bluuebunny/crossref_metadata_2025 huggingface dataset:
The Specificity of Viral Sialidases
Authors: CORFIELD Anthony P. , WEMBER Margret , SCHAUER Roland , ROTT Rudolf
Published: July 2010

The action of sialidases from Newcastle disease virus (NDV), influenzaA2 virus (IA2V) and fowl plague virus (FPV) on sialyloligosaccharide substrates containing α2 ‐ 3, α2 ‐ 6 or α2 ‐ 8 linkages was studied.

In all cases 2‐ 3‐linked sialic acids were preferentially released. Compared with II6Neu5AcLac, all 2–6‐ linked substrates, including sialyl‐N‐acetyllactosamine and its asparaginyl derivative, a urinary hexasaccharide and Neu5Ac(2–6)GalNAc were cleaved at improved rates by NDV and less by FPV sialidases. In the case of IA2V sialidase the asparaginyl oligosaccharide was very poorly cleaved, illustrating a variation in viral strain specificity.

A decrease in relative rates was observed in the order NDV >IA2V > FPV for substrates with 2–3 linkages relative to II6Neu5AcLac. The greatest relative rate was 470‐fold higher. The 2–3‐linked sialyl‐N‐acetyl‐lactosaminylasparagine and IV3Neu5AcLcOse4 were poor substrates for the IA2V sialidase, but the rates were greater than with the 2–6‐linked substrates.

The ganglioside substrate II3Neu5AcLacCer showed lower activity than its oligosaccharide analogue, but neither II3Neu5AcGgOse4Cer nor its oligosaccharide were substrates.

The Km values for 2–6‐linked substrates were generally of the order 10 mM while those for the 2–3‐ linked substrates were approximately 1 mM. The V values were consistently higher for the 2–3‐linked substrates. IV3Neu5AcLcOse4 showed high Km and very high V values, while the 2–8‐linked disialyllactose showed this trend only with NDV enzyme, the IA2V and FPV sialidases exhibiting high Km and low V values.

The results are discussed in the light of the current knowledge of viral sialidase specificity and relative to the binding of virus particles to cell surfaces.
A Cleavage Site of Ribonuclease F
Authors: GUREVITZ Michael , WATSON Ned , APIRION David
Published: July 2010

A new endoribonuclease activity, RNase F, was partially purified from Escherichia coli cells. This activity can specifically cleave a precursor RNA molecule (of species 1) isolated from T4‐infected cells [N. Watson & D. Apirion (1981) Biochem. Biophys. Res. Commun. 103, 543–551], The cleavage results in products which are very similar to RNA molecules found in the cell, generating a 3′‐phosphate and a 5′‐hydroxyl groups. The cleavage takes place between a cytosine and an adenine moiety, within a possible loop and stem structure; the cut is in the border between the double‐stranded and single‐stranded regions of this structure.

The specificity of this enzyme could be the introduction of a cleavage near the 3′ ends o f tRNA molecules and other RNAs like species 1 which could resemble tRNA in their three‐dimensional structure.
Quantification of Pathways of Glucose Utilization and Balance of Energy Metabolism of Rabbit Reticulocytes
Authors: SIEMS Werner , MÜLLER Marianne , DUMDEY Renate , HOLZHÜTTER Hermann‐Georg , RATHMANN Jörg , RAPOPORT Samuel Mitja
Published: July 2010

In this work it is demonstrated that glucose constitutes the main substrate of energy metabolism of rabbit reticulocytes under aerobic conditions in the presence of 5 mM glucose. Amino acids and fatty acids are minor sources of energy. The shares of processes utilizing glucose in reticulocytes were estimated from tracer experiments. A new mathematical technique used permits the derivation of closed terms for the specific radioactivity of single positions of C atoms of the metabolites of the citrate cycle. By means of regression analysis, the undeter‐mined flux rates in the citrate cycle were calculated.

On the basis of the data an overall balance sheet of glucose utilization and of ATP generation is given. About 45% of the glucose of reticulocytes is catabolized via the citrate cycle, about the same percentage yields lactate. Only 2% of the glucose was oxidized in the oxidative pentose pathway whereas the remainder is used for the formation of serine and glycine required for hemoglobin synthesis.

These results are related to knowledge about the main processes utilizing ATP in reticulocytes, i.e. the synthesis of hemoglobin and the energy‐dependent proteolysis. Our approach to the investigation of metabolic relations in the reticulocytes can be applied to other tissues in which equilibria between large metabolite pools play a role
Rates of Flux through the Pentose Cycle in Perfused Rat Liver
Authors: KUEHN Alexander , SCHOLZ Roland
Published: July 2010

A method for the determination of substrate flux through the pentose cycle was developed employing [l‐14C]glucose in experiments with perfused rat livers. The method consists first of a kinetic analysis which differentiates between the production of 14CO2 from [l‐14C]glucose via the pentose cycle and via the citrate cycle and, second of a calculation of the specific radioactivity of the hexose monophosphate pool from measured rates of glycolysis and the specific radioactivity of lactate released into the perfusate. The method was validated by experiments comparing the results of tracer infusions with [l‐14C]glucose, [6‐14C]glucose and [3‐14C]pyruvate.

In livers from fed rats perfused with 10 mM glucose, the rate of substrate flux through the pentose cycle was around 0.2 μmol ×min−1× g−1; it was about 20% of the substrate flux via glycolysis. The kinetic data were inconsistent with the existence of an L‐type pentose cycle in liver.
Cucumber Mosaic Virus RNA 3
Authors: GOULD Allan R. , SYMONS Robert H.
Published: July 2010

The complete sequence of the 2193 residues of RNA 3 (Mr 746000) of cucumber mosaic virus (Q strain) was determined by a combination of chemical and enzymic sequencing techniques utilizing cloned DNA fragments. The nucleotide sequence of RNA 3 also gave the complete sequence of 1027 residues of RNA 4 (Mr 349000), which codes for the viral coat protein and is derived from the 3′ end; there are 53 untranslated nucleotides at the 5′ end of RNA 4. The nucleotide sequence provided the amino acid sequences of the two proteins coded for by RNA 3: the 5′‐terminal 3A protein with 333 amino acids (Mr 36700) and the 3′‐terminal viral coat protein with 236 amino acids (Mr 26200). These two coding regions are in the same reading frame, are separated by an intercistronic region of 123 nucleotides and are flanked by two untranslated regions of 94 nucleotides at the 5′ terminus and of 263 nucleotides at the 3′ terminus.

Secondary structure models are postulated for parts of the RNA 3 sequence. These are considered to be important in the control of the translation and replication of RNA 3 and in the processing of RNA 3 to give RNA 4.
Complete Primary Structure of Protein Phosphatase Inhibitor‐1 from Rabbit Skeletal Muscle
Authors: AITKEN Alastair , BILHAM Terry , COHEN Philip
Published: July 2010

The complete primary structure of protein phosphatase inhibitor‐1 has been determined. The protein consists of a single polypeptide chain of 165 residues, molecular weight 18640. The threonine residue that must be phosphorylated for activation is at position 35 and the active cyanogen bromide peptide, CB‐1, comprises residues 2–66. The N‐terminal methionine is acetylated and 40% of the inhibitor‐1 molecules lack the C‐terminal dipeptide Ala‐Val. Serine‐67 is substantially phosphorylated in vivo, but this phosphoserine residue does not appear to influence the activity of inhibitor‐1.
A Transition‐State Analysis of the Enzyme Catalysis in the Affinity Labelling of Horse‐Liver Alcohol Dehydrogenase by Bromoacids
Authors: BRANDSNES Øystein , DAHL Knut H. , McKINLEY‐McKEE John S.
Published: July 2010

The effect of temperature on the inactivation of liver alcohol dehydrogenase and on the alkylation of a model thiol free in solution by bromoacetate, 2‐bromopropionate, 3‐bromopropionate and 2‐bromo‐3‐(5‐imidazolyl)‐propionate has been studied and the thermodynamic activation parameters calculated.

All the bromoacids had a favourable entropy of activation in reaction with the enzyme compared to the model reaction with the thiolate anion of cysteine. This results from the formation of a reversible bromoacid‐enzyme complex prior to the irreversible inactivation. The enthalpy of activation is however unfavourable, due to the lower intrinsic reactivity of the metal‐thiol in the enzyme, compared to the thiolate anion of cysteine.

The difference in free energy of activation between the enzyme reaction and the model reaction was used to measure catalysis. The efficiency of the enzyme catalysis of alkylation increased in the order: bromoacetate < 2‐bromopropionate < 3‐bromopropionate < 2‐bromo‐3‐(5‐imidazolyl)propionate.

Promotion by imidazole of enzyme inactivation by bromoacetate is a pure enthalpy effect. This is due to imidazole when bound to the active‐site metal improving the intrinsic reactivity of the metal‐thiol of Cys‐46.
Coordinate Regulation of Synthesis of Ribosomal Proteins during Encystation of Acanthamoeba castellanii
Authors: SCHULZE Ingrid , JANTZEN Helga
Published: July 2010

The synthesis of ribosomal proteins has been examined during growth and encystment of Acanthamoeba castellanii. Cells have been radiolabelled with [35S]methionine and ribosomal proteins have been extracted either from ribosomes or from total cell extracts. The results show that there is less synthesis of ribosomal proteins relative to total cell proteins soon after transfer of growing cells into non‐nutrient medium, suggesting that the synthesis of all ribosomal proteins is under coordinate control. Furthermore, synthesis of ribosomal RNA and ribosomal proteins seems to be regulated coordinately, since within 1 h there is 70–80% decline in synthesis of both.

Translation of total RNA in vitro indicates that the relative decrease in the synthesis of ribosomal proteins during development is not only due to a relative decrease in the content of ribosomal protein mRNA. However, a considerable (about 10‐fold) and coordinate decrease in the relative content of ribosomal protein mRNA occurs during encystment. These data suggest a difference in the control of the synthesis of total proteins and ribosomal proteins as well as in the regulation of the content of total mRNA and ribosomal protein mRNA.

The comparison of patterns of ribosomal proteins indicates that during development no qualitative changes occur. The content of one protein of the small ribosomal subunit is low in growing cells and increases during encystation. Since the differences in the concentration of this protein are neither due to changing levels of the corresponding mRNA nor to changing rates of synthesis, changing affinities of this protein to ribosomes in vivo are suggested.
Amino‐Acid Sequence of the Vitamin‐K‐Dependent Part of Protein Z
Authors: HØJRUP Peter , ROEPSTORFF Peter , PETERSEN Torben E.
Published: July 2010

After tryptic digestion of protein Z a peptide corresponding to residues 1–43 has been isolated and sequenced. Smaller peptides were generated by treatment of the peptide with pepsin and then separated by high‐performance liquid chromatography. The purity of the peptides was sufficient for mass spectrometric measurement. Thirteen γ‐carboxyglutamic acid residues have been identified and the peptide shows strong homology to the corresponding domains of the classical vitamin‐K‐dependent blood clotting proteins.
Structural and Immunological Characterization of Type IV Collagen Isolated from Chicken Tissues
Authors: MAYNE Richard , WIEDEMANN Hanna , DESSAU Waltraud , VON DER MARK Klaus , BRUCKNER Peter
Published: July 2010

Previously, a type IV collagen fraction was isolated from chicken gizzard and further fractionated into three components called F1, F2 and F3 [Mayne, R. and Zettergren, J. G. (1980) Biochemistry, 19, 4065–4072]. F1 and F2 were consistently isolated in a 2:1 proportion, and the existence of a small native fragment of structure (F1)2F2 was proposed. In the present series of experiments, a type IV collagen fraction was isolated from the chicken kidney and shown to consist almost entirely of F1 and F2 which were again present in a 2:1 proportion. Identical one‐dimensional peptide maps for F1 and F2 from both sources were obtained by polyacrylamide gel electrophoresis of peptides obtained after cleavage with cyanogen bromide or Staphylococcus aureus V8 protease. The denaturation temperature of a preparation containing F1 and F2 in native form was determined by optical rotatory dispersion and a single melting curve was observed with a melting temperature of 33°C. This result provides further supportive evidence that F1 and F2 exist as a native fragment (F1)2F2. Antibodies were prepared in rabbits against a type IV collagen fraction isolated from chicken gizzard, and immunofluorescent staining of a wide variety of basement membranes was demonstrated. Experiments were performed in which various type IV collagen fractions were observed in the electron microscope after rotary shadowing. The lengths of (F1)2F2 and F3 were 147 nm and 174 nm respectively, the sum of these lengths (321 nm) corresponding closely to the length of the major triple‐helical domain of type IV collagen (326–328 nm). A specific cleavage site was located at a distance of 215 nm from the 7‐S domain which, together with the length of (F1)2F2, gives a total length of 362 nm. This value corresponds closely to the maximum length of the arms which originate from the 7‐S domain (355 nm) when type IV collagen was solubilized with a low concentration of pepsin. The results suggest that (a) type IV collagen isolated from the chicken gizzard is closely related, if not identical, to type IV collagen isolated from other tissues; (b) there is a single type IV collagen molecule of chain organization [α1(IV)]2α2(IV); (c) the order of the pepsin‐resistant fragments within a type IV molecule is 7S‐F3‐(F1)2F2.
Adenosine Diphosphate Ribosylation of Chicken‐Erythrocyte Histones H1, H5 and High‐Mobility‐Group Proteins by Purified Calf‐Thymus Poly(adenosinediphosphate‐ribose) Polymerase
Authors: POIRIER Guy G. , NIEDERGANG Claude , CHAMPAGNE Madeleine , MAZEN Alice , MANDEL Paul
Published: July 2010

Poly(ADP‐ribosylation) of histones H1, H5 and non‐histone chromosomal high‐mobility‐group proteins HMG 1, 2, 14 and 17 from chicken erythrocytes by purified calf thymus poly(ADP‐ribose) polymerase was studied using acid/urea/Triton gel electrophoresis and autoradiography.

With histone H1, besides ADP‐ribosylated H1 supporting short chains of polymer, the appearance of H1 ‘dimer’ was observed and this reaction was dependent on NAD concentration and incubation time. In addition, highly modified and/or aggregated species of histone H1 were observed.

Histone H5 was slightly ADP‐ribosylated at low NAD concentrations. At higher NAD concentrations or after longer incubations the formation of H5 ‘dimer’ and of more modified forms of H5 could be observed.

HMG 1 and HMG 2 were found to be ADP‐ribosylated, the reaction being dependent on NAD concentration and time. Here again some discrete intermediates appeared. HMG 14 and HMG 17 were only slightly ADP‐ribosylated under our experimental conditions.

These results indicate that the purified DNA‐independent poly(ADP‐ribose) polymerase can catalyse the formation of H1 ‘dimer’ as in nuclei and nucleosomes and that H5 and HMG proteins can also be ADP‐ribosylated and produce well‐defined higher complexes. These modifications of nuclear proteins may provide a means of localized conformational changes of the chromatin structure in vivo.
Methionyl‐tRNA Synthetase from Escherichia coli
Authors: BARKER David G. , EBEL Jean‐Pierre , JAKES Ross , BRUTON Chris J.
Published: July 2010

A 3300‐base segment of Escherichia coli chromosomal DNA, cloned into pBR322, will complement a methionine auxotroph in which the lesion is a defective methionyl‐tRNA synthetase with a much reduced affinity for methionine. Crude extracts of these transformants contain elevated levels of a protein which has a subunit molecular weight of 66000, methionyl‐tRNA synthetase aminoacylation activity in vitro and which cross‐reacts with anti‐(methionyl‐tRNA synthetase) antibodies. This polypeptide is very slightly larger than the well‐characterised and crystallised tryptic fragment of methionyl‐tRNA synthetase.

A DNA sequence of 1750 residues at one end of the cloned insert codes for a non‐terminated open reading frame in which we can locate a large number of methionyl‐tRNA synthetase tryptic and chymotryptic peptides. We have also sequenced 300 nucleotides upstream of this coding segment where we find a large invert repeat in the putative methionyl‐tRNA synthetase promoter region.
Identification of Nucleosides in Hydrolysates of Transfer RNA by High‐Resolution Mass Spectrometry
Authors: PANG Henrianna , SMITH David L. , CRAIN Pamela F. , YAMAIZUMI Kazuko , NISHIMURA Susumu , McCLOSKEY James A.
Published: July 2010

High‐resolution mass spectrometry has been investigated as a technique for identification of modified nucleosides in unfractionated hydrolysates of transfer RNA. The method is based on recognition of predetermined sets of exact mass values which are characteristic of individual nucleoside components. Mass spectra are photographically recorded in a non‐time‐resolved fashion, so that differing rates of nucleoside vaporization are of no consequence. Experimental parameters of sensitivity, spectrometer resolution and rates of vaporization were studied. Under typical conditions, 1–4 μg of tRNA are hydrolyzed, converted to volatile trimethylsilyl derivatives, and mass spectra of the resulting mixture recorded during 3 min at resolution 20000; 75–100% of the minor nucleosides are usually identified in a single recording. The method is complementary to conventional methods of identification which rely on chromatographic mobilities, and can in principle be generally applied to recognition of biologically or chemically modified bases in nucleic acid.
A Refined Quaternary Structure of Androctonus australis Hemocyanin
Authors: SIZARET Pierre‐Yves , FRANK Joachim , LAMY Josette , WEILL Jacques , LAMY Jean N.
Published: July 2010

The quaternary structure of the (4 × 6)‐mer hemocyanin from the scorpion Androctonus australis previously published [Lamy, J., Bijlholt, M. M. C., Sizaret, P.‐Y., Lamy, J., and van Bruggen, E. F. J. (1981) Biochemistry, 20, 1849–1856] has been refined. The relative positions in the half molecule of subunits Aa 3 A and Aa 3B compared to those of Aa 3C and Aa 5B have been established by double labeling of the (2 × 6)‐mer with binary mixtures of subunit‐specific Fab fragments. The results show that subunits Aa 3B and Aa 5B are located in the same hexamer while Aa 3A and Aa 3C are in the other half of the (2 × 6)‐mer.

The choice of the enantiomer was deduced from a careful examination of electron micrographs of the native molecule. Finally a position was assigned to each of the 24 subunits on the flip and flop faces as defined by Van Heel and Frank [Ultramicroscopy, 6, 187–194 (1981)].
Cobra Venom Acetylcholinesterase: Nature of Charge Isoforms
Authors: RABA Raivo , AAVIKSAAR Aavo
Published: July 2010

Charge isoforms of cobra (Naja naja oxiana) venom acetylcholinesterase, separated by isoelectric focusing, differ only by the number of free carboxyl groups of glutamic and/or aspartic acid side‐chains in the enzyme molecule. The isoforms appear to be produced by a post‐translational deamidation of accessible glutamin and/or asparagine residues. The isoforms have identical catalytic specificities towards characteristic acetylcholinesterase substrates.
Affinity Labeling of Glyceraldehyde‐3‐phosphate Dehydrogenase from Sturgeon and Bacillus stearothermophilus by 3‐Chloroacetylpyridine–adenine Dinucleotide
Authors: BRANLANT Guy , EILER Brigitte , WALLEN Leif , BIELLMANN Jean‐François
Published: July 2010

The inactivation of glyceraldehyde‐3‐phosphate dehydrogenase from Bacillus stearothermophilus and sturgeon by 3‐chloroacetylpyridine–adenine dinucleotide, which is not active as a coenzyme, obeys pseudo‐first‐order kinetics. The mechanism of inactivation corresponds to an affinity label. Four moles of inactivator are incorporated per mole of tetramer for both enzymes. All the kinetic results strongly support the idea that neither enzyme exhibits negative cooperativity for the non‐covalent binding of 3‐chloroacetylpyridine–adenine dinucleotide.

NAD+ strongly protects both glyceraldehyde‐3‐phosphate dehydrogenases from inactivation. In the presence of NAD+, the inactivation process seems also to exhibit pseudo‐first‐order kinetics. The fact that each kinetic experiment can be described by only one pseudo‐first‐order rate constant is unexpected.

The results of thiol titration are in favor of the modification of the essential Cys‐149, although another thiol group per monomer, probably Cys‐153, becomes titratable on both native alkylated enzymes. This latter result suggests that the alkylation of the Cys‐149 by the 3‐chloroacetylpyridine–adenine dinucleotide should affect the tertiary structure of these enzymes.
Biosynthesis of Blood‐Group I and i Substances
Authors: BLANKEN Willem M. , HOOGHWINKEL Gerrit J. M. , VAN DEN EIJNDEN Dirk H.
Published: July 2010

Highly purified bovine colostrum β1‐N‐acetylglucosaminide β1→4 galactosyltransferase was used to investigate the galactosylation of the synthetic, branched trisaccharide GlcNAcβ1→3(GlcNAcβ1→6)Gal, which is the branching point in blood‐group I antigenic structures. Two galactose residues could readily be incorporated from UDP‐galactose into the trisaccharide, yielding a pentasaccharide with the following structure: Galβ1→4GlcNAcβ1→3(Galβ1→4GlcNAcβ1→6)Gal. From a partially completed incubation an intermediate tetrasaccharide was isolated, the structure of which was investigated by use of an acetolysis method, involving high‐pressure liquid chromatography and double labelling techniques. It appeared that this intermediate consisted for more than 95% of one of two possible structures: GlcNAcβ1→3(Galβ1→4GlcNAcβ1→6)Gal. This reveals that the enzymatic galactosylation of the trisaccharide proceeds in a highly preferred order, in which the 1→6‐linked N‐acetylglucosamine residue is galactosylated first and thus that the galactosyltransferase displays a high degree of ‘branch specificity’. Kinetic data suggest that galactosylation of the 1→6 linked N‐acetylglucosamine in the trisaccharide enhances the acceptor properties of the 1→3‐linked N‐acetylglucosamine residue.
Synthesis of Peptidoglycan in vivo in Methicillin‐Resistant Staphylococcus aureus
Authors: WYKE Anne W. , WARD J. Barrie , HAYES Michael V.
Published: July 2010

The cell‐wall composition and degree of cross‐linking of peptidoglycan in a strain of Staphylococcus aureus (strain MR‐1) which is highly resistant to methicillin were similar to those of other strains of S. aureus. When the organism was grown in the presence of very low concentrations of methicillin (equivalent to 3 · 10−4× minimum growth‐inhibitory concentration [MGIC] there was a large decrease in the degree of cross‐linking of the peptidoglycan. Increasing concentrations of methicillin (up to 1.25 · 10−2× MGIC) caused a further decrease in cross‐linkage but thereafter a minimum value was reached. This remained unchanged even after growth of the organisms in much higher concentrations of the antibiotic up to 0.3 × minimum growth‐inhibitory concentration.

S. aureus MR‐1 was able to grow normally for many generations under these conditions and reduction in cross‐linkage of peptidoglycan was the only change detected in wall chemistry. Growth in the presence of methicillin (up to 0.3 × MGIC) (or other β‐lactam antibiotics) did not lead to an imbalance in the biosynthesis of peptidoglycan since no soluble polymers were secreted into the growth medium and nucleotide‐linked precursors did not accumulate intracellularly.

High concentrations of β‐lactam antibiotics (5 × MGIC) were bacteriostatic not bactericidal and this may be related to an apparent deficiency in the endogenous autolytic enzymes of strain MR‐1.

Studies of the penicillin‐binding proteins after growth in the presence of methicillin suggest that one of these proteins remains resistant to very high concentrations of the antibiotic. We propose that this protein acts as the primary transpeptidase responsible for the incorporation of newly synthesised peptidoglycan into the growing wall.
A Shuttle Mechanism for DNA‐Protein Interactions
Authors: ZAHRADKA Peter , EBISUZAKI Kaney
Published: July 2010

Previously it had been shown that poly(ADP‐ribose) polymerase requires DNA for its activity and that this enzyme is auto‐poly(ADP‐ribosyl)ated. The studies reported here indicate that this self‐modification inhibits the enzyme and decreases its affinity for DNA, as shown by sucrose gradient density centrifugation. The coupling of poly(ADP‐ribose) polymerase with poly(ADP‐ribose) glycohydrolase reactivates the polymerase by degrading poly(ADP‐ribose) and restoring the polymerase‐DNA complex. The assay of polymerase in the presence of glycohydrolase was made possible by use of a double‐label assay involving release of 14C‐labelled nicotinamide and the incorporation of 3H‐labelled ADP‐ribose from NAD+. These results provide the basis for a shuttle mechanism in which the polymerase can be moved on and off DNA by the action of these two enzymes. Mg2+ and histone HI appear to activate the polymerase by increasing the affinity of the polymerase for DNA.
Phosphorylation and Inactivation of Yeast Fructose‐Bisphosphatase in vivo by Glucose and by Proton Ionophores
Authors: MAZÓN Maria J. , GANCEDO Juana M. , GANCEDO Carlos
Published: July 2010

Addition of glucose to yeast cells causes a phosphorylation and an inactivation of the gluconeogenic enzyme fructose‐bisphosphatase [Mazón, M. J., Gancedo, J. M., and Gancedo, C. (1982) J. Biol. Chem. 257, 1128–1130], We report here that the addition of the proton ionophores 2,4–dinitrophenol and carbonylcyanide m‐chlorophenylhydrazone to yeast cells produces the same effect as that of glucose. Both glucose and ionophores produced: (a) phosphorylation and inactivation of fructose‐bisphosphatase, (b) an immediate rise in the intracellular concentration of cAMP, (c) an instant inhibition of the transport of amino acids driven by the membrane potential. It is proposed that the effect of glucose on fructose‐bisphosphatase involves as a first step the depolarization of the plasma membrane resulting in an increase of the intracellular concentration of cAMP. This in turn would stimulate phosphorylation of fructose‐bisphosphatase.
A Sensitive Peptide Mapping Method
Authors: CHANG Jui‐Yoa , KNECHT René , BALL Roland , ALKAN Sefik S. , BRAUN Dietmar G.
Published: July 2010

Peptide mixtures, precolumn‐derivatized with dimethylaminoazobenzene isothiocyanate, have been separated by reversed‐phase high‐performance liquid chromatography to generate a dimethylaminoazobenzene thiocarbamoyl peptide map. The eluted peptide derivatives are detected in the visible region with a sensitivity of 2–5 pmol and can be collected for direct structural analysis. This technique was applied to compare the sequence homology of two immunoglobulin light chains which were derived from two anti‐azobenzenearsonate monclonal antibodies, namely 10K44‐7A1 and 10K26‐12A1. The complete variable region sequences of 10K44‐7A1 and J0K26‐12A1 light chains were established based on the sequence analysis of tryptic peptides, intact light chains and reference sequences obtained previously [Siegelman M. and Capra, J. D. (1981) Proc. Natl Acad. Sci. USA, 78, 7679–7683], Altogether, three amino acid substitutions have been detected within complementary determining regions 1 and 2, and framework region 3, all requiring only a single base change at the DNA level. This new technique provides detection limits and the feasibility of analysing peptides which are not obtainable with conventional techniques.
Purification and Properties of Protein Methylase II from Wheat Germ
Authors: TRIVEDI Leena , GUPTA Arun , KI PAIK Woon , KIM Sangduk
Published: July 2010

Protein methylase II (S‐adenosylmethionine:protein‐carboxyl O‐methyltransferase, EC 2.1.1.24) was purified from wheat germ approximately 1200‐fold with a yield of 4.6% by employing gel filtration. The enzyme, using S‐adenosyl‐l‐methionine, methyl‐esterifies the free carboxyl group of protein. Since the enzymatic product was unstable and hydrolyzed non‐enzymatically to yield methanol, methanol was identified from the hydrolysate by the formation of the methyl ester of 3, 5‐dinitrobenzoate. The pH optimum for the wheat germ enzyme was 7.0 in contrast to the 6.0 for the methylase II of calf brain. The wheat germ enzyme had a molecular weight of 41000 compared to 25000 for the corresponding rat erythrocyte enzyme [S. Kim (1975) Arch. Biochem. Biophys. 161, 652–657]. The enzyme also differed with the mammalian enzyme in protein substrate preference: the mammalian enzyme showed equal preference to histone and immunoglobulin G while the wheat germ enzyme transferred a methyl group 4.5 times more to histone than to immunoglobulin G. The Km values for histone and S‐adenosyl‐l‐methionine were 0.2 mM and 5 μM. S‐Adenosyl‐l‐homocysteine and its analogues, sinefungin and A9145C, were competitive inhibitors with Ki values 1.5 μM, 0.4 μM and 0.1 μM, respectively.

To investigate the identity of endogenous substrate(s) for protein methylase II, the crude wheat germ extract was incubated with S‐adenosyl‐l‐[methyl‐3H]methionine and the methylated proteins were analyzed by acid/urea and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Among other methyl acceptors in the wheat germ, histone polypeptides were the major endogenous substrates.
Structure of the Wall Polysaccharide Isolated from a Group of Corynebacteria
Authors: ABOU‐ZEID Christiane , VOILAND Annie , MICHEL Georges , COCITO Carlo
Published: July 2010

Leprosy‐derived corynebacteria or LDC is a group of ‘diphtheroid’ bacteria isolated from leprosy patients. Their cell wall was previously shown to contain, in addition to a peptidoglycan of the meso‐diaminopimelic type and to mycolic acids of the corynomycolic type, a polysaccharide fraction. The present work relates the preparation and analysis of the wall polysaccharides of three LDC strains and two reference organisms Mycobacterium smegmatis and Corynebacterium hoffmannii. These polymers were obtained by mild alkali hydrolysis of purified cell walls, and were purified to homogeneity by column chromatography on DEAE‐cellulose and Sephadex G‐75 columns. By this procedure, two fractions containing, respectively, neutral polysaccharides and polysaccharide‐peptidoglycan complexes, were obtained. The former, which was composed of arabinose and galactose in M. smegmatis, contained, in addition, mannose in LDC cells, and mannose plus glucose in C. hoffmannii. The presence of arabinose, galactose and mannose in a constant ratio in the wall polysaccharide preparations of three LDC isolates suggests that arabinogalactomannan is the polysaccharide component of the envelope of this group of corynebacteria. A structural analysis was accomplished by methylation analysis and periodate oxidation. On the basis of the analytical data, a model for the primary structure of the polymer has been proposed, which is similar to that available for the arabinogalactan of Mycobacterium tuberculosis. According to the proposed model, the main chain of LDC polysaccharide is made of galactopyranose, galactofuranose and arabinofuranose. The latter is present in both the branching points and the lateral chains of the polymer. Mannose in the pyranose form, and arabinose in the furanose form, are located at the extremities. In conclusion, the inference that arabinogalactan is the wall polysaccharide of microorganisms belonging to genera Corynebacterium, Mycobacterium, Nocardia (CMN group) is not generally valid, since the corresponding polymers in some corynebacteria have additional monosaccharide components.
Characterization of the Region on Protein L7/L12 Involved in Binding to Ribosomal Particles
Authors: SCHOP Enno N. , MAASSEN J. Antonie
Published: July 2010

Tryptic digestion of reductively methylated protein L7/L12 yields a large tryptic fragment, which comprises amino acids 1–59. At the most, two molecules of this fragment can bind to a 50‐S ribosomal particle, deprived of protein L7/L12. Besides, binding of each single 1–59 fragment competes with binding of one dimeric L7/L12 molecule. Molecular weight studies on the fragment reveal a monomeric structure. Digestion of the 1–59 fragment with carboxypeptidase Y leads to the formation of a 1–55 fragment. The binding characteristics of the latter fragment are similar to those of the 1–59 fragment.

The results suggest that a monomeric stretch of L7/L12, comprising the first 55 amino acids, is sufficient for attaching L7/L12 to the ribosome.
Diffusion‐Controlled Reactions of Enzymes
Authors: ZHOU Guo‐Qiang , ZHONG Wei‐Zhu
Published: July 2010

In the study of enzyme‐catalysed mechanisms, it is often necessary to calculate the upper limit of enzyme reaction, which is usually used as an important criterion to identify whether an assumed enzyme‐catalysed mechanism is reasonable or not. Basically, in the existing methods, there are two kinds of models, the Alberty‐Hammes‐Eigen model and the Chou model, for calculating the upper limit of enzyme reaction. In this paper, an analysis and comparison between these two models are made. It is pointed out that the magnitude of the van der Waals' binding energy between enzyme and substrate molecules will play a key role in deciding whether there is a significant difference or not for the results calculated from these two models. Through such a comparison, the role of the major protein outside the active site of an enzyme molecule also becomes obvious: if the van der Waals' binding energy is very small, the major protein will act like a ‘wall’, blocking the flow of some substrate molecules to the active site; while if the van der Waals' energy is greater than 3 kT (where k is the Boltzmann constant and T the absolute temperature), the major protein will behave like a ‘accelerator’, speeding up the flow of the substrate molecules to the active site around the enzyme molecule.
Binding and Exchange of Nucleotides on the Chloroplast Coupling Factor CF1
Authors: GIRAULT Guy , GALMICHE Jean‐Michel , LEMAIRE Claire , STULZAFT Oscar
Published: July 2010

On the soluble part of the coupling factor (CF1), extracted from spinach chloroplasts, three nucleotide‐binding sites are identified. Three ADP are bound per CF1 when the enzyme is incubated with ADP either with or without Mg2+. Two ADP and one ATP are bound per CF1 when the enzyme is incubated with a limiting concentration of ATP, in the presence of Mg2+. At high ATP concentration, in the presence of Mg2+, one free ATP exchanges with one bound ADP and two ATP and one ADP remain bound per CF1. When Mg2+ is omitted from the incubation medium of ATP and CF1, only two ADP and around 0.5 ATP are bound per CF1.

The three nucleotide binding sites of CF1 fall into two different and independent categories according to the ability of the bound nucleotides to be exchanged with free nucleotides. On one site the bound ADP is difficult to exchange. On the other two sites, the bound nucleotides, ADP or ATP, are readily exchangable.

We propose that the two exchangeable sites form the catalytic part of the enzyme where ATP is hydrolyzed. When ATP concentration is high enough, in the presence of Mg2+, one ATP displaces one bound ADP and allows the ATP hydrolysis to proceed. We propose too that the site where ADP is difficult to exchange may represent the ‘tight’ ADP‐binding site, different from the catalytic ones, which becomes exchangeable on the CF1in vivo when the thylakoid membranes are energized by light, as stressed by Bickel‐Sandkötter and Strotman [(1976) FEBS Lett. 65, 102–106].
Two‐step Interaction of α‐Chymotrypsin with a Fluorescent Inhibitor
Authors: RODIER Francis Jean Lucien , ILGENFRITZ Georg
Published: July 2010

Bovine α‐chymotrypsin interacts with a dansylated inhibitor, 1‐dimethylaminonaphthalene‐5‐sulfonyl‐d‐tryptophan ethyl ester, to give a stable and strongly fluorescent derivative. The kinetics of the interaction of the enzyme with this inhibitor has been investigated by the temperature‐jump method. It is shown that the complex formation proceeds in two steps, a binding step and a slow isomerisation, as deduced from a study of the concentration dependence of the relaxation times. The rate constants for the binding process are k12= 7.9 × 105 M−1 s−1 and k21= 1.36 × 103 s−1, giving a binding constant of 585 M−1. This last value, compared to the overall binding constant determined statically of 1740 M−1 leads to the determination of the kinetic parameters for the slow isomerisation k23= 19 s−1 and k32= 9 s−1. Both values have the same order of magnitude as the rate constants found for the pH‐induced relaxation of some pancreatic proteins. This slow isomerisation can be related to internal motions of the active center of the enzyme.
Dissociation of Apolipoprotein AI from Apoprotein‐Lipid Complexes and from High‐Density Lipoproteins
Authors: ROSSENEU Maryvonne , VAN TORNOUT Philippe , LIEVENS Marie‐José , SCHMITZ Gerd , ASSMANN Gerd
Published: July 2010

The dissociation of the apoprotein AI (apoAI) from the apoAI‐dimyristoylglycerophosphocholine complex and from human high‐density lipoproteins (HDL), was induced by incubation with guanidinium hydrochloride at concentrations between 0 M and 7 M.

The kinetics and extent of denaturation were followed by monitoring both the fluidity of the lipid phase by fluorescence polarization measurements and the protein conformation by measuring the tryptophanyl fluorescence emission.

The association with lipids protects apoAI against denaturation both in HDL and in the apoAI‐phospholipid complex. The results of the kinetic and end‐point measurements suggest that the denaturing effect of guanidine hydrochloride on the apoAI‐lipid complex and on HDL is a two‐step process. It involves the dissociation of the apoAI‐phospholipid bond, as evidenced by fluidity measurements: this effect is maximal between 3 M and 4 M guanidine hydrochloride. The conformational change of the apoAI protein into a randomly coiled structure with the tryptophanyl residues exposed to the solvent is maximal between 4 M and 6 M guanidine hydrochloride.

HDL and the apoAI‐phospholipid complex have a closely similar behaviour towards denaturation by guanidine hydrochloride indicating that the phospholipid‐apoAI association in HDL is primarly responsible for the stability of the lipoprotein molecule.
Transcription of Sea‐Urchin Mesenchyme Blastula
Authors: SPINELLI Giovanni , CASANO Caterina , GIANGUZZA Fabrizio , CIACCIO Mirella , PALLA Franco
Published: July 2010

Mesenchyme blastula sea‐urchin embryos were heat‐shocked at 31°C and pulse‐labelled with [3H]uridine. The nuclear RNA was fractionated on glyoxal/agarose gels and each RNA fraction hybridized with cloned early blastula histone DNA. The results showed that after heat shock there is an accumulation of histone RNA molecules larger than the messenger and a decrease, with respect to the control, of 9‐S histone RNA. Chasing of the heat shock RNA by incubation of the embryos with unlabelled uridine and cytidine restores the radioactivity, as well as the hybridization profiles, of control embryos. Furthermore saturation curves obtained by hybridizing histone DNA with labelled 9‐S RNA, showed an absence of the histone messenger in the cytoplasm of heat‐shocked embryos, whereas it is present in both control and chased embryos. These results are consistent with the hypothesis that the accumulated histone RNA in heat‐shocked embryos is the precursor of mature messengers.
Secondary Structure of the lac Repressor Headpiece
Authors: SCHNARR Manfred , MAURIZOT Jean‐Claude
Published: July 2010

The secondary structure of the short tryptic headpiece of the lac repressor has been investigated by the analysis of its infrared and circular dichroic spectra. For the latter we used the method of Provencher and Glöckner [Biochemistry (1981) 20, 33–37], which seems to be at present the most successful for the determination of the β content of proteins. Nevertheless our results indicate that in the case of the lac repressor headpiece this method overestimates the amount of β structure. We find that the headpiece contains an important helical content of about 50%, depending slightly on the ionic strength. A decomposition of the infrared spectrum in a sum of Gaussian curves reveals clearly the absence of a vibrational band around 1630 cm−1 excluding thus the presence of a multi‐stranded β‐pleated sheet. The only β structure compatible with the infrared results seems to be a two‐stranded antiparallel β sheet, as judged from our results on the β‐sheet model‐compound gramicidin S. The unusually strong intensity of the amide I' band is in favour of the existence of such a structure. The quantitative analysis of both infrared and circular dichroism spectra indicates the presence of a certain (but different) amount of β structure. Comparing these results with several secondary structure predictions, part of the helical residues should be located between Leu‐45 and (at least) Arg‐35, and an eventual two‐stranded β sheet should be situated in the N‐terminal part of the headpiece.
Structural Prediction of Membrane‐Bound Proteins
Authors: ARGOS Patrick , RAO J. K. Mohana , HARGRAVE Paul A.
Published: July 2010

A prediction algorithm based on physical characteristis of the twenty amino acids and refined by comparison to the proposed bacteriorhodopsin structure was devised to delineate likely membrane‐buried regions in the primary sequences of proteins known to interact with the lipid bilayer. Application of the method to the sequence of the carboxyl terminal one‐third of bovine rhodopsin predicted a membrane‐buried helical hairpin structure. With the use of lipid‐buried segments in bacteriorhodopsin as well as regions predicted by the algorithm in other membrane‐bound proteins, a hierarchical ranking of the twenty amino acids in their preferences to be in lipid contact was calculated. A helical wheel analysis of the predicted regions suggests which helical faces are within the protein interior and which are in contact with the lipid bilayer.
Isolation and Characterization of Nucleic‐Acid‐Like Polymers Containing Glucose from Ehrlich Ascites Tumor Cells
Authors: KAGAWA Haruo , KAGAWA Kazuko
Published: July 2010

Novel nucleic‐acid‐like polymers containing glucose, glucose ‘nucleic acid’, were isolated from cytoplasmic particles of Ehrlich ascites tumor cells. It is assumed that the basic structure of the polymers is similar to those of known nucleic acids except that their sugar constituents are glucose derivatives instead of ribose or deoxyribose.

When the glucose ‘nucleic acid’ was incubated with venom phosphodiesterase, four kinds of glucose ‘nucleotides’ containing bases of cytosine, adenine, guanine and thymine were obtained.

When the polymers were incubated with both venom phosphodiesterase and alkaline phosphatase, inorganic phosphates of glucose ‘nucleotides’ were removed and each nucleotide was converted to a glucose ‘nucleoside’.
Mode of Interaction of Polyoxyethyleneglycol Detergents with Membrane Proteins
Authors: LE MAIRE Mare , KWEE Sianette , ANDERSEN Jens P. , MØLLER Jesper V.
Published: July 2010

Binding of dodecyloctaethyleneglycol monoether (C12E3) and purified Triton X‐100 to various integral membrane proteins was studied by chromatographic procedures. Binding capacity decreased in the following order: bovine rhodopsin > photochemical reaction center > sarcoplasmic reticulum Ca2+‐ATPase. The detergents were bound in different amounts to the proteins and less than corresponding to the aggregation number of the pure micelles. Appreciable binding of C12E8 to Ca2+‐ATPase was observed far below the critical micelle concentration, consistent with interaction of the membrane protein with non‐micellar detergent. Model calculations indicate that the detergents cannot combine with the membrane proteins, forming an oblate ring similar to that of pure detergent micelles, such as has been previously proposed for e.g. cytochrome b5 [Robinson and Tanford (1975) Biochemistry, 14, 365–378]. Other arrangements (prolate and monolayer rings), in which all detergent molecules are in contact with the protein, are considered as alternatives for covering the hydrophobic surface of the membrane protein with a continuous layer of detergent.
Metal‐Nucleotide Structure at the Active Sites of the Mammalian Hexokinases
Authors: DARBY Martyn K. , TRAYER Ian P.
Published: July 2010

The diastereomers of adenosine 5′‐O‐(2‐thiotriphosphate) (ATPβS) in the presence of Mg2+, Co2+ and Cd2+ have been used to determine the stereospecificity of the metal‐nucleotide binding site of rat muscle hexokinase type II and rat liver glucokinase by the method developed by Jaffe and Cohn [J. Biol. Chem. 254, 10839–10845 (1979)]. The kinetic parameters, Km and V, for the mammalian hexokinase reaction have been determined for ATPβS in the presence of the three divalent metal ions. In the presence of Mg2+, both enzymes exhibit a preference for the B diastereomer of ATPβS (V ratio, B/A · 20). With Cd+, the stereospecificity is reversed and the A diastereomer is the preferred substrate, suggesting direct coordination of S on the β‐P to this metal ion. Co2+ exhibits a decreased specificity for the B diastereomer over Mg2+. This decreasing order of stereo‐specificity for the B isomer reflects primarily the decreasing ratios of nucleotide complexes coordinated to O rather than S on the β‐P as the metal ion is changed from Mg2+ to Co2+ to Cd2+. The kinetic parameters for the hexokinases have also been determined for adenosine 5′‐O‐(1‐thiotriphosphate) (ATPαS) using the same three metal ions as activators. The A diastereomer is the preferred substrate regardless of the metal ion. This absence of reversal of stereospecificity for metal‐ATPαS suggests that the α‐P is not involved in coordinating the metal on the enzyme, unlike the β‐P. That is, the structural constraints of the ATP‐binding site on the enzyme overcome the preferred coordination of Cd2+ to S. Given the greater stability of bidentate metal‐ATP complexes over monodentate, these data are interpreted as indicating that MgATP binds to the mammalian hexokinases as the βγ‐bidentate complex in the screw sense geometry, as has been found for the yeast hexokinase (Jaffe and Cohn, reference cited above).
Synthesis and Hydrolysis of 1,3‐β‐Xylosidic Linkages by Endo‐1,4‐β‐xylanase of Cryptococcus albidus
Authors: BIELY Peter , VRŠANSKÁ Mária
Published: July 2010

Purified extracellular endo‐1,4‐β‐xylanase (EC 3.2.1.8) of the yeast Cryptococcus albidus was found to catalyze not only the known 1,4‐β‐transfer, but an alternative transglycosylation reaction leading to the formation of 1,3‐β‐glycosidic linkages. From a mixture of products of β‐xylanase degradation of phenyl β‐D‐xylopyranoside three xylooligosaccharide fractions, differring chromatographically from the 1,4‐β‐linked products, were isolated by preparative paper chromatography. Their structure was elucidated by mass spectrometry, 13C‐NMR spectroscopy and enzymic hydrolysis by β‐xylanase and β‐xylosidase. The isomeric xylotriose was identified as 3‐O‐β‐D‐xylopyranosyl‐4‐O‐β‐D‐xylopyranosyl‐D‐xylose. The fraction of isomeric tetrasaccharides was found to be represented mainly by 4‐O‐β‐D‐xylopyranosyl‐3‐O‐β‐D‐xylopyranosyl‐4‐O‐β‐D‐xylopyranosyl‐D‐xylose. The xylooligosaccharides containing one 1,3‐β‐linkage were also produced on the enzyme treatment of 1,4‐β‐xylotriose and 1,4‐β‐xylan. When treated with the enzyme responsible for their synthesis, the isomeric xylooligosaccharides were hydrolyzed at the 1,3‐β‐linkage, despite the fact the enzyme does not attack 1,3‐β‐xylan. The results are interpreted in the relation to the characterized four‐subsite substrate‐binding site of the enzyme.