for this antigen.

Chemical analyses showed enterobacterial common antigen to be a linear polymer of 1,4‐linked N‐acetyl‐d‐glucosamine and N‐acetyl‐d‐mannosaminuronic acid units esterified to a small extent by palmitic and acetic acids. These amino sugar and fatty acid components represent about 65–70% of the material. The presence of additional still unknown lipid components which might account for the missing 30% and for the low solubility of the isolated antigen in water is discussed. The molecular weight of the readily soluble part of the inhomogeneous isolated enterobacterial common antigen was determined by sedimentation studies in an analytical ultracentrifuge using methanol as solvent; it was found to be 2700.
Serological and Immunological Properties of Isolated Enterobacterial Common Antigen
Authors: MÄNNEL Daniela , MAYER Hubert
Published: March 2005

Enterobacteriaceae common antigen (sometimes abbreviated to ECA) isolated from Salmonella montevideo by a combination of phenol/water and phenol/chloroform/petroleum ether extraction procedures and further purified by ion‐exchange chromatography was investigated relative to its serological and immunological properties.

The isolated material was highly active in coating erythrocytes for agglutination with antisera specific for enterobacterial common antigen and in inhibition of hemagglutinating systems established for this antigen. With some high‐titered antisera to enterobacterial common antigen the material precipitated in a characteristic bird‐wing pattern after immunoelectrophoresis at pH 8.6. Alkalitreated material gave a single precipitin line indicating that it was more uniform than the untreated enterobacterial common antigen, which was probably partly cross‐linked. An additional weak precipitin line, located close to the antiserum trough, was occasionally observed. Specific serum absorptions with genetically defined ECA‐negative rfe− or rff− mutants did not influence the precipitation pattern at all, whereas similar absorptions with living or heat‐killed ECA‐positive strains removed all precipitating antibodies.

Esterification of the carboxylic groups of the D‐mannosaminuronic acid residues in enterobacterial common antigen leads to a reversible loss of its precipitating capacity, proving that this aminuronic acid is an essential part of the serological determinant of this antigen.

Attempts to immunize rabbits with the isolated, soluble enterobacterial common antigen were unsuccessful. High titers were, however, elicited in rabbits and NMRI mice when the immunization was carried out with the antigen attached to acetic‐acid‐hydrolyzed S. minnesota R595 bacteria according to the technique of Galanos. The specificity of the immune response was ascertained by absorbing the sera with closely related pairs of ECA‐positive and ECA‐negative enterobacterial strains.

The data show that the material isolated as enterobacterial common antigen from S. montevideo has retained the serological and immunological characteristics of the antigen which was first described and defined by Kunin.
Cytochrome c from Schizosaccharomyces pombe
Authors: CLAISSE Maurice L. , SIMON‐BECAM Anne‐Marie
Published: March 2005

Cytochrome c from the fission yeast Schizosaccharomyces pombe has been purified. Its chromato‐graphic and spectral properties are reported and compared to those of iso‐1‐cytochrome c from baker's yeast; the amino‐acid composition is described.

Schiz. pombe cytochrome c has a much lower affinity for Amberlite IRP64 than Sacch. cerevisiae iso‐1‐cytochrome c. Its α absorption band splits into three maxima (cα1, cα2, and cα3) at −190°C; this is unusual in yeasts, as shown by the low‐temperature whole‐cell absorption spectra which were examined in various yeast genera, species, and strains.

A minor component can be separated by Amberlite chromatography. It exhibits the same low‐temperature splitting of the α absorption band as the main fraction and it has a similar amino‐acid composition with a notable exception: it is an unmethylated form of the cytochrome.
The Interaction of Human Hemoglobin with Erythrosin
Authors: KALOUSEK Ivan , JANDOVÁ Danuše , VODRÁŽKA Zdeněk
Published: March 2005

The interactions of erythrosin with deoxyhemoglobin, oxyhemoglobin, carbonmonoxyhemoglobin, methemoglobin, cyanomethemoglobin and hemoglobin α and β chains have been studied by using the equilibrium dialysis, the difference and circular dichroic (CD) spectra and stopped‐flow method. The values of equilibrium and kinetic parameters, as well as CD characteristics, show that in addition to a number of weak binding sites hemoglobin contain four, relatively strong binding sites, one per chain. The properties of the strong binding sites depend on the ligand of the heme group and the charge of the heme group is not directly responsible for this fact. Consequently the properties of deoxyhemoglobin and methemoglobin, differing from the mutually close properties of the other derivatives, confirm that the state of the heme group affects the conformation of hemoglobin molecules in solution. These results are in a good agreement with the classification established on the heme iron spin state.
The Structure of Carbohydrate Chains of Blood‐Group Substance
Authors: DEREVITSKAYA Varvara A. , ARBATSKY Nicolai P. , KOCHETKOV Nicolai K.
Published: March 2005

Twenty individual higher reduced oligosaccharides, having from seven to eleven monosaccharide units, were isolated after sodium borohydride degradation of blood‐group substance H from pig stomach linings. Anion‐exchange high‐pressure liquid chromatography appears to be a very convenient and effective method for this kind of higher oligosaccharide mixtures separation. The oligosaccharide structures were determined by means of periodate oxidation, methylation analysis, partial acid and enzymic hydrolysis. It has been found that all the oligosaccharides investigated can be divided into four series. The oligosaccharides belonging to each series have the common oligosaccharide fragment to which terminal l‐fucose and/or N‐acetyl‐d‐glucosamine residues are attached. Comparison of all the oligosaccharide structures, including tri, penta and hexasaccharides described earlier, shows that the lower oligosaccharides represent the structural element of the higher oligosaccharides.
Translational Regulation of Expression of the Brome‐Mosaic‐Virus RNA Genome in vitro
Authors: ZAGÓRSKI Włodzimierz
Published: March 2005

Total brome mosaic virus (BMV) RNA, comprising four mRNA molecules, was translated in the wheat germ system. It was shown that at a low ratio of total BMV RNA to cell‐free extract all four BMV genes were expressed. At a high excess of total BMV RNA over the cell‐free extract, the only BMV gene translated was the coat protein cistron (RNA 4). The effect was due to diminished initiation of synthesis of non‐coat proteins in the presence of a high excess of template over 23000 ×g extract. This leads to exclusion from the translational machinery of all but coat protein genes. The effect is responsible for regulation of expression of BMV genes in vitro and can be of importance for enhancement of coat protein synthesis as expected in the late phase of virus development in vivo.
Calsequestrin, Myosin, and the Components of the Protein‐Glycogen Complex in Rabbit Skeletal Muscle
Authors: CAUDWELL Barry , ANTONIW John F. , COHEN Philip
Published: March 2005

The protein‐glycogen particle preparation isolated from rabbit skeletal muscle extracts by mild acidification and differential centrifugation shows eight major protein‐staining bands when examined by polyacrylamide gel electrophoresis, five of which have previously been identified as the glycogen debranching enzyme, the α and β subunits of phosphorylase kinase, glycogen phosphorylase and glycogen synthase [Taylor, C., Cox, A. J., Kernohan, J. C. & Cohen, P. (1975) Eur. J. Biochem. 51, 105–115].

The other three major proteins in this preparation have now been identified. Two of the proteins were identified as the heavy chain of myosin and actin respectively, based on their electrophoretic migration, their insolubility in buffers of low ionic strength, and their solubility in buffers containing 0.6 M KCl.

The third protein has been found to be calsequestrin, a major calcium‐binding protein of the sarcoplasmic reticulum. This conclusion was based on the unusual chromatographic behaviour of the protein on DEAE‐cellulose at pH 7.5, its electrophoretic mobility, amino acid composition, and immunological cross reactivity.

A simple method for the isolation of calsequestrin as a by‐product of the purification of glycogen synthase has been developed, by which 70–80 mg of the protein were routinely isolated from 5000 g of muscle (6 rabbits) within five days. The protein was homogeneous by the criterion of polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. The molecular weight determined by the latter technique was 41000 and the absorbance index A280 nm1%, was 11.7. The protein had a very low sedimentation coefficient of 1.97 S indicating that it is either very asymmetric or a largely unfolded protein, in the buffers used in these experiments, a conclusion supported by its abnormal gel filtration behaviour.

Since the three hitherto unidentified components of the protein‐glycogen particle preparation are not enzymes of glycogen metabolism and merely constituents of particulate material (actomyosin and fragments of the sarcoplasmic reticulum) which have coprecipitated with the protein‐glycogen complex, over 95% of the protein attached to glycogen is accounted for by just four enzymes.
Compound I Formation with Turnip Peroxidases and Peroxybenzoic Acids
Authors: JOB Dominique , JONES Peter
Published: March 2005

The kinetics of formation of the compounds I of the turnip peroxidase isoenzymes 1 and 7 with peroxybenzoic acid and a series of substituted peroxybenzoic acids (p‐OCH3, p‐CH3, p‐Cl, m‐Cl, m‐NO2 and p‐NO2) were studied at 25°C. The pH profiles of the observed second‐order rate constants, after correction for the alkaline transitions of the enzymes, indicate that isoenzyme 1 reacts exclusively with unionized peroxy acids whereas with isoenzyme 7, although the major contribution involves reaction with unionized peroxy acid, an additional reaction with peroxy anion is observed. In contrast to the behaviour of horse‐radish peroxidase, (where the rate constants for unhindered unionized peroxybenzoic acids exceed that with H2O2 and the reactions are probably diffusion controlled) the rate constants, kHA, for isoenzyme 1 reaction with unionized peroxy acids are all lower than that with H2O2 and the activation energies indicate chemical control. For isoenzyme 7 the value of kHA with the most weakly acidic peroxybenzoic acids exceeds the rate constant with H2O2. For both isoenzyme 1 and isoenzyme 7 the values of kHA are very sensitive to peroxy acid acidity and in both cases log kHA shows good correlation with pKHA.

The results are compared with each other and with previous data for both turnip and horse radish peroxidases. It is suggested that the multifactorial influences upon the kinetics of compound I formation include (a) diffusion, (b) substrate hydrophobicity, the hydrophobic binding affinity of the active sites of the enzymes and perhaps hydrogen bonding interactions, (c) a substrate‐charge‐type discrimination elicited by the protein at the entrance to the active site, (d) substrate substituent inductive effects which suggest the importance of the generation of highly nucleophilic peroxy anions from neutral hydroperoxides within the active site. These influences are of very variable relative importance in enzymes from different sources and in different isoenzymes from the same source.
The Influence of Prolonged Ethanol Intake on the Levels and Turnover of Alcohol and Aldehyde Dehydrogenases and of Brain (Na + K)‐ATPase of Rats
Authors: GUERRI Consuelo , WALLACE Ruth , GRISOLIA Santiago
Published: March 2005

Levels of liver alcohol dehydrogenase, liver mitochondrial aldehyde dehydrogenase and brain (Na + K)‐stimulated ATPase were determined in control rats and in rats fed an ethanol liquid diet for 7 weeks. The alcohol dehydrogenase level did not change with the diet; however, the levels of mitochondrial aldehyde dehydrogenase and of the (Na + K)‐ATPase were increased 2.5‐fold and 2‐fold respectively.

Also, the levels of these enzymes from brain and liver fractions of rats given a 20% ethanol solution as the sole drinking fluid were determined at intervals from 4 to 18 weeks. A progressive increase was found in the level of aldehyde dehydrogenase and of ATPase.

To ascertain whether synthesis or degradation of these enzymes were responsible for the marked increase in activity the turnover of several liver fractions and of the indicated enzymes were measured by the dual‐labelled isotope technique. Long‐term ethanol administration did not affect the rate of turnover of the liver cell fractions except for the mitochondrial fraction. This fraction showed a marked decrease in the rate of degradation. The degradation constant of the liver alcohol dehydrogenase did not change; however, the rate of degradation of liver mitochondrial aldehyde dehydrogenase (low‐Km) and of brain (Na + K)‐ATPase was decreased.
Synthesis of Carp Proinsulin in Xenopus Ocytes
Authors: RAPOPORT Tom A. , THIELE Bernd J. , PREHN Siegfried , MARBAIX Gérard , CLEUTER Yvette , HUBERT Evelyne , HUEZ Georges
Published: March 2005

Total poly(A)‐containing RNA from Brockmann bodies of carp (Cyprinus carpio) directs the synthesis of authentic carp proinsulin in Xenopus oocytes. Neither preproinsulin nor further processing of pronisulin to insulin can be detected in the oocytes.
Adenosine 3′:5′‐Monophosphate in Perinatal Rat Liver
Authors: DIMARCO Peter N. , OLIVER Ivan T.
Published: March 2005

Developmental changes in the concentration of adenosine 3′:5′‐monophosphate (cyclic AMP) and the effects of glucagon and epinephrine were studied in the perinatal rat liver. Hepatic cyclic AMP concentration doubled tha last day gestation. After birth, the cyclic AMP concentraion continuced to increase and maximal levels were observed on the fifthe postnatal day. Surgical deliveray of foetuses on days 20, 21 and 22 of gestation resulted in a rapid increase in cyclic AMP concentration. Maximal concentrations were reached within one hour of delivery in the day‐21 and day‐22 foetuses. However with surgically delivered day‐20 foetuses, the cyclic AMP concentration increased for at least two hours. Glucagon and epinephrine increased the hepatic cyclic AMP concentration in rats deliver3d surgically on days 20, 21 and 22 of gestation and in postnatal rats. Maximal stimulation by epinephrine was observed in 2‐day‐old rats. Maximal stimulation by glucagon was observed in 10‐day‐old rats. The rsults support the hypothesis that cyclic AMP is the intracelluar effector for the synthesis of some enzymes in perinatal rat. The cyclic AMP concentration in the perinatal rat liver in vivo appears to be controlled by changes in the relative concentrations of plasma glucagon and insulin.
Perinatal Changes in Liver Corticosterone, Serum Insulain and Plasma Glucagon and Corticosterone in the Rat
Authors: DI MARCO Peter N. , GHISALBERTI Ann V. , MARTIM Clayton E. , OLIVER Ivan. T.
Published: March 2005

This report describes some hormonal changes occuring in the rat during the last 3 days of gestation and the first 6 h after surgical deliversy on days 20–22uu ofgestation. In the foetus, the concentration of glucagon increased 2‐fold and that of insulin decreaed from 4.7 to 3.5 ng/ml between days 21 and 22. Consequently the insulin/glucagon molar ratio decreased from 15.6 on day 21 to 5.9 on day 22. After delivery on dasys 21 and 22 of gestation plama glucagon concentration increased 2‐fold and reeached a maximim at 30 min after birth. In addition, the concentration of serum insulin decreased to about 50% of the foetal values by 30 min post partum. Consequentaly the insulin/glucagon ratio decreased to a minimum 30 min after delivery. There was no sinificant change in the concentration of total, that bound to corticosteroid‐binding globulin and free corticosterone in the plasma nor in the concentration of liver corticosterone for 6 h after delivery on day 22 of gestation. Only free plasma corticosterone concentration increased after delivery on day 21 and peaked one hour after birth. The concentration of both pancreatic hommones and corticosterone showed a different development pattern after premauature delivery on day 20 of gestatin. Plasma glucagon increased and plasma insulin decreased for 2 h. Consequentaly the insulin/glucagon ratio decreased from 8.8 at birth to 2.4 at 2 h post partum. The concentration liver corticosterone as well as total, bound and free corticosterone in the plsma incrreased to maximal levels within 1–2 h ofdelivery. The results support the proposal that the relative concentration of insulin and glucagon in the blood regulate the adenosine 3′:5′‐monophosphate concentration and thus enzyme induction in the liver of the perinatal rat. However, the changes in corticosterone concentration wer not consistent with the view that steroids are a primary signal for tyrosine sminotransferase induciton in the newborn rat.
Inhibitory Effects on Lipid Metabolismn in Cultured Cells of ML‐236B, a Potent Inhibitor of 3‐Hydroxt‐3‐methylglutaryl‐Cornzyme‐A Reductase
Authors: KANEKO Osoa , HAZAMA‐SHIMADA Yoko , ENDO Akira
Published: March 2005

Effects of ML‐236B, a potent competiove inhibitor 3‐hydrocy‐3‐methyglutaryl‐CoA reductase, on the lipiod metabolism were studied using mouse L cells and human smin fibrobalsts. ML‐236B completely inhibited sterol sythesis from both [14C]acetate and [14C]octanoate in these cells at a tconcentration of 0.5 μg/ml (11.117 μM). withour affecting thqat form [14C]mevalonate. Syntheses of other lipids adn macromolecules like DNA, RNA and protein were not affected at concentrations up to 5 μg/ml. Concentrations of ML‐236B giving 50% inhibitionof sterol synthesis for [14C]acetate were as low homozygote with familial hypercholesteroloemia. The ML‐236B‐mediated inhibition of sterol syunthesis was readitly reversivle. The complkete inhibition of endogenous sterol synthesis at a higher concentration (5μg/ml) of ML‐236‐B caused a marked inhibition of cell growth even in the presence of exogenous cholesterol contained in while gfatel calf serum as lipoproteins. This inhivition of growth was prevented by teh presence in the culture medium of mevalonate, but not by acetate, thus indficating that ML‐236B inhibit cell growth by specifically interferring with mevalonate wynthesis. It is further concluded thjat slight activity of endogenous sterol synthesis, which provides enfogenous copounds generated from meavalonate, may be essential to the growth of cells even when sufficeient amounts of cholesterol are availavel to the cells.
The Regulation of Glycogen Metaabolism
Authors: NIMMO Gilllian A. , COHEN Philip
Published: March 2005

Inhibitor‐1 is a protein which inhibits phosphorylase phosphatase only when it has been phosphorvlated by cyclic‐AMP‐dependent protein kinase [Huang, F. L. and Glinsmann. W. H. (1976) Eur. J. Biochem. 70, 419–426].

Inhibitor‐1 was purified by a heat treatment at 90°C, precipitation with ammonium sulphate, chromatography on DEAE‐cellulose, gel filtration on Sephadex G‐100, and finally recromatography of the phosp0horylated protein on DEAE‐cellulose. The protein was purified 4000‐fold and 1.5 mg per 1000 g muscle was obtained in seveven days corresponding to an overall yield of 15–20%. The purified protein was in a state approaching homogeneity as judged by the criteria of polyacrylamide‐gel electrophoresis and ultracentrifugal analysis. The concentration of inhibitor‐1 in vivo was calculated to be 1.5 μMM, which is at least as high as the concentrationof phosphorylase phosphatase.

The amino acid composition of inhibitor‐1 showed several unusual features. Glutamic acid and proline accounted for nearly one third of the residues, tyrosine, tryptophan and cysteine were absent, and the content of aromatic amino acids was very low.

The molecular weight measured by sedimentation equilibraium centrifugation was 19 200 and by amino acid analysis was 20 800. These values were lower than the mol. wt of 26000 determined empirically by gel electrophoresis in the presence of sodium dodecyl sulphate, and much lower than the apparent molecular weitht of 60000 estimated by filtraation on Sephadex G‐100. The gel filtration behaviour, stability to heating at 100°C and amino acid composition suggest than inhibitor‐1 may possess little ordered structure.

The phosphorylated form of inhibitor‐1contained close to one molecule of covalently bound phosphate per mole of protein, which is consistent with previous finding of a unique decapeptide sequence at the site of phosphorylation, Ile‐Arg‐Arg‐Arg‐Arg‐Pro‐Thr(p‐Pro‐Ala‐Thr‐[Cohen, P., Rylatt, D. B. and Nimmo, G. A. (1977) FEEBS Lett. 76, 182–186].

The phosphorylated form of inhibitor‐1 inhibited phosphorylase activity (0.02 U) by 50% at a concentration of only 7.0 nM in the standard assay, but the phosphorylated decapeptide was 1000–2000 times less effective as an inhibitor.
Purification and Regulation of Glucose‐6‐Phosphate Dehydrogenase from Obligate Methanol‐Utilizing Bacterium Methylomonas M15
Authors: STEINBACH Rolf A. , SAHM Hermann , SCHÜTTE Horst
Published: March 2005

Glucose‐6‐phosphate dehydrogenase has been purified 192‐fold from Methylomonas M15 grown on methanol as sole source for carbon and energy. The purification procedure involved N‐cetyl‐N,N,N‐trimethylammonium bromide precipitation, ion‐exchange chromatography and affinity chromatography on blue‐dextran‐Sepharse. The final enzyme preparations were homoveneous as judged by acrylamide gel electrophoresis and by sedimentation in an ultracentrifuge. The molecular weight of the enzyme was calculated to be 108000 ± 5000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels indicates that the enzyme is a dimer composed of two probably identical subunits each having a molecular weight of 55000.

The enzyme exhibits activity with either NAD or NADP as electron acceptor. Mg2+ or an other biblent cation is not essential for enxyme activity. The Kmvalues were found to be 0.2 mM for NAD, 0.014 mM for NADP, 0.29 mM for glucose 6‐phosphate with NAD and 0.11 mM for glucose 6‐phosphate with NADP as electron acceptor. The reduced coenzymes NADPH and NADH as well as ATP are powerful inhibitors of the enzyme.

The intracellular levels of glucose 6‐phosphate, ATP, NAD and NADP were measured and found to be approximately 2.5 mM, 1.5 mM, 1 mM and 2 mM respectively, during exponential growth. From a consideration of the substrate pool sizes and types of inhibitors, it is concluded that this enzyme may function in two roles in the cell: NADH reproduction for energetics and NADPH production for reductive biosynthesis.
γ‐Glutamyl Transpeptidase: Sidedness of Its Active Site on Renal Brush‐Border Membrane
Authors: HoRIUCHI Seikoh , INOUE Masayasu , MORINO Yoshimasa
Published: March 2005

Sidedness of γ‐glutamyl transpeptidase, an integral glycoportein of rat renal brusch border membrane, was investigated and the active siyte of the membrane‐bound enzyme was shown to face to the luminal side of the renal tubules on the basis of the two following observations. First, γ‐glutamyl transpeptidase activity exhibited by purified brush border membrane vesicles was effectively inhibited by an S‐acetyldextran polymer (molecular weight: 215 000) derivative ofglutathione, which is believed to be impermeable through plasma membranes. Secondly, the membrane‐bound γ‐glutamyl transpeptidase was quantitatively released by treating vesicles with papain immobilized to Sephadex G‐10 beads.

Strict stoichiometry of affinity labeline of rat renal γ‐glutamyl transpeptidase, which possesses one active site per molecule [Inoue, M., Horiuchi, S., and Morino, Y. (1977) Eur. J. Biochem. 73, 335–342; Tate, S. S., and Meister, A. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 931–935], was utilized to compare the molecular size of the detergent‐solubilized enzyme with that of the papain‐solubilized enzyme. Thus, 1 mol redioactive 6‐diazo‐5‐oxo‐l‐norleucine was covalently incorporated into 102000 g detergent‐solubilized enzyme and 64000 g papain‐solubilized enzyme. Both enzyme foorms of γ‐glutamyl transpeptidase are composed of two non‐identical subunits, the large and the small subunit. Molecular weightgs of these subunits were determined in sodium dodecylsulfate/polyacrylamide gel electrophresis from the retardation coefficients obtained from Ferguson plots. The result indicated the size difference between these two enzyme forms could be largely accounted for by the size difference in the large subunits. Thus, iot is likely that the oligomeric γ‐glutamyl transpeptidase was anchored into the brush nborder membrane mainly via te large subunit of tghe enzyme.
Homologous RNA Polymerase binding to Escherichia coli DNA
Authors: PEDONE Francesco , BALLARIO Paola , DI MAURO Ernesto
Published: March 2005

The number and the distribution of the sites of Escherichia coli DNA that from stable complexes with the homologous RNA polymerase (class A sites according to Hinkle and Chamberlin [3]) have been investigated. Almost all the DNA can bind RNA polymerase, even when fragmented at short (undergenic) size; this general non‐promoter‐specific binding is highly labile and is not temperature‐dependent. The range of RNA polymerase DNA ratios that give rise to the stable temperature‐dependent complexes was examined.

The amount and the distribution of class A complexes were studied analysing the dissociation of complexes formed by RNA polymerase on DNA fragments of various length. The E. coli genome appears to form 3.8 × 103 stable complexes; the majority of these complexes shows a short‐range distribution (800–1200 base pairs). The rest is more widely spaced (1200–6000 base pairs).
A Structural Model of Cholinergic Synaptic Vesicles from the Electric Organ of Torpedo marmorata Deuduced from Density Measurements at Different Osmotic Pressures
Authors: BREER Heinz , MORRIS Stephen J. , WHITTAKER Victor P.
Published: March 2005

Density measurements made on cholinergic synaptic vesicles from the electric organs of Torpedo marmorata at different osmotic pressures are consistent with the following structural model of the vesicle.

The particle behaves like a sphere 80–100 nm in diameter bounded by a semi‐permeable membrane. The bulk of its soluible constituents are in true solution at physiologica osmolalities. The limiting mambrance is approximately 4–5‐nm thick, suggesting that it contains large areas of phospholipid bilayer exposed to its bathing medium.

The limiting membrane takes up abouit 26% (v/v) of the particle, a further 34% (v/v) of which is osmotically active water and 31% (v/v) hydrated core material at 800 mosmol/l. The byouant density of the membrane is 1.132 g · cm−3. the density of the hydrated core material is approximately 1.05 g · cm−3.

The membrane is selectively permeable to small molecules when subjected to hypo‐osmotic stress. It is proposed that this occurs by the formation of small transient pores in the lipid bilayer of the membrane, which are induced by stretching caused by the osmotic pressure change.
Synthesis of Lens Crystallins in Xenopus Oocytes as Determined by Quantitative Immunoprecipitation
Authors: ASSELBERGS Fred A. M. , VAN VENROOIJ Walther J. , BLOEMENDAL Hans
Published: March 2005

Total poly(A)‐containing calf lens mRNA was microinjected into Xenopus oocytes and synthesis of α, β, and γ‐crystallins was demonstrated. By a method of quantitative immunoprecipitation the rate of translation of purified 14‐S αA2‐crystallin mRNA was compared with virtually the same molar amounts of mRNA, taking into account the larger size of the αA2‐crystallin mRNA. Kinetics of translation were also very similar and both mRNAs were translated with similar rate and efficiency for at least two days. It was estimated that 20–30 polypeptide chains per hour per mRNA molecule were synthesized.
Purification and Partial Characterization of Three Extracellular from Cellulomunas sp.
Authors: BÉGUIN Pierre , EISEN Harvey
Published: March 2005

Three extracellular cellulases have been purified from cultures of Cellulomonas. One was found in solution in the cell‐free supernatant and two others were found to be bound to the cellulose added as a carbon source. The free enzyme and one of the cellulose‐bound enzymes bind to Sephadex. The two cellulose‐bound enzymes are glycosylated. The three enzymes behave as endocellulases towards solble carboxymethyl‐cellulose and have little activity on cellulose powder.
Structural Studies of Iron and Cobalt Tetrasulfonated Phthaslcyanine‐Globin Complexes
Authors: PRZYWARSKA‐BONIECKA Helena , TRYNDA Liliana
Published: March 2005

The structue of the complexes of iron and cobalt tetrasulfonated phthlaocyanines with globin has been investigated by circular dichroism (CD), electron paramagnetic resonance (EPR) and polyacrylamide gel electrophoresis.

Electrophoretic investigations and the molecular weight estimation indicates that the model complexesin the solutionss are dimers.

It is evident from the results of CD measurements that the incorporation of the iron or cobalt tetrasulfonated phthalocyanine into apohemoglobin significantly increases the helical structure oif the protein and causes an appearance of the induced Soret and visible Cotton effects. Unlike methemoglobin, several discrete transition energies in the CD Soret band of FE(III)L‐globin are oberved which suggests an inequivalence of the subunits within this complex. This suggestion is supported by EPR studies, which show that the iron atoms in Fe(III)L‐globin are in two low electronic states. Ele3ctronic structures of the cobalt ions in Co(II)L‐globin and oxyCo(II)L‐globin are similsar to those of coboglbin asnd oxycoboglobin, repsectively, as is proved by EPR results. On this basis we conclude that the oxygen adduct of Co(II)L‐globin can be described as a superoxide ion corrdinated to a formaslly cobaltic phthalocyanine compound.