bluuebunny/crossref_metadata_2025 huggingface dataset:
The quaternary structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii
Authors: HANEMAAIJER Roeland , WESTPHAL Adrie H. , VAN DER HEIDEN Tini , DE KOK Arie , VEEGER Cees
Published: March 2005

After limited proteolysis of the dihydrolipoyl transacetylase component (E2) of Azotobacter vinelandii pyruvate dehydrogenase complex (PDC), a C‐terminal domain was obtained which retained the transacetylase active site and the quaternary structure of E2 but had lost the lipoyl‐containing N‐terminal part of the chain and the binding sites for the peripheral components, pyruvate dehydrogenase and lipoamide dehydrogenase. The C‐terminus of this domain was determined by treatment with carboxypeptidase Y and shown to be identical with the C‐terminus of E2. Together with the previously determined N‐terminus and the known amino acid sequence of E2, a molecular mass of 27.5 kDa was calculated. From the molecular mass of the native catalytic domain, 530 kDa, and the symmetry of the cubic structures observed on electron micrographs, a 24‐meric structure is concluded instead of the 32‐meric structure proposed previously.

From the effect of guanidine hydrochloride on the light‐scattering of intact E2 it was concluded that dissociation occurs in a two‐step reaction resulting in particles with an average mass 1/6 that of the original mass before the N → D transition takes place. Cross‐linking experiments with the catalytic domain indicated that the multimeric E2 is built from tetramers and that the tetramers are arranged as a dimer of dimers.

A model for the quaternary structure of E2 is given, in which it is assumed that the tetrameric E2 core of PDC is formed from each of the six morphological subunits located at the lateral face of the cube. Binding of peripheral components to a site that interferes with the cubic assembly causes dissociation, resulting in the unique small PDC of A. vinelandii.
Preparation and kinetic properties of 5‐ethylphenazine – poly(ethylene glycol) – NAD+ conjugate, a unique catalyst having an intramolecular reaction step
Authors: YOMO Tetsuya , SAWAI Haruyo , URABE Itaru , OKADA Hirosuke
Published: March 2005

5‐Ethylphenazine ‐ poly(ethylene glycol) ‐ NAD+ conjugate (EP+‐PEG‐NAD+) was prepared by linking 1‐(3‐carboxypropyloxy)‐5‐ethylphenazine (I) to poly(ethylene glycol)‐bound NAD+ (PEG‐NAD+) and its kinetic properties were studied. As a reference compound, poly(ethylene glycol)‐bound 5‐ethylphenazine derivative (III) was also prepared and the effects of poly(ethylene glycol) on the reaction rate of the 5‐ethylphenazine moiety with NADH was investigated. The second‐order rate constant, k1, of the reaction of III with NADH is 2.78 mM−1 s−1 and is about 1.7 times that of 1‐(3‐ethoxycarbonylpropyloxy)‐5‐ethylphenazine (II) with NADH. A similar effect of the attached poly(ethylene glycol) was observed for the reaction of PEG‐NADH with I or II. The second‐order rate constants, k2 and k3, of the reactions of the reduced form of III with oxygen and with 3‐(4′,5′‐dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium ion, respectively, were k2= 1.22 mM−1 s−1 and k3= 32 mM−1 s−1; the k2 value is not changed but the k3 value is decreased by the attachment of the polymer.

EP+‐PEG‐NAD+ works as a unique catalyst having an intramolecular reaction step within its turnover cycle in a coupled multi‐step reaction system containing malate dehydrogenase, malate, EP+‐PEG‐NAD+, a tetrazolium salt and oxygen. The first‐order rate constant, k4, of the intramolecular reaction was 1.1 s‐1. The effects of the covalent linking of the 5‐ethylphenazine and the NAD+ moieties were estimated by comparing the value of k4 with that of k1 for the reaction of III with NADH; the effective concentration of the NADH moiety for the 5‐ethylphenazine moiety on the same EP+‐PEG‐NADH molecule (or vice versa) was calculated to be 0.40 mM from the ratio of k4/k1. The values of the rate constants in the coupled multi‐step reaction system enable us to understand the dynamic features of the system and the characteristics of EP+‐PEG‐NAD+ as a catalyst are discussed.
Purification and characterization of 20‐hydroxy‐leukotriene B4 dehydrogenase in human neutrophils
Authors: GOTOH Yoichi , SUMIMOTO Hideki , MINAKAMI Shigeki
Published: March 2005

Leukotriene B4 (LTB4) is converted to 20‐hydroxy‐LTB4 (20‐OH‐LTB4) which is subsequently oxidized to 20‐carboxy‐LTB4 (20‐COOH‐LTB4). The oxidation of the hydroxy LTB4 to the carboxy LTB4 by human neutrophils was associated with the reduction of NAD+ and required both cytosolic and microsomal fractions. We isolated a cytosolic protein which oxidized the hydroxy LTB4 in the presence of NAD+ and the microsomal fraction. It was homogeneous on SDS/PAGE, with a subunit molecular mass of 37 kDa, and may be a dimeric protein with two identical or similar subunits because its molecular mass, estimated by Sephadex G‐100 column chromatography, was about 80 kDa. The protein was an alcohol dehydrogenase with high affinity for ω‐hydroxy fatty acids, such as 12‐hydroxylaurate and 16‐hydroxypalmitate. We conclude that the cytosolic protein oxidizes 20‐OH‐LTB4 to 20‐oxo‐LTB4 and the microsomal fraction oxidizes the oxo‐LTB4 to the carboxy‐LTB4, based on the finding that the activity which oxidizes ω‐hydroxy fatty acids is present only in the cytosol fraction, while that which oxidizes hydrophobic aldehydes is found only in the microsomal fraction and that the stoichiometry of the formation of 20‐COOH‐LTB4 to the reduction of NAD+ was 1:2. The affinity of the dehydrogenase for 20‐OH‐LTB4 may be higher than that for 12‐hydroxylaurate (Km= 70 μM), because the latter inhibited the oxidation of the former by only 40%, at a concentration of 12‐hydroxylaurate 10 times higher than that of 20‐OH‐LTB4. The enzyme activity was not affected by pyrazole and 4‐methylpyrazole at millimolar concentrations. These characteristics indicate that the dehydrogenase is a unique type of alcohol dehydrogenase.
Mechanism of action of bombesin on amylase secretion
Authors: BRUZZONE Roberto
Published: March 2005

The mode of action of bombesin on amylase secretion was investigated in rat pancreatic acini. Bombesin induced a dose‐dependent increase in inositol 1,4,5‐trisphosphate and cytosolic free Ca2+. The threshold concentration capable of inducing both effects was 0.1 nM and the half‐maximal dose of the peptide for Ca2+ mobilization was approximately 0.6 nM. By contrast, amylase release was approximately 30 times more sensitive than inositol 1,4,5‐trisphosphate production and Ca2+ mobilization to bombesin action, with 1 pM being the first stimulatory concentration and a half‐maximal effect at approximately 20 pM. The ability of low bombesin doses to trigger enzyme secretion was unaffected by chelation of extracellular Ca2+ with EGTA. In order to test whether the stimulation of amylase release was truly a Ca2+ ‐independent response, the intracellular Ca2+ stores were depleted by pretreating acini with EGTA plus ionomycin, the Ca2+ ionophore. Under these conditions bombesin was still capable of eliciting a significant twofold enhancement of the secretory activity. These results indicate that bombesin, an agonist thought to activate secretion mainly through mobilization of Ca2+ from intracellular stores, elicits amylase release at low concentrations, independently of a concomitant rise in cytosolic free Ca2+. The relevance of these findings to the physiological regulation of pancreatic exocrine secretion is discussed.
Modulation of the affinity of the single‐stranded DNA‐binding protein of Escherichia coli (E. coli SSB) to poly(dT) by site‐directed mutagenesis
Authors: BAYER Irene , FLIESS Anja , GREIPEL Joachim , URBANKE Claus , MAASS Günter
Published: March 2005

A vector for site‐directed mutagenesis and overproduction of the Escherichia coli single‐stranded‐DNA‐binding protein (E. coli SSB) was constructed. An E. coli strain carrying this vector produces up to 400 mg pure protein from 25 g wet cells. The vector was used to mutate specifically the Phe60 residue of E. coli SSB. Phe60 had been proposed to be located near the single‐stranded‐DNA‐binding site.

Substitution of the Phe60 residue by Val, Ser, Leu, His, Tyr and Trp gave proteins with no or only minor conformational changes, as detected by NMR spectroscopy.

The affinity of the mutant E. coli SSB proteins for single‐stranded DNA decreased in the order Trp > Phe (wild‐type) > Tyr > Leu > His > Val > Ser, leading to the conclusion that position 60 is a site of hydrophobic interaction of the protein with DNA.
Nucleotide sequence of the glyceraldehyde‐3‐phosphate dehydrogenase gene from the mesophilic methanogenic archaebacteria Methanobacterium bryantii and Methanobacterium formicicum
Authors: FABRY Stefan , LANG Jutta , Niermann Thomas , Vingron Martin , HENSEL Reinhard
Published: March 2005

The genes for glyceraldehyde‐3‐phosphate dehydrogenase (gap genes) from the mesophilic methanogenic archaebacteria Methanobacterium formicicum and Methanobacterium bryantii were cloned and sequenced. The deduced amino acid sequences show 95% identity to each other and about 70% identity to the glyceraldehyde‐3‐phosphate dehydrogenase from the thermophilic methanogenic archaebacterium Methanothermus fervidus. Although the sequence similarity between the archaebacterial glyceraldehyde‐3‐phosphate dehydrogenase and the homologous enzyme of eubacteria and eukaryotes is low, an equivalent secondary‐structural arrangement can be deduced from the profiles of the physical parameters hydropathy, chain flexibility and amphipathy.

In order to find possible thermophile‐specific structural features of the enzyme from M. fervidus, a comparative primary‐sequence analysis was performed. Amino acid exchanges leading, to a stabilization of the main‐chain conformation, could be found throughout the sequence of the thermophile enzyme. Striking features of the thermophile sequence are the preference for isoleucine, especially in β‐sheets, and a low arginine/lysine ratio of 0.54.
Studies on uroporphyrinogen decarboxylase of etiolated Euglena gracilis Z
Authors: JUKNAT Adela Ana , SEUBERT Andreas , SEUBERT Sigrid , IPPEN Hellmut
Published: March 2005

A 423‐fold purified fraction of uroporphyrinogen decarboxylase (EC 4.1.1.37) showing a specific activity of 770 units/mg protein has been employed in order to study some properties in etiolated Euglena gracilis Z.

Uroporphyrinogen decarboxylase has a relative molecular mass of 54000, an optimum pH of 7.2 and exhibits Michaelis‐Menten kinetics, employing both uroporphyrinogen I and uroporphyrinogen III as substrates.

Anaerobic conditions seem not to be necessary for uroporphyrinogen decarboxylase activity. Neither EDTA nor cysteine affected enzyme activity, whereas dithiothreitol produced a remarkable activation of coproporphyrinogen formation.

Kinetic data employing both substrates showed an accumulation of porphyrinogen (i.e. hexa‐ and heptaporphyrin) containing six or seven COOH groups, depending on the uroporphyrinogen concentration used.

An unusual elution profile of the intermediates on Sephacryl S‐200 was found.
Two different intrachain cAMP sites in the cAMP‐dependent protein kinase of the dimorphic fungus Mucor rouxii
Authors: PAVETO Cristina , PASSERON Susana , CORBIN Jackie D. , MORENO Silvia
Published: March 2005

cAMP sites of the cAMP‐dependent protein kinase from the fungus Mucor rouxii have been characterized through the study of the effects of cAMP and of cAMP analogs on the phosphotransferase activity and through binding kinetics. The tetrameric holoenzyme, which contains two regulatory (R) and two catalytic (C) subunits, exhibited positive cooperativity in activation by cAMP, suggesting multiple cAMP‐binding sites. Several other results indicated that the Mucor kinase contained two different cooperative cAMP‐binding sites on each R subunit, with properties similar to those of the mammalian cAMP‐dependent protein kinase. Under optimum binding conditions, the [3H]cAMP dissociation behavior indicated equal amounts of two components which had dissociation rate constants of 0.09 min−1 (site 1) and 0.90 min−1 (site 2) at 30°C. Two cAMP‐binding sites could also be distinguished by C‐8 cAMP analogs (site‐1‐selective) and C‐6 cAMP analogs (site‐2‐selective); combinations of site‐1‐ and site‐2‐selective analogs were synergistic in protein kinase activation. The two different cooperative binding sites were probably located on the same R subunit, since the proteolytically derived dimeric form of the enzyme, which contained one R and one C component, retained the salient properties of the untreated tetrameric enzyme. Unlike any of the mammalian cyclic‐nucleotide‐dependent isozymes described thus far, the Mucor kinase was much more potently activated by C‐6 cAMP analogs than by C‐8 cAMP analogs. In the ternary complex formed by the native Mucor tetramer and cAMP, only the two sites 1 contained bound cAMP, a feature which has also not yet been demonstrated for the mammalian cAMP‐dependent protein kinase.
Enzyme Nomenclature. Recommendations 1984
Authors: WEBB Edwin C.
Published: March 2005

This document contains 275 entries for new enzymes which have come to light since the finalization of the 1984 edition at the end of November, 1983, and the publication of Supplement 1 (Eur. J. Biochem. 157, 1–26 (1986). The 51 entries marked with an asterisk are revisions of the corresponding entries in Enzyme Nomenclature (1984), published by Academic Press, New York.

References containing a letter (e.g. 123a) are listed at the end of this article; numbers without letters refer to the Reference List in Enzyme Nomenclature (1984).

Comments and suggestions may be sent to any member of NC‐IUB or JCBN: J. F. G. Vliegenthart (Chairman), J. R. Bull, C. R. Cantor, M. A. Chester, C. Liébecq (as representative of the IUB Committee of Editors of Biochemical Journals), G. P. Moss, J. Reedijk, W. Saenger, N. Sharon, or P. Venetianer, to its Secretary, A. Cornish‐Bowden, CNRS‐CBM2, 31 Chemin Joseph‐Aiguier, B. P. 71, 13402 Marseille Cedex 9, France or to E. C. Webb, 1/221 Kings Road, Mundingburra, Townsville, Australia, 4812.
Characterization of a 48‐kDa nucleic‐acid‐binding fragment of nucleolin
Authors: SAPP Martin , RICHTER Arndt , WEISSHART Klaus , CAIZERGUES‐FERRER Michelle , AMALRIC Francois , WALLACE Michael O. , KIRSTEIN Mark N. , OLSON Mark O. J.
Published: March 2005

Nucleolin (C23 or 100 kDa) is an abundant single‐stranded‐nucleic‐acid‐binding nucleolar protein proposed to be involved in the early stages of ribosome assembly. A stable 48‐kDa fragment of the protein was produced either by proteolytic activity present in nucleolar extracts or by added trypsin. The hydrodynamic and DNA‐binding properties of the 48‐kDa fragment were compared with the parent molecule. Protein sequencing indicated that the fragment begins at residue 282; amino acid composition of the fragment including 10 – 12 methylated arginine residues suggested that the fragment contains the entire COOH‐terminal two‐thirds of the protein. The 48‐kDa fragment was more globular than nucleolin, as indicated by a lower frictional coefficient (1.3 vs. 2.0 for nucleolin) and a similar sedimentation coefficient (4.1 – 4.3S) in spite of the reduction in molecular mass. Although the 48‐kDa fragment retained single‐stranded‐DNA‐binding activity, the binding capacity and the ability to reassociate DNA were about fivefold and sixfold lower, respectively, than nucleolin. Similarly, tenfold higher concentrations of the 48‐kDa fragment were required to form nucleoprotein aggregates. These results suggest that nucleolin contains a globular COOH‐terminal domain for nucleic‐acid binding and a NH2‐terminal region which is involved in protein‐protein interactions and modulating nucleic‐acid‐binding activity.
A structural requirement in the subsite F of lysozyme
Authors: MURAKI Michiro , MORIKAWA Masaaki , JIGAMI Yoshifumi , TANAKA Hideaki
Published: March 2005

Arginine 115 in the subsite F of human lysozyme (peptidoglycan N‐acetylmuramoylhydrolase, EC 3.2.1.17) was replaced with lysine, histidine, glutamine or glutamine acid by site‐directed mutagenesis. The conversions which conserve positive charge, Arg115 to Lys or His (at acidic pH), have little affected on either the kinetic parameters for Micrococcus lysodeikticus cells or the activity against glycol chitin, nor on the cleavage patterns of hexa(N‐acetylglucosamine) [(GlcNAc)6] and penta(N‐acetylglucosamine) [(GlcNAc)5]. On the other hand, the conversions which cause loss of the positive charge, Arg115 to His (neutral and alkaline pH), Gln or Glu, not only reduced the activity against glycol chitin but also changed the cleavage patterns for (GlcNAc)6 and (GlcNAc)5. These results suggest that Arg115 is structurally required not for the specific hydrogen bonding interaction with a sugar residue but for the positively charged character in the construction of subsite F in human lysozyme.
Characterization of two murine (2′‐5′)(A)n‐dependent endonucleases of different molecular mass
Authors: BISBAL Catherine , SALEHZADA Tamim , LEBLEU Bernard , BAYARD Bernard
Published: March 2005

RNase L is considered as the major if not unique target of (2′‐5′)(A)n and therefore as an important intracellular mediator of interferon action. It behaves as an 185‐kDa species in various cell extracts when analyzed by gel filtration. SDS/PAGE analysis of the polypeptides covalently labelled with a (2′‐5′)(A)4‐3′‐[32P]p Cp probe reveals a single 80‐kDa species, thus attesting a multimeric form of the 185‐kDa protein. At variance with such data, mouse spleen extracts reveal an additional 40‐kDa polypeptide with (2′‐5′)(A)n‐dependent ribonucleolytic activity. This seemingly new form of RNase L migrates as a 40‐kDa polypeptide when analyzed under native or denaturing conditions. It bears some structural similarity with the larger‐molecular‐mass RNase L as revealed by partial proteolysis. It is probably not generated through proteolytic degradation of the 185‐kDa RNase L during extract preparation, although its physiological significance is unknown. Indeed various protease inhibitors do not significantly alter the ratio of 40‐kDa and 185‐kDa (2′‐5′)(A)n‐dependent ribonucleases; moreover, the (2′‐5′)(A)n‐ binding capacity of the 40‐kDa polypeptide is more stable than that of the 185‐kDa one.
Purification and properties of five different forms of human procarboxypeptidases
Authors: PASCUAL Roger , BURGOS Francisco J. , SALVA Miquel , SORIANO Fernando , MENDEZ Enrique , AVILES Francesc X.
Published: March 2005

Three different procarboxypeptidases A and two different procarboxypeptidases B have been isolated for the first time, in a pure and native state, from human pancreatic extracts. These proteins were purified in one or two quick steps by anion‐exchange HPLC. All these forms have been biochemically characterized. Two of the procarboxypeptidases A, the A1 and A2 forms, are obtained in a monomeric state while the other, the A3 form, is obtained as a binary complex of a procarboxypeptidase A with a proproteinase E. This complex is stable in aqueous buffers at various ionic strengths and develops carboxypeptidase A and proteinase E activities in the presence of trypsin. The A1 and A2 forms show clear differences in electrophoretic mobility in SDS/polyacrylamide gels, isoelectric point, proteolytic activation process with trypsin and susceptibility to thermal denaturation. In contrast, these properties are similar in the A1 and A3 (binary complex) forms. On the other hand, with respect to the properties listed above, the B1 and B2 forms differ from each other mainly in isoelectric point. An overall comparison of the above properties reveals the unusual character of the A2 form, midway between the other A and B forms. N‐terminal extended sequence analysis carried out on these proenzymes confirm that they constitute different isologous forms.
Comparative X‐ray and Fourier‐transform‐infrared investigations of conformational properties of bacterial and synthetic lipid A of Escherichia coli and Salmonella minnesota as well as partial structures and analogues thereof
Authors: LABISCHINSKI Harald , NAUMANN Dieter , SCHULTZ Christian , KUSUMOTO Shoichi , SHIBA Tetsuo , RIETSCHEL Ernst Th. , GIESBRECHT Peter
Published: March 2005

By using the X‐ray and infrared spectroscopic characteristics of various synthetic analogues and partial structures of lipid A in the dried state, a comparison of these compounds with their natural counterparts was undertaken. As judged by their X‐ray diffraction and infrared spectroscopic features, the compounds tested could be divided into two main groups. The first group covered those samples synthesized in accordance with a previously assumed structure, while those synthesized in accordance with present knowledge on the lipid‐A primary structure formed the second group. Members of the first group were characterized by a liquid‐like, α‐type arrangement of their fatty acyl chains in a non‐lamellar supramolecular structure, while all members of the second group formed bilayered phases with a much more ordered, β,‐type conformation of their fatty acyl chains. Synthetic Escherichia coli‐type lipid A (compound 506), proved to be essentially identical to its natural counterpart with respect to those conformational properties accessible by our methods. The synthetic hepta‐acyl species of Salmonella minnesota lipid A (compound 516) revealed an unexpected conformational behaviour, whereby a fattyacyl‐chain packing could be detected which was different from the hexagonal arrangement found for all other compounds of the second group.
Defective processing and binding of low‐density lipoprotein receptors in fibroblasts from a familial hypercholesterolaemic subject
Authors: KNIGHT Brian L. , GAVIGAN Sean J. P. , SOUTAR Anne K. , PATEL Dilip D.
Published: March 2005

The properties of the low‐density lipoprotein (LDL) receptor were studied in skin fibroblasts from a homozygous familial hypercholesterolaemic subject, MM (MM cells), who exhibits a defect in the processing of the precursor form of the receptor. Despite the prolonged half‐life of the precursor (3 h), essentially all was eventually processed to the mature form, which was degraded with a half‐life of approximately 6 h. The receptor content of the MM cells, determined by radioimmunoassay, was slightly lower than normal and by immunoblotting it was estimated that 55% was present as the mature form at equilibrium. The mature receptor reached the cell surface and was internalized and recycled apparently normally, with about 60% accessible to pronase at any time. Immunoblotting, immunoassay and surface labelling with 125I all indicated that the cells contained twice as much surface receptor protein as would be expected from the binding of LDL.

Maximum heparin‐releasable LDL binding to MM cells was 20% of normal and the apparent affinity for LDL was reduced. The cells did not show the marked increase in affinity and reduction in binding exhibited by normal cells when cooled to 4°C. Also, neither the apparent affinity of the receptors nor the maximum binding at 37 C was greatly affected by an antibody that has been shown to reduce the affinity and halve the binding of normal cells. The results suggest that the mutation in the LDL receptor gene of these cells affected the ability of the receptors to bind LDL in a similar manner to the antibody, possibly by promoting aggregation of receptors on the cell surface, and halving the amount of LDL bound.
Hemoglobin from the Antarctic fish Notothenia coriiceps neglecta
Authors: D'AVINO Rossana , di PRISCO Guido
Published: March 2005

Antarctic fishes live at a constant temperature of −1.8°C, in an oxygen‐rich environment. In comparison with fishes that live in temperate or tropical waters, their blood contains less erythrocytes and hemoglobin. A study was initiated on the structure and function of Antarctic fish hemoglobin. The erythrocytes of the Antarctic benthic teleost Notothenia coriiceps neglecta, of the family Nototheniidae, have been shown to contain two hemoglobins, accounting for about 90% and 5% of the total content. These hemoglobins have been isolated, and obtained in crystalline form. They are tetramers and contain two pairs of globin chains. The globin chains of each hemoglobin have been purified and characterised. The two hemoglobins appear to have one of the two globin chains in common. The Root and Bohr effects have been investigated in erythrocytes, ‘stripped' hemolysates and pure hemoglobins, indicating that the functional properties are finely regulated by pH and allosteric effectors.
The histidine residue of codon 715 is essential for function of elongation factor 2
Authors: OMURA Fumihiko , KOHNO Kenji , UCHIDA Tsuyoshi
Published: March 2005

Several mutant cDNAs of elongation factor 2 (EF‐2) were constructed by site‐directed mutagenesis and their products expressed in mouse cells were investigated. Amino acid substitution for the histidine residue of codon 715, which is modified post‐translationally to diphthamide, resulted in non‐functional EF‐2 and this substitution did not render EF‐2 resistant to Pseudomonas aeruginosa exotoxin A, which inactivates EF‐2 transferring ADP‐ribose to the diphthamide residue. These non‐functional EF‐2s with replacements of the histidine‐715 residue showed various extents of inhibition of protein synthesis by competing with functional EF‐2 in vivo. These results suggest that histidine‐715 is essential for the translocase activity of EF‐2 and that the region around diphthamide functions in recognition of, and/or binding to ribosomes. Substitution of proline for the alanine‐713 residue and substitution of glutamine for the glycine‐717 residue converted EF‐2 to partially toxin‐resistant forms. Two‐dimensional gel analysis with fragment A of diphtheria toxin of these toxin‐resistant EF‐2s revealed that their ADP‐ribosylations by toxin were much less than that of wild‐type EF‐2.
Tubulin and MAP2 regulate the PCSL phosphatase activite
Authors: JESSUS Catherine , GPROS Jozef , CAYLA Xavier , HERMANN Jacques , HENDRIX Peter , OZON René , MERLEVEDE Wilfried
Published: March 2005

Tubulin can stimulate specifically the aryl phosphatase activity of the low‐Mr polycation‐stimulated (PCSL) phosphatase, measured as p‐nitrophenyl phosphatase activity, or using reduced carboxamidomethylated and maleylated (RCM) lysozyme, phosphorylated on tyrosyl residues, as a substrate. This stimulation is independent of the degree of polymerization of tubulin (A50= 60 nM) and is due to an increase in Vmax. It is mechanistically different from the ATP‐induced activation and resistant to heat and trypsin treatment. Chymotrypsin destroys the stimulatory effect of tubulin. The polycation‐stimulated phosphorylase phosphatase activity is inhibited by tubulin, probably by a polycation/polyanion interaction. The microtubule‐associated protein. MAP2. is inhibitory to the p‐nitrophenyl phosphatase activity and tubulin can elimiate this inhibitory effect. MAP2 also inhibits the polycation‐stimulated phosphorylase phosphatase activity.
Specificity of antibodies to sea anemone toxin III and immunogenicity of the pharmacological site of anemone and scorpion toxins
Authors: BAHRAOUI El Mostafa , AYEB Mohamed EL , GRANIER Claude , BERESS Laslo , ROCHAT Hervé
Published: March 2005

Toxin III (ATX III) of the sea anemone (Anemonia sulcata) is a polypeptide containing 27 amino acid residues.

It has no sequence similarity with other toxins (ATX I and II) from the same species, or with scorpion toxins, although they apparently act in a similar manner by prolonging action potentials. The specificity of ATX III antibodies was characterized using ATX III, ATX I, native and chemically modified ATX II, and scorpion α‐toxins. The results obtained suggest that a regior of ATX III, partially or totally overlapping the pharmacological site shared with ATX I and ATX II, is immunogenic. It includes a guanidino and at least two carboxylate groups. The corresponding region is not immunogenic in ATX I and ATX II. Anti‐(ATX III) antibodies recognize the similar regions of ATX I and ATX II and apparently do not recognize scorpion toxins.
Correlation between enzyme activity and hinge‐bending domain displacement in 3‐phosphoglycerate kinase
Authors: SINEV Michael A. , RAZGULYAEV Oleg I. , VAS Mária , TIMCHENKO Alexander A. , PTITSYN Oleg B.
Published: March 2005

Diffuse X‐ray‐scattering data give evidence for large‐scale structural change in pig muscle 3‐phosphoglycerate kinase upon substrate binding. Simultaneous binding of 3‐phosphoglycerate and MgATP either to the unmodified enzyme or to its active methylated derivative leads to about an 0.1‐nm decrease in radius of gyration. These data coincide well with the previous data for yeast 3‐phosphoglycerate kinase.

When, instead of methylation, the two reactive thiol groups of pig muscle 3‐phosphoglycerate kinase are carboxamidomethylated, the enzyme becomes inactive and the radii of gyration of its ‘apo’ and ‘holo’ forms do not differ within limits of experimental error.

Thus, a correlation exists between the activity of 3‐phosphoglycerate kinase and its substrate‐induced largescale conformational change. This correlation is a strong argument in favor of the functional importance of domain locking in the reaction catalyzed by 3‐phosphoglycerate kinase.
Purification and characterization of trypsin from the poikilotherm Gadus morhua
Authors: ÁGEIRSSON Bjarni , FOX Jay W. , BJARNASON Jón B.
Published: March 2005

A serine protease shown to be trypsin was purified from the pyloric caeca of Atlantic cod (Gadus morhua), and resolved into three differently charged species by chromatofocusing (pI 6.6, 6.2 and 5.5). All three trypsins had similar molecular mass of 24.2 kDa. N‐terminal amino acid sequence analysis of cod trypsin showed considerable similarity with other known trypsins, particularily with dogfish and some mammalian trypsins. The apparent Km values determined at 25°C for the predominant form of Atlantic cod trypsin towards p‐tosyl‐L‐arginine methyl ester and N‐benzoyl‐L‐arginine P‐nitroanilide were 29 μM and 77 μM respectively, which are notably lower values than those determined for bovine trypsin (46 μM and 650 μM respectively). The difference was particularly striking when the amidase activity of the enzymes was compared. Furthermore, the Kcat values determined for the Atlantic cold trypsins were consistently higher than the values determined for bovine trypsin. The higher catalytic efficiency (Kcat/Km) of Atlantic cod trypsin as compared to bovine trypsin may reflect an evolutionary adaptation of the poikilothermic species to low environmental temperatures.
The kinetics of the thermal deactivation of transglutaminase from guinea‐pig liver
Authors: NURY Sylvie , MEUNIER Jean‐Claude , MOURANCHE Annette
Published: March 2005

By incubating native (N) transglutaminase from guinea‐pig liver at various temperatures and assaying it at 25°C, two steps in the irreversible deactivation process to the denatured form (D) have been found. The fitting of the data to the equations of two possible models (the two‐steps model and the two‐isoenzymes model) is only compatible with the first one (N→X→D). It is shown that the structure of the active intermediate, X, depends on the deactivation temperature and on the thermal history of the enzyme. This may mean that transglutaminase exists in a large number of microstates. Surprisingly, the activation energy of deactivation is lower than that of activity (36.6 ± 3.4 against 47.2 ± 2.2 kJ · mol−1). By deactivating transglutaminase at a constant temperature (55°C) and assaying it at variable temperatures, the activation energy of the intermediate, (X55), has been determined to be 40.2 ± 5 kJ · mol−1, of the same order of magnitude as the native form.

Among several agents assayed, only Ca2+ had a positive effect on the thermal stability of this enzyme. At 40°C, transglutaminase was quite stable in the presence of Ca2+ (in its absence, the half‐life was 65 min) and at 45°C, its thermostability had been considerably increased, the half‐life being raised from 47 min to 275 min.
Involvement of antizyme in stabilization of ornithine decarboxylase caused by inhibitors of polyamine synthesis
Authors: MURAKAMI Yasuko , NISHIYAMA Masaki , HAYASHI Shin‐ichi
Published: March 2005

Contrary to previous findings, ornithine decarboxylase (ODC) was stabilized by treatment of cells with DL‐α‐difluoromethylornithine, an enzyme‐activated irreversible inhibitor of ODC. Both this inhibitor and cyclohexylamine, a spermidine synthase inhibitor known to stabilize ODC, caused decreases in the antizyme/ODC ratio by increasing ODC content and conversely decreasing antizyme content. The relationship between cellular polyamine levels and antizyme content indicated that spermidine is the most important polyamine for antizyme induction. These results suggest that antizyme is involved in the mechanism underlying the stabilization of ODC by inhibitors of polyamine synthesis and support the hypothesis that cellular polyamines regulate ODC degradation via antizyme.
The primary structure of alcohol dehydrogenase from Drosophila lebanonensis
Authors: VILLARROYA Angel , JUAN Elvira , EGESTAD Börje , JÖRNVALL Hans
Published: March 2005

Insect alcohol dehydrogenase is highly different from the well‐known yeast and mammalian alcohol dehydrogenases. The enzyme from Drosophila lebanonesis has now been characterized by protein analysis and was found to have a 254‐residue protein chain with an acetyl‐blocked N‐terminal Met. Comparisons with the structures of the enzyme from other species allows judgement of the extent of variability within the insect alcohol dehydrogenases. They have diverged to a considerable extent; two forms analyzed at the protein level differ at 18% of all residues, and all the known Drosophila alcohol dehydrogenase structures reveal differences at 72 positions. Some deviations, against a background similarity, in the extent of changes are noted among the parts corresponding to different exons. The structural variation within Drosophila is about as large as the one for the mammalian zinccontaining alcohol dehydrogenase. Consequently, the results illustrate Drosophila relationships and establish great variations also for group of alcohol dehydrogenases lacking zinc.
Properties of D‐amino‐acid oxidase from Rhodotorula gracilis
Authors: PILONE SIMONETTA Mirella , POLLEGIONI Loredano , CASALIN Paola , CURTI Bruno , RONCHI Severino
Published: March 2005

The flavoprotein D‐amino‐acid oxidase was purified to homogeneity from the yeast Rhodotorula gracilis by a highly reproducible procedure. The amino acid composition of the protein was determined; the protein monomer had a molecular mass of 39 kDa and contained one molecule of FAD. The ratio between A274/A455 was about 8.2. D‐Amino‐acid oxidase from yeast showed typical flavin spectral perturbations on binding of the competitive inhibitor benzoate and was reduced by D‐alanine under anaerobiosis. The enzyme reacted readily with sulfite to form a covalent reversible adduct and stabilized the red anionic form of the flavin semiquinone on photoreduction in the presence of 5‐deazariboflavin; the 3,4‐dihydro‐FAD from was not detectable after reduction with sodium borohydride. Thus D‐amino‐acid oxidase from yeast exhibited most of the general properties of the dehydrogenase/oxidase class of flavoproteins; at the same time, the enzyme showed some peculiar features with respect to the same protein from pig kidney.
Mechanism of action of cysteinyl leukotrienes on glucose and lactate balance and on flow in perfused rat liver
Authors: IWAI Masaru , JUNGERMANN Kurt
Published: March 2005

Rat livers were perfused at constant pressure via the portal vein with media containing 5 mM glucose, 2mM lactate and 0.2 mM pyruvate.

Leukotrienes C4 and D4 enhanced glucose and lactate output and reduced perfusion flow to the same extent and with essentially identical kinetics. They both caused half‐maximal alterations (area under the curve) of carbohydrate metabolism at a concentration of about 1 nM and of flow at about 5 nM. The leukotrine‐C4/D4 antagonist CGP 35949 B inhibited the metabolic and hemodynamic effects of 5 nM leukotrienes C4 and D4 with the same efficiency, causing 50% inhibition at about 0.1 μM.

Leukotriene C4 elicited the same metabolic and hemodynamic alterations with the same kinetics as leukotriene D4 in livers from rats pretreated with the γ‐glutamyltransferase inhibitor, acivicin.

The calcium antagonist, nifedipine, at a concentration of 50 μM did not affect the metabolic and hemodynamic changes caused by 5 nM leukotriene D4. The smooth‐muscle relaxant, nitroprussiate, at a concentration of 10 μM reduced flow changes, without significantly affecting the metabolic alterations.

Leukotriene D4 not only reduced flow; it also caused an intrahepatic redistribution of flow, restricting some areas from perfusion. Thus leukotrienes increased glucose and lactate output directly in the accessible parenchyma and, in addition, indirectly by washout from restricted areas during their reopening upon termination of application.

The phospholipase A2 inhibitor, bromophenacyl bromide, but not the cyclooxygenase inhibitor, indomethacin, at a concentration of 20 μM reduced the metabolic and hemodynamic effects of 5 mM leukotriene D4.

Stimulation of the sympathetic hepatic nerves with 2‐ms rectangular pulses at 20 Hz and infusion of 1 μM noradrenaline increased glucose and lactate output and decreased flow, similar to 10 nM leukotrienes C4 and D4. The kinetics of the metabolic and hemodynamic changes caused by the leukotrienes differed, however, from those due to nerve stimulation and noradrenaline.

The leukotriene‐C4/D4 antagonist, CGP 35949 B, even at very high concentrations (20 μM) inhibited the metabolic and hemodynamic alterations caused by nerve stimulation or noradrenaline infusion only slightly and unspecifically.

The present results suggest that (a) leukotriene C4 and leukotriene D4 modulate hepatic metabolism and hemodynamics with equal kinetics and efficiencies specifically, via leukotriene C4/D4 receptors; (b) they act without being dependent upon calcium uptake via slow calcium channels, in part indirectly, by stimulating the formation of other phospholipase A2 produts and (c) they do not have a major role in the actions of sympathetic hepatic nerves and circulating noradrenaline. Metabolic regulation by leukotrienes, which are formed only in non‐parenchymal cells, constitutes another example of a complex intra‐organ cell—cell communication between non‐parenchymal and parenchymal cells.
Nuclear basic protein transition during sperm differentiation
Authors: CHAUVIÈRE Muriel , MARTINAGE Arlette , BRIAND Gilbert , SAUTIÈRE Pierre , CHEVAILLIER Philippe
Published: March 2005

The remodeling of nucleoproteins during dog‐fish spermiogenesis involves two successive nuclear protein transitions: the first from somatic‐type histones to transition proteins during the nuclear elongation of spermatids and the second leading to protamine‐DNA association in mature spermatozoa. The chromatin of elongating spermatids contains two transition proteins called S1 and S2. The amino acid sequence of protein S1, a polypeptide of 87 residues was determined previously [Chauvière, M., Martinage, A., Briand, G., Sautière, P. & Chevaillier, Ph. (1987) Eur. J. Biochem. 169, 105–111]. In the present paper, we report the elucidation of the primary structure of the minor transition protein S2 established by automated Edman degradation of the protein and of its fragments generated by cleavage at methionine and aspartate residues. S2 contains 80 residues and has a molecular mass of 9726 Da. S2 is mainly characterized by a high content of basic amino acids mostly represented by lysine, a relatively high level of hydrophobic residue, the presence of six phosphorylatable residues and the lack of cysteine. Its amino acid sequence shows that the N‐terminal half is highly basic, while the acidic residues are located in the C‐terminal part of the protein where more diversity in amino acids is noticed.

The two transition proteins S1 and S2 share striking structural similarities. Few but significative similarities have been detected with the mammalian transition protein TP1 [Kistler, W. S., Noyes, C., Hsu, R. & Heinrikson, R. L. (1975) J. Biol. Chem. 250, 1847–1853], suggesting similar functions for all these proteins in chromatin remodeling during sperm differentiation. By contrast, the two dog‐fish spermatid‐specific proteins are structurally unrelated to sperm protamines and cannot be considered as their precursors.
Solution conformation of sialosylcerebroside (GM4) and its NeuAc(α2→3)Galβ sugar component
Authors: POPPE Leszek , DABROWSKI Janusz , von der LIETH Claus‐Wilhelm , NUMATA Masaaki , OGAWA Tomoya
Published: March 2005

The solution conformations of GM4 ganglioside [NeuAc(α2→3)Gal(β→1)Cer] in (2H3C)2SO and its component disaccharide in 2H2O were investigated with the aid of 1H‐nuclear magnetic resonance spectroscopy (nuclear Overhauser effect, analysis of coupling constants) and by energy‐minimum calculations. The existence of three low‐energy conformers obtained by theoretical calculations was supported by experimental findings in the case of GM4, whereas the disaccharide appears to exist as a mixture of two conformers.
Characterization of the interleukin‐1‐induced tyrosine phosphorylation of a 41‐kDa plasma membrane protein of the human tumor cell line K 562
Authors: MARTIN Michael , LOVETT David H. , SZAMEL Marta , RESCH Klaus
Published: March 2005

A 41‐kDa protein, which was specifically phosphorylated upon incubation with natural purified murine interleukin 1, was recently identified by us [Martin, M., Lovett, D. H. and Resch, K. (1986) Immunobiology 171, 165–169] in highly purified plasma membranes from the human tumor cell line K 562. An in vitro assay was used to investigate and characterize the phosphorylation induced by interleukin 1, possibly involved in signal transduction and generation. Plasma membranes were incubated with radiolabeled ATP in the presence of purified natural murine interleukin 1, or recombinant human interleukin 1α and the pattern of phosphoproteins was studied after separation by SDS/PAGE and subsequent autoradiography. A 41‐kDa protein (pp41) was specifically phosphorylated on a tyrosine residue in the presence of interleukin 1 in a dose‐ and time‐dependent manner. The protein showed a weak background phosphorylation in the absence of monokine. Phosphorylation took place very efficiently at 0°C, whereas phosphatases were not active at that temperature. At 37°C, a rapid dephosphorylation was observed which was inhibited specifically by Zn2+ and vanadate. The interleukin‐1‐specific induction of the phosphorylation could also be observed after detergent solubilization of the plasma membranes. Affinity labeling with an ATP analogue revealed an ATP‐binding and cleaving site at 41 kDa. Interleukin 1 did not induce the phosphorylation of p41 in plasma membranes obtained from a subclone of K 562, which did not respond to interleukin 1 with growth inhibition, as was reported recently for the K 562 mother line [Lovett, D. H., Kozan, B., Hadam, M., Resch, K. and Gemsa, D. (1986) J. Immunol. 136, 340–347]. These data suggest that the interleukin‐1 receptor is functionally linked to a protein‐tyrosine kinase, which is implicated in its biological function.
Regulation of the cytoplasmic pH by a proton‐translocating ATPase in Streptococcus faecalis (faecium)
Authors: SUZUKI Takashi , KOBAYASHI Hiroshi
Published: March 2005

Earlier work from this laboratory led to the proposal that the cytoplasmic pH of streptococcal cells is regulated solely by changes in the amount and activity of a proton‐translocating ATPase, F1F0 complex [Kobayashi, H., Suzuki, T. & Unemoto, T. (1986) J. Biol. Chem. 261, 627–630]. We have now examined the proposal with the aid of computer simulation. We find that an increase in the amount of the H+‐ATPase is necessary for pH regulation and is sufficient to maintain a constant steady‐state cytoplasmic pH. An increase in H+‐ATPase activity is insufficient by itself to maintain a constant cytoplasmic pH, but suppresses the initial fluctuation of the pH. When both variations were allowed, the simulated cytoplasmic pH remained constant despite large perturbations, suggesting that this regulatory system has ample capacity to compensate for pH changes. The present work shows that a computer simulation is a useful way to examine a model for biological regulatory system; application of the simulation to other regulatory systems is discussed.
cDNA sequence of adrenodoxin reductase
Authors: HANUKOGLU Israel , GUTFINGER Tamar
Published: March 2005

Adrenodoxin reductase is an NADP dependent flavoenzyme which functions as the reductase of mitochondrial P 450 systems. We sequenced two adrenodoxin reductase cDNAs isolated from a bovine adrenal cortex cDNA library. The deduced amino acid sequence shows no similarity to the sequence of the microsomal P 450 systems or other known protein sequences. Nonetheless, by sequence analysis and comparisons with known sequences of dinucleotide‐binding folds of two NADP‐binding flavoenzymes, two regions of adrenodoxin reductase sequence were identified as the FAD‐ and NADP‐binding sites. These analyses revealed a consensus sequence for the NADP‐binding dinucleotide fold (GXGXXAXXXAXXXXXXG, in one‐letter amino acid code) that differs from FAD and NAD‐binding dinucleotide‐fold sequences. In the data base of protein sequences, the NADP‐binding‐site sequence appears solely in NADP‐dependent enzymes, the binding sites of which were not known to date. Thus, this sequence may be used for identification of a certain type of NADP‐binding site of enzymes that show no significant sequence similarity.
Structure and biological activity of basement membrane proteins
Authors: TIMPL Rupert
Published: March 2005

Collagen type IV, laminin, heparan sulfate proteoglycans, nidogen (entactin) and BM‐40 (osteonectin, SPARC) represent major structural proteins of basement membranes. They are well‐characterized in their domain structures, amino acid sequences and potentials for molecular interactions. Such interactions include self‐assembly processes and heterotypic binding between individual constituents, as well as binding of calcium (laminin. BM‐40) and are likely to be used for basement membrane assembly. Laminin, collagen IV and nidogen also possess several cell‐binding sites which interact with distinct cellular receptors. Some evidence exists that those interactions are involved in the control of cell behaviour. These observations have provided a more defined understanding of basement membrane function and the definition of new research goals in the future.
Purification of ATP diphosphohydrolase from bovine aorta microsomes
Authors: YAGI Kiyohito , ARAI Yukinori , KATO Norikatsu , HIROTA Kouichi , MIURA Yoshiharu
Published: March 2005

ATP diphosphohydrolase (EC 3.6.1.5) hydrolyzes pyrophosphate bonds of nucleoside di‐ and triphosphates in the presence of divalent cations. We purified the enzyme from the vessel wall of bovine aortas. The procedure gave a homogeneous preparation of ATP diphosphohydrolase for the first time from an animal source. Bovine aorta microsomes were treated with 50 mM bicarbonate buffer (pH 10.0) containing 0.025% Triton X‐100. The enzyme was then solubilized from the microsomes with 0.5% Triton X‐100 and purified to homogeneity by DEAE‐Sepharose CL‐6B chromatography and 5′AMP‐Sepharose 4B affinity chromatography. The apparent molecular mass of the pure enzyme was 110 kDa. The activity recovered was 6% of that of the microsomes. The enzyme was more active with Ca2+ than Mg2+. The sensitivity of ADPase activity to divalent cations was higher than that of ATPase activity. The enzyme had broad substrate specificity to nucleoside di‐ and triphosphates.