bluuebunny/crossref_metadata_2025 huggingface dataset:
An evaluation of the role of a pyroglutamyI peptidase, a post‐proline cleaving enzyme and a post‐proline dipeptidyI amino peptidase, each purified from the soluble fraction of guinea‐pig brain, in the degradation of thyroliberin in vitro
Authors: BROWNE Padraig , O'CUINN Gerard
Published: March 2005

The degradation of thyroliberin (<Glu‐His‐Pro‐NH2) to its component amino acids by the soluble fraction of guinea pig brain is catalysed by four enzymes namely a pyroglutamate aminopeptidase, a post‐proline cleaving enzyme, a post‐proline dipeptidyl aminopeptidase and a proline dipeptidase.

1. The pyroglutamate aminopeptidase was purified to over 90% homogeneity with a purification factor of 2868‐fold and a yield of 5.7%. In addition to catalysing the hydrolysis of thyroliberin, acid thyroliberin and pyroglutamate‐7‐amido‐4‐methylcoumarin the pyroglutamate aminopeptidase catalysed the hydrolysis of the peptide bond adjacent to the pyroglutamic acid residue in luliberin, neurotensin, bombesin, bradykinin‐potentiating peptide B, the anorexogenic peptide and the dipeptides pyroglutamyl alanine and pyroglutamyl valine. Pyroglutamyl proline and eledoisin were not hydrolysed.

2. The post‐proline cleaving enzyme was purified to apparent electrophoretic homogeneity with a purification factor of 2298‐fold and a yield of 10.6%. The post‐proline cleaving enzyme catalysed the hydrolysis of thyroliberin and N‐benzyloxycarbonyl‐glycylproline‐7‐amido‐4‐methylcoumarin. It did not catalyse the hydrolysis of glycylproline‐7‐amido‐4‐methylcoumarin or His‐Pro‐NH2.

3. The post‐proline dipeptidyl aminopeptidase was partially purified with a purification factor of 301‐fold and a yield of 8.9%. The post‐proline dipeptidyl aminopeptidase catalysed the hydrolysis of His‐Pro‐NH2 and glycylproline‐7‐amido‐4‐methylcoumarin but did not exhibit any post‐proline cleaving endopeptidase activity against thyroliberin or N‐benzyloxycarbonyl‐glycylproline‐7‐amido‐4‐methylcoumarin.

4. Studies with various functional reagents indicated that the pyroglutamate aminopeptidase could be specifically inhibited by 2‐iodoacetamide (100% inhibition at an inhibitor concentration of 5μM), the post‐proline cleaving enzyme by bacitracin (IC50= 42 μM) and the post‐proline dipeptidyl aminopeptidase by puromycin (IC50= 46 μM). Because of their specific inhibitory effects these three reagents were key elements in the elucidation of the overall pathway for the metabolism of thyroliberin by guinea pig brain tissue enzymes.
Isolation, purification and characterization of the ATPase complex from the thermophilic cyanobacterium Synechococcus 6716
Authors: LUBBERDING Henk J. , ZIMMER Guido , VAN WALRAVEN Hendrika , SCHRICKX Jaap , KRAAYENHOF Ruud
Published: March 2005

The ATPase complex is isolated and purified from membrane vesicles of the thermophilic cyanobacterium Synechococcus 6716 by octyl glucoside and cholic acid by a modification of the procedure for its extraction from spinach chloroplasts. The complex is purified by differential centrifugation and ammonium sulfate precipitation and by gel filtration on Sepharose 6B. The purified fraction, without any phycocyanin contamination, shows ATP hydrolysis activity and Pi/ATP exchange activity of 1564 and 350 nmol · min−1· mg protein−1, respectively. N, N′‐Dicyclohexylcarbodiimide inhibits the ATP hydrolysis activity of this purified fraction. On polyacrylamide gels most typical F1 ATPase polypeptides are identified, but the low‐molecular weight polypeptides visible cannot be ascribed to the Fo part of the complex with certainty; non‐identified bands around 30kDa are also present.
Evidence that the Yb subunits of hepatic glutathione transferases represent two different but related families of polypeptides
Authors: BEALE Dennis , MEYER David J. , TAYLOR John B. , KETTERER Brian
Published: March 2005

Three soluble rat liver glutathione (GSH) transferases A, C and one referred to as ‘D’, all of which are dimers of Yb subunits [Bass et al. (1977) Biochim. Biophys. Acta, 492, 163–175], have been compared with respect to C‐terminal amino acids and tryptic peptide maps. GSH transferases A and ‘D’ gave different tryptic peptide maps and different C‐terminal amino acids, Iysine and proline respectively. In each case the number of tryptic peptides is about half of that expected from their Iysine and arginine content, and there are 2 mol C‐terminal amino acid/mol enzyme. This indicates that GSH transferases A and ‘D’ represent two different Yb homodimers, which we refer to here as Y1bY1b and Y2bY2b respectively. GSH transferase C is the corresponding heterodimer Y1bY2b since it gives all the tryptic peptides which arise from GSH transferase A and GSH transferase ‘D’ and also contains both C‐terminal Iysine and proline. These results provide a structural basis to similar conclusions drawn by Mannervik and Jensson [(1980) J. Biol. Chem. 257, 9909–9912] based on enzymic and immunological comparisons.

Tryptic peptide maps show that GSH transferases A and ‘D’ have considerable homology since there are 23 peptides common to both, 12 peptides unique to A and 8 peptides unique to ‘D’. Even so GSH transferase A is selectively induced by a phenobarbitone regime. It is, therefore, concluded that Y1b and Y2b are derived from separate but related genes. A similar conclusion has been drawn concerning the Ya and Yc subunits [Beale et al. (1982) Eur. J. Biochem. 126, 459–463], and a comparison of amino acid compositions, presented here, further suggests a genetic relationship between both pairs of subunits.
Conformational studies of trinucleoside bisphosphates 2. Potential energy calculations
Authors: LEE Che‐Hung
Published: March 2005

Semi‐empirical potential energy calculations were carried out for the conformations of ApApA, a trinucleoside bisphosphate (trimer), as proposed in the preceding paper in this journal. Energy minimals were obtained for these conformers, i.e. conformers I‐I, I‐II, III‐I′ and I′‐I′ (the first Roman numeral represents the conformation of the ApAp‐ moiety and the second one the ‐pApA moiety of ApApA), of the nearest‐neighbor interactions, and conformers B1, B2 and B3 of bulged structures. The results show that the detailed conformations of the component dinuclotide (dimer) moieties in I‐I, I‐II, III‐I′ and I′‐I′ are close (± 10°) to the corresponding stable conformations of dinucleotides in solution proposed by Lee and coworkers. The bulged conformations B1, B2 and B3 have open forms for each dinucleotide moiety. Their preferred local conformations are anti, γ+, β+, ɛ+ (except for Apɛin B3, ɛ−), and 3′endo (except for Ap‐ in B3, 2′endo) for the glycosidic bond, C4′‐C5′, C5′‐O5′, C3′‐O3′ and the ribose moiety, respectively. The conformation of the phosphodiester linkage in ‐pApA moiety is ζt2, αt3 for all the three bulged conformers, while that in ApAp‐is ζ−1, αt2 for B1, ζ+1α−2 for B2, and ζ+1, α+2 for B3. The calculated composite chemical shifts of the base protons from ring‐current effects agree with the corresponding observed trimerization shifts. Conformers I‐I and I′‐I′ are helix‐like structures and correspond to the right‐handed and the ‘vertical‐stacked’ helices, respectively. The structure of I‐II is similar to that observed in the ApApA single crystals. The bulged conformations may serve as models for frameshift mutations.
Synthesis and properties of NBD‐n‐acylcholines, fluorescent analogs of acetylcholine
Authors: MEYERS Hans‐Wilhelm , JÜRSS Rolf , BRENNER Hans R. , FELS Gregor , PRINZ Heino , WATZKE Heidrun , MAELICKE Alfred
Published: March 2005

We have synthesized a homologous series of fluorescent analogs of acetylcholine, N‐7‐(4‐nitrobenzo‐2‐oxa‐1,3‐diazolyl)‐ω‐amino‐n‐alkanoic acid β(N,N,N‐trialkylammonium) ethylesters (NBD‐n‐acylcholines) and report here on their physiological and biochemical properties.

(1) All NBD‐n‐acylcholines trimethylated at the cholinergic nitrogen are agonists of acetylcholine at the frog neuromuscular junction. Their potencies in depolarizing frog muscle cells decrease with decreasing chain length.

(2) The affinities of binding to the purified receptor from Electrophorus electricus also decrease with decreasing chain length with a large drop in affinity for the derivatives n= 4 and n= 3.

(3) The rate constants of association to acetylcholine receptor and to acetylcholine esterase are of the order of 108 M−1s−1 and do not vary significantly with the chain length of the NBD‐n‐acylcholines. In contrast, the dissociation rate constants decrease with increasing chain length.

(4) The quenching of fluorescence of NBD‐n‐acylcholines accompanying binding to purified receptor and esterase from E. electricus appears to be due to the formation of a hydrogen bond between the ω‐amino group as donor and an unidentified acceptor group in a hydrophobic pocket of the protein.

With their advantageous fluorescence properties, their simple pharmacology, and their clear structure‐function relationships, these compounds are useful tools for the study of cholinergic mechanisms.
Redox‐linked proton translocation in the b–c1 complex from beef‐heart mitochondria reconstituted into phospholipid vesicles
Authors: LORUSSO Michele , GATTI Domenico , BOFFOLI Domenico , BELLOMO Elisabetta , PAPA Sergio
Published: March 2005

Possible involvement of polypeptides of b–c1 complex of beef‐heart mitochondria in its redox and protonmotive activity has been investigated, by means of chemical modification of amino acid residues in the soluble as well as in the phospholipid‐reconstituted b–c1 complex. Treatment of the enzyme with tetranitromethane (C(NO2)4) or with ethoxyformic anhydride (EFA), that modify reversibly tyrosyl and hystidyl residues respectively, resulted in a marked inhibition of electron transport from reduced quinols to cytochrome c. This was accompanied, in b–c1 reconstituted into phospholipid vesicles, by a parallel inhibition of respiratory‐linked proton translocation; the H+/e− stoichiometry remained unchanged. Treatment of b–c1 complex with DCCD, that specifically modifies carboxylic groups of glutammic or aspartic residues caused a marked depression of proton translocation in b–c1 vesicles, under conditions where the rate of electron flow in the coupled state, was enhanced. As a consequence the H+/e− stoichiometry was lowered.

SDS gel electrophoresis and [14C]DCCD‐labelling of the polypeptides of the b–c1 complex showed a major binding of 14C‐DCCD to the 8‐kDa subunit of the complex and possible cross‐linking, induced by DCCD treatment, of polypeptide(s) in the 8‐kDa band and the 12‐kDa band, with the Fe‐S protein of the complex, with the appearance of a new polypeptide band with an apparent molecular mass of about 40 kDa.

Involvement of polypeptides of low molecular mass, for which no functional role was so far described, and possibly of the Fe‐S protein in the redox‐linked proton translocation in b–c1 complex is suggested.
Molecular characterization of the gene coding for major outer membrane protein OmpA from Enterobacter aerogenes
Authors: BRAUN Gabi , COLE Stewart T.
Published: March 2005

The ompA gene from Enterobacter aerogenes was subcloned into a low‐copy‐number plasmid vector and the resultant plasmid, pTU7En, used to study its expression in Escherichia coli K12. Although the gene was strongly expressed and large amounts of OmpA protein were present in the outer membrane its product was not functionally identical to the E. coli polypeptide. In particular, the E. aerogenes OmpA protein was unable to confer sensitivity to OmpA‐specific phages of E. coli. When the primary structure of the protein was deduced from the nucleotide sequence of its gene it was found that three domains differed extensively from the corresponding regions of the E. coli protein. As two of these are known to be exposed on the cell surface we inferred that these alterations are responsible for differences in the biological activity of the two proteins.
A survey of H10‐ and H5‐like protein structure and distribution in higher and lower eukaryotes
Authors: SMITH Bryan J. , HARRIS Martin R. , SIGOURNAY Catherine M. , MAYES Elaine L. V. , BUSTIN Michael
Published: March 2005

A survey of H1° and H5‐like proteins has been conducted through a range of higher and lower eukaryotic species. All mammals examined possessed H1° proteins, although in variable amounts, and the protein's structure was well conserved, though not invariant. The testis‐specific histone Hlt (from rat) did not have an H1°‐like structure and it appears that H1° does not occur in spermatocytes in any form. The results also show that Xenopus laevis contains H1°‐like proteins, but lower, non‐vertebrate eukaryotes (a crustacean, two fungi and a plant tissue) do not possess H1° or H5 proteins. The evidence suggests that H1° and H5 proteins may be considered as belonging to one family, distinct from H1 types. This H1°/H5 family may well be ‘replacement histone’ variants of H1. The results do not support suggestions of roles such as repression of DNA synthesis or of transcription for H1°/H5 proteins.
Chemical modification of hydroxynitrile lyase by selective reaction of an essential cysteine‐SH group with α,β‐unsaturated propiophenones as pseudo‐substrates
Authors: JAENICKE Lothar , PREUN Jochem
Published: March 2005

3‐Oxo‐3‐phenylpropyne and 3‐oxo‐3‐phenylpropene were synthesized as active‐site‐directed irreversible inhibitors of the bitter almond hydroxynitrile lyase (EC 4.1.2.10), an FAD‐protein. The substrate and competitors (e.g. benzoate) decrease the rate of the inhibitor‐mediated deactivation of the enzyme. By excess addition of either one of the two inhibitors, the deactivation process is shown to be pseudo‐first order. The reaction with equimolar amounts of 3‐oxo‐3‐phenylpropyne with the enzyme is accompanied by a shift in the ultraviolet spectrum of the inhibitor, allowing direct measurement of the enzyme‐inactivation process. The spectral change has second‐order kinetics. Incubation with 3‐oxo‐3‐[p‐3H]phenylpropyne or 3‐oxo‐3‐[1‐14C]phenylpropene shows a one‐to‐one stoichiometry for the inhibitor‐enzyme reaction. Dissociation of the 3‐oxo‐3‐[p‐3H]phenylpropyne‐inactivated holoenzyme with acid ammonium sulfate yields a labeled apoenzyme; the inhibitor does not react with free or enzyme‐bound FAD. After boranate reduction and exhaustive hydrolysis of the 3‐oxo‐3‐[1‐14C]phenylpropene‐inactivated enzyme, a labeled cysteine derivative was isolated which was identified by chromatographic and mass spectroscopic comparison with synthetic references as l‐2‐amino‐4‐thia‐dl‐7‐hydroxy‐7‐phenylheptanoic acid, the reduced, linear addition product of the inhibitor to a cysteine‐SH group.
Structure of a novel flavin chromophore from Avena coleoptiles, the possible “blue light” photoreceptor
Authors: GHISLA Sandro , MACK Rudi , BLANKENHORN Gunter , HEMMERICH Peter , KRIENITZ Eberhard , KUSTER Thomas
Published: March 2005

A yellow chromophore has been isolated from Avena coleoptiles grown in the dark. It had previously been shown by Zenk [Zenk, M. H. (1967) Z. Pflanzenphysiol. 56, 122–140] to be a flavin of still unidentified structure, and had been suggested to be the blue light photoreceptor in this organism. The structure of this flavin (F1‐X) has been identified as 5′‐malonylriboflavin by a combination of physicochemical techniques, and by its identity with a sample obtained synthetically. The 5′‐malonylester linkage is relatively labile towards hydrolysis and photolysis, it isomerizes to an equilibrium mixture containing probably the 4′ isomer. The electronic spectra of 5′‐malonylriboflavin (absorption and fluorescence) are practically identical to those of normal riboflavin (vitamin B2).
Interaction of a glutathione S‐conjugate with glutathione reductase Kinetic and X‐ray crystallographic studies
Authors: BILZER Manfred , KRAUTH‐SIEGEL R. Luise , SCHIRMER R. Heiner , AKERBOOM Theo P. M. , SIES Helmut , SCHULZ Georg E.
Published: March 2005

S‐Conjugates of glutathione influence the glutathione/glutathione disulfide (GSH/GSSG) status of hepatocytes in at least two ways, namely by inhibition of GSSG transport into the bile [Akerboom et al. (1982) FEBS Lett. 140, 73–76] and by inhibition of the enzyme GSSG reductase (EC 1.6.4.2).

The interaction of GSSG reductase with a well‐studied conjugate, namely S‐(2,4‐dinitrophenyl)‐glutathione and its electrophilic precursor 1‐chloro‐2,4‐dinitrobenzene are described. For short exposures both compounds are reversible inhibitors of the enzyme, the Ki values being 30 μM and 22 μM respectively. After prolonged incubation, 1‐chloro‐2,4‐dinitrobenzene blocks GSSG reductase irreversibly, which emphasizes the need for rapid conjugate formation in situ.

As shown by X‐ray crystallography the major binding site of S‐(2,4‐dinitrophenyl)‐glutathione in GSSG reductase overlaps the binding site of the substrate, glutathione disulfide. However, the glutathione moiety of the conjugate does not bind in the same manner as either of the glutathiones in the disulfide.
Effect of nickel on activity and subunit composition of purified hydrogenase from Nocardia opaca 1b
Authors: SCHNEIDER Klaus , SCHLEGEL Hans G. , JOCHIM Karla
Published: March 2005

The NAD‐reducing hydrogenase of Nocardia opaca 1b was found to be a soluble, cytoplasmic enzyme. N. opaca 1b does not contain an additional membrane‐bound hydrogenase. The soluble enzyme was purified to homogeneity with a yield of 19% and a final specific activity of 45 μmol H2 oxidized min−1 mg protein−1.

NAD reduction with H2 was completely dependent on the presence of divalent metal ions (Ni2+, Co2+, Mg2+, Mn2+) or of high salt concentrations (0.5–1.5 M). The most specific effect was caused by NiCl2, whose optimal concentration turned out to be 1 mM. The stimulation of activity by salts was the greater the less chaotrophic the anion. Maximal activity was achieved in 0.5 M potassium phosphate. Hydrogenase was also activated by protons. The pH optimum in 50 mM triethanolamine/HCl buffer containing 1 mM NiCl2 was 7.8–8.0. In the absence of Ni2+, hydrogenase was only active at pH values below 7.0. The reduction of other electron acceptors was not dependent on metal ions or salts, even though an approximately 1.5‐fold stimulation of the reactions by 0.1–10 μM NiCl2 was observed. With the most effective electron acceptor, benzyl viologen, a 50‐fold higher specific activity was determined than with NAD.

The total molecular weight of hydrogenase has been estimated to be 200000 (gel filtration) and 178000 (sucrose density gradient centrifugation, and sodium dodecyl sulfate electrophoresis) respectively. The enzyme is a tetramer consisting of non‐identical subunits with molecular weights of 64000, 56000, 31000 and 27000. It was demonstrated by electrophoretic analyses that in the absence of NiCl2 and at alkaline pH values the native hydrogenase dissociates into two subunit dimers. The first dimer was dark yellow coloured, completely inactive and composed of subunits with molecular weights of 64000 and 31000. The second dimer was light yellow, inactive with NAD but still active with methyl viologen. It was composed of subunits with molecular weights of 56000 and 27000.

Immunological comparison of the hydrogenase of N. opaca 1b and the soluble hydrogenase of Alcaligenes eutrophus H16 revealed that these two NAD‐linked hydrogenases are partially identical proteins.
The effect of single base‐pair mismatches on the duplex stability of d(T‐A‐T‐T‐A‐A‐T‐A‐T‐C‐A‐A‐G‐T‐T‐G) · d(C‐A‐A‐C‐T‐T‐G‐A‐T‐A‐T‐T‐A‐A‐T‐A)
Authors: TIBANYENDA Nicodemus , DE BRUIN Simon H. , HAASNOOT Cornelis A. G. , VAN DER MAREL Gijs A. , VAN BOOM Jacques H. , HILBERS Cornelis W.
Published: March 2005

The stability and dynamics of the duplex d(T‐A‐T‐T‐A‐A‐T‐A‐T‐C‐A‐A‐G‐T‐T‐G) · d(C‐A‐A‐C‐T‐T‐G‐A‐T‐A‐T‐T‐A‐A‐T‐A) has been studied by means of ultraviolet‐melting, temperature‐jump relaxation kinetics, stopped‐flow and NMR spectroscopy. In addition, the influence of the mismatches A · A, G · T, A · C and T · C, incorporated in this double helix through the introduction of non‐complementary bases in the second strand, on these parameters has been investigated. The thermodynamic parameters characterizing the melting of the duplexes have been determined. Interestingly, a substantial decrease was observed for the values of the melting enthalpy when proceeding from 0.015 M to 0.1 M NaCl solutions. All duplexes that contain mismatches have melting temperatures below that of the totally complementary double helix. On the basis of NMR experiments and differences in the free enthalpy values between the totally complementary double helix and the duplexes with mismatches, it could be concluded that some degree of stacking of the two mispaired bases between the neighbouring base pairs is maintained. At 1 M NaCl the enthalpy and entropy of the helix‐to‐coil transition of the totally complementary double helix are in good agreement with values calculated on the basis of the thermodynamic data of Borer et al. [Borer; Ph. N., Dengler, B. & Tinoco, I. (1974) J. Mol. Biol. 86, 843–853] which were derived for RNA. The kinetics of the complementary duplex and duplexes with G · T and A · C mismatches were studied by means of stopped‐flow and temperature‐jump techniques. The rate constants of formation are the same for the three double helices. The decrease in stability of the duplexes with mismatches is therefore entirely due to an increase of the dissociation constant. In temperature‐jump experiments very often a fast relaxation process is observed in addition to the relaxation characterizing the disruption of the double helix. This fast relaxation process is customarily attributed to base destacking in the single helix. By combining temperature‐jump relaxation kinetics with NMR melting experiments, it is shown that at the low temperature side of the melting transition this fast relaxation process is caused by rapid changes in the double‐helical structure.
Vibrational normal modes of folded prolyl‐containing peptides
Authors: LAGANT Philippe , VERGOTEN Gérard , FLEURY Guy , LOUCHEUX‐LEFEBVRE Marie‐H.
Published: March 2005

Normal mode analysis of standard β turns has been performed using the refined force field obtained from type‐II β‐turn‐like conformation frequencies. Effects of various potentials are discussed and theoretical predictions are compared to the experimental results of various authors.
Cyclic changes of the rate of phospholipid synthesis during synchronous growth of Escherichia coli
Authors: JOSELEAU‐PETIT Danièle , KEPES François , KEPES Adam
Published: March 2005

The problem of the coordination between cyclic events in the DNA assembly line and the cell envelope assembly line was approached with the technique of synchronized cultures. Escherichia coli strains ML 30, K12 3300, K12 PC2, K12 BB2014 and B/rF were synchronized by repeated cycles of mass doubling followed by short phosphate starvation periods. Steady‐state balanced growth was obtained by subsequent incubation in non‐limiting growth conditions for one or more generation times.

Several successive cell cycles were monitored for mass increase and cell number, while the rate of DNA synthesis and the rate of phospholipid synthesis were usually measured with more than one method.

In all strains, and in strain ML 30 in five different growth media giving doubling times from 20–110 min, a discontinuity in the rate of synthesis of phosphatidylethanolamine and of phosphatidylglycerol was observed. These two major phospholipid components of inner and outer membranes were synthesized at a constant rate per cell for a large portion of the cell cycle and the rate of synthesis of both increased twofold at the same time. This cyclic program was reproducible not only in successive cell cycles, but also in separate experiments with the same strain, in the same medium. In contrast, differences in timing were observed with different strains, and in the same strain with different carbon sources. In particular, the simultaneity of the increase in phospholipid synthesis either with DNA initiation or with cell division could not be observed as a rule.
Purification of chick nuclear thyroid‐hormone‐receptor protein
Authors: HEINTZ Judith F. , CAREW Lyndon B. , MURNANE Amy , HEINTZ Roger L.
Published: March 2005

A crude nuclear thyroid‐hormone‐receptor protein preparation from chick liver (an ammonium sulfate fractionation of high‐ionic‐strength‐solubilized chromatin proteins) binds both triiodothyronine and thyroxine with high affinity. This crude preparation has characteristics similar to preparations from a variety of animal tissues, reported by several different laboratories, and is used for the further purification of the receptor protein. For this purification an affinity chromatography medium, 4‐[N‐(3,5,3′‐triiodothyronine)‐2‐amino‐3‐hydroxypropoxy]‐butylpropoxy‐Sepharose ether, is used to take advantage of the observation that hydroxymercuribenzoic acid causes a reversible dissociation of the complex between triiodothyronine and the receptor protein. The hydroxymercuribenzoate treatment greatly increases this rate of dissociation at low temperatures compared with other methods, such as free triiodothyronine competition or an increase in ionic strength or pH. This procedure results a in purified fraction (1000–10000‐fold with respect to binding triiodothyronine), which has a molecular mass of approximately 65 kDa and which retains a high degree of the original thyroid‐hormone‐binding activity.
Monoclonal anti‐glycoprotein antibody that blocks cell adhesion in Polysphondylium pallidum
Authors: TODA Katsumi , BOZZARO Salvatore , LOTTSPEICH Friedrich , MERKL Rainer , GERISCH Günther
Published: March 2005

Polyclonal and monoclonal antibodies were prepared against a glycoprotein (gp 64) of Polysphondylium pallidum previously shown to act as a target site of adhesion‐blocking Fab prepared from antisera against whole membranes of aggregation‐competent cells. The purified glycoprotein, with a nominal Mr of 64000, could be fractionated into two subspecies, gp 64I and gp 64II with apparent Mr of 66000 and 60000, as determined in 7.5% sodium dodecyl sulfate/polyacrylamide gels.

Rabbit antibodies against purified gp 64 reacted not only with the two subspecies but also with many other membrane proteins. Almost all the cross‐reactivity could be abolished by absorption of the antibodies with extensively purified gp 64. All monoclonal antibodies obtained by screening with gp 64 showed similar cross‐reactivity. One monoclonal antibody specifically precipitating gp 64 was selected by screening with antigen that had been pretreated with anhydrous hydrogen fluoride for removal of carbohydrates.

Fab from polyclonal anti‐(gp 64) sera as well as one monoclonal Fab completely blocked cell adhesion of aggregation‐competent P. pallidum cells. A carbohydrate fraction prepared by treatment of gp 64 with proteases and hydrazine completely neutralized the adhesion‐blocking Fab. The product of hydrazinolysis contained less than 3% of the original peptide as based on the glucosamine recovered, but the specific neutralizing activity of the carbohydrate was essentially the same as that of the glycoprotein. In conclusion, monoclonal as well as polyclonal adhesion‐blocking Fab reacted with carbohydrates; gp 64 shared the relevant carbohydrate moieties with other membrane proteins.
Target size analysis and molecular properties of Ca2+ channels labelled with [3H]verapamil
Authors: GOLL Alexandra , FERRY David R. , GLOSSMANN Hartmut
Published: March 2005

[3H]Verapamil was employed to label the drug receptor sites within the Ca2+‐channel in skeletal muscle microsomes which are coupled in a negative heterotropic allosteric manner to the previously characterized 1,4‐dihydropyridine receptors. At 2°C the Kd of a high‐affinity receptor site is 45 nM and the maximum density of binding sites is 37 pmol/mg of protein. Established subcellular fractionation procedures were used to isolate transverse tubule membranes from rabbit and guinea‐pig skeletal muscle. [3H]Verapamil, d‐cis‐[3H]diltiazem as well as 1,4‐[3H]dihydropyridine receptors copurify with t‐tubule membranes. The ratio of high‐affinity verapamil: 1,4‐dihydropyridine d‐cis‐diltiazem Ca2+ channel receptor sites is 4:2:1. The verapamil drug receptors are heat‐labile and have essential sulfhydryl groups since they are inactivated by p‐chloromercuriphenylsulfonic acid and N‐ethylmaleimide. The receptors recognize the main classes of Ca2+ antagonists and agonists in a stereoselective manner. Divalent cations (Mn2+ > Ca2+ > Mg2+) are inhibitory. Target size analysis with high‐energy electrons was performed and the Mr of the verapamil drug receptor site is 110000.
Catabolite repression in yeasts is not associated with low levels of cAMP
Authors: ERASO Pilar , GANCEDO Juana M.
Published: March 2005

The relationship between levels of cAMP and catabolite repression in yeasts has been investigated. Strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe and Kluyieromyces fragilis were used. The yeasts were grown on different carbon sources to attain various degrees of repression. Galactose repressed as much as glucose, while maltose was less effective. Full derepression was achieved with ethanol.

The enzymes tested were fructose‐bisphosphatase, malate dehydrogenase, glutamate dehydrogenase (NAD dependent), cytochrome oxidase and isocitrate lyase (this last enzyme was found to be absent in Schizosaccharomyces).

The levels of cAMP were 2–3 times higher in the repressed conditions than in the derepressed ones. It is therefore concluded that in yeasts catabolite repression is not mediated by a lowering of the intracellular concentration of cAMP.
Coupling of ATP synthesis and methane formation from methanol and molecular hydrogen in Methanosarcina barkeri
Authors: BLAUT Michael , GOTTSCHALK Gerhard
Published: March 2005

The addition of methanol to a cell suspension of Methanosarcina barken resulted in an increase of the intracellular ATP concentration from 1 nmol/mg to 10 nmol/mg protein and in the formation of a proton‐motive force Δp of‐130 mV. Δp consisted of more than 90% of the membrane potential ΔΨ. These values were similar under N2 and under H2. The addition of the uncoupler tetrachlorosalicylanilide to the above system under N2 led to a drastic decrease of both, the ATP concentration and the Δp and to a stop of methanogenesis. With methanol and H2, however, methane formation continued, although the effect of the uncoupler on the ATP pool and on Δp was a under N2. The proton‐translocating ATPase inhibitor N,N‐dicyclohexylcarbodiimide caused a rapid exhaustion of the ATP pool and a discontinuation of methane synthesis, whereas Δp was unaffected. Inhibition of methane formation under these conditions could be relieved by the addition of the uncoupler tetrachlorosalicylanilide. These results demonstrate that methane formation according to the equation CH3OH + H2→ CH4+ H2O was coupled to ATP synthesis by a chemiosmotic mechanism and was under the control of ΔΨ: Methane formation only proceeded if the ΔΨ generated was used for ATP synthesis or if an uncoupler was present. Under N2, methane formation according to the equation 4CH3OH → CO2+ 3 CH4+ 2H2O was abolished by an uncoupler, because one step in the oxidation of methanol to 1CO2 apparently depended on an energized state of the membrane.
Interconversion from 3Fe into 4Fe clusters in the presence of Desulfovibrio gigas cell extracts
Authors: MOURA José J. G. , LeGALL Jean , XAVIER António V.
Published: March 2005

Desulfovibrio gigas ferredoxin II (FdII) contains a single 3Fe cluster [Huynh, B. H., Moura, J. J. G., Moura, I., Kent, T. A., LeGall, J., Xavier, A. V., and Münck, E. (1980) J. Biol. Chem. 255, 3242–3244]. In the oxidized state the protein exhibits an intense electron paramagnetic resonance (EPR) signal at g= 2.02. Upon one‐electron reduction the center becomes EPR silent. In the presence of D. gigas crude cell extracts, devoid of acidic electron carriers and supplemented with pyruvate and FdII, an EPR signal typical of reduced [4Fe–4S] centers is obtained. The appearance of this signal correlates with the beginning of stimulation of the phosphoroclastic reaction as judged by the production of H2. These results, supported by the occurrence of easy chemical conversion of the 3Fe cluster of D. gigas ferredoxin into 4Fe structures [Moura, J. J. G., Moura, I., Kent, T. A., Lipscomb, J. D., Huynh, B. H., LeGall, J., Xavier, A. V., and Munch, E. (1982) J. Biol. Chem. 257, 6259–6267], suggest that cluster conversion takes place in conditions close to the situation in vivo. This cluster interconversion is discussed in the context of some of the relevant metabolic pathways of Desulfovibrio spp.
Prolipoprotein modification and processing in Escherichia coli
Authors: GIAM Chou‐Zen , CHAI Toby , HAYASHI Shigeru , WU Henry C.
Published: March 2005

An Escherichia coli mutant (lpp‐14‐1), with an alteration of glycine to aspartic acid at the 14th amino acid residue of the prolipoprotein signal sequence, has previously been shown to contain unmodified and unprocessed prolipoprotein in its cell envelope. Both the wild‐type and the lpp‐14‐1 alleles of the lpp gene have been cloned onto a phage λ vector. Two pseudorevertant alleles of lpp‐14‐1 (14R21 and 6a) have been isolated, cloned and sequenced. Amino acid sequences, deduced from the DNA sequences of the two revertant lipoprotein alleles, and biochemical characterization of the revertant lipoproteins, show that a conversion of the aspartic acid (residue 14) to asparagine completely restores the modification and processing of the 14R21 revertant prolipoprotein, while a change of the threonine‐16 to isoleucine‐16 partially enhances the modification and processing of the 6a prolipoprotein, which retains the aspartate‐14 substitution. Secondary structure analysis of the revertant prolipoprotein signal sequenccs according to the Chou and Fasman rules revealed that the specific coil region in residues 14 and 15, and the β‐short structure in residues 16–18 of signal sequence may be important for prolipoprotein modification. These results suggest essential roles of both a unique secondary structure and hydrophobicity in residues 14‐18 of prolipoprotein signal sequence for the proper recognition by the glyceryl transferase.
The regulatory properties of isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase from Escherichia coli ML308 and the roles of these activities in the control of isocitrate dehydrogenase
Authors: NIMMO Gillian A. , NIMMO Hugh G.
Published: March 2005

Isocitrate dehydrogenase kinase can use ATP but not other nucleoside triphosphates as a phosphate donor. It responds hyperbolically to both ATP and isocitrate dehydrogenase.

The kinase is inhibited sigmoidally by low concentrations of DL‐isocitrate and hyperbolically by ADP, AMP, NADPH, phosphoenolpyruvate and several other effectors.

Isocitrate dehydrogenase phosphatase requires a nucleotide for activity; ADP and ATP are the best activators. The phosphatase responds hyperbolically to ADP or ATP, to Mg2+ ions and to phosphorylated isocitrate dehydrogenase.

The phosphatase is activated twofold to threefold by AMP, oxaloacetate, pyruvate, phosphoenolpyruvate, 2‐oxoglutarate and DL‐isocitrate. It is inhibited hyperbolically by NADPH.

The pH optima and the Km values for substrates of the kinase and the phosphatase are reported.

We propose that the role of the phosphorylation of isocitrate dehydrogenase during growth of Escherichia coli on acetate is to render this enzyme rate‐limiting in the citric acid cycle; this should cause an increase in the level of isocitrate and divert the flux of carbon through the glyoxylate bypass.

We suggest that the phosphorylation state of isocitrate dehydrogenase in intact cells is controlled by the levels of isocitrate, phosphoenolpyruvate, NADPH and the adenine nucleotides. This theory can explain many recent observations on the control of the activity of isocitrate dehydrogenase.
Characterization of the vacuolar ATPase activity of the crassulacean‐acid‐metabolism plantKalanchoë daigremontianaReceptor modulating
Authors: SMITH J. Andrew C. , URIBE Ernest G. , BALL Erika , HEUER Sabine , LÜTTGE Ulrich
Published: March 2005

Plants performing crassulacean acid metabolism show a large nocturnal accumulation of malic acid in the vacuole of the photosynthetic cells. It has been postulated that an H+‐translocating ATPase energizes the transport of malic acid across the tonoplast into the vacuole. In the present work we have characterized the ATPase activity associated with vacuoles of the crassulacean‐acid‐metabolism plantKalanchoë daigremontianaand compare it with other phosphohydrolases.

Vacuoles were isolated by polybase‐induced lysis of mesophyll‐cell protoplasts. The vacuoles had a high activity of unspecific acid phosphatase (pH optimum 5.3). The acid phosphatase was strongly inhibited by ammonium molybdate (with 50% inhibition at about 0.5 mmol m3‐), but was not completely inhibited even at much higher ammonium‐molybdate concentrations. In contrast, the vacuolar ATPase activity, assayed in the presence of 100 mmol m−3ammonium molybdate, had a pH optimum of 8.0. ATP was the preferred substrate, but GTP, ITP and ADP were hydrolyzed at appreciable rates. The mean ATPase activity at pH 8.0 was 14.5 mmol h−1(103vacuoles)−1an average 13% of which was attributable to residual acid‐phosphatase activity. Inorganic‐pyrophosphatase activity could not be demonstrated unambiguously.

The vacuolar ATPase activity was M2+‐dependent, had an apparentKmfor MgATP2‐of 0.31 mol m−3, and was 32% stimulated by 50 mol m3‐KCl. Of the inhibitors tested, oligomycin slightly inhibited the vacuolar ATPase activity and diethylstilbestrol and NO−3were both markedly inhibitory. Dicyclohexylcarbodiimide and tributyltin were also strongly inhibitory. Tributyltin caused a 50% inhibition at about 0.3 mmol m−3. This is taken as evidence that the vacuolar ATPase might function as an H+‐translocating ATPase.

It is shown that the measured activity of the vacuolar ATPase would be of the right order to account for the observed rates of nocturnal malic‐acid accumulation inK. daigremontiana.
Studies on the transmembrane disposition of the neural cell adhesion molecule N‐CAM
Authors: GENNARINI Gianfranco , ROUGON Geneviève , DEAGOSTINI‐BAZIN Hermine , HIRN Michel , GORIDIS Christo
Published: March 2005

The N‐CAMs are a group of surface glycoproteins involved in adhesive interactions of neurones. Related molecules of the mouse nervous system, identified in our laboratory, have been called BSP‐2 and shown to act as ligands in adhesion of neuroblastoma cells. Results presented in this report show that they are immunochemically identical with N‐CAM. A monoclonal anti‐(N‐CAM) antibody, that recognized a determinant accessible only after permeabilization of intact cells, was used to define the mode of association of the N‐CAMs with the plasma membrane. This antibody bound a 35000‐Mr fragment in lysates of trypsin‐treated neuroblastoma cells. It is concluded that the antibody reacts with a transmembrane or cytoplasmic domain of the molecules.

The same antibody recognized the Mr‐180000 and Mr‐140000 proteins but not the Mr‐120000 chain, which copurify from adult mouse brain. The latter polypeptide was detected in the cytosol and could be partially released from brain membranes by osmotic shock. Part or all of the Mr‐120000 protein may thus lack a transmembrane segment.

Our conclusion that the N‐CAM forms of higher Mr are transmembrane proteins was further corroborated by our finding that they contain phosphoserine residues, which can be labeled with (32P)phosphate in intact neuroblastoma cells.
Content and localization of FMN, Fe‐S clusters and nickel in the NAD‐linked hydrogenase of Nocardia opaca 1b
Authors: SCHNEIDER Klaus , CAMMACK Richard , SCHLEGEL Hans G.
Published: March 2005

By preparative polyacrylamide gel electrophoresis at pH 8.5, and in the absence of nickel ions, two types of subunit dimers of the NAD‐linked hydrogenase from Nocardia opaca 1b were separated and isolated, and their properties were compared with each other as well as with the properties of the native enzyme.

The intact hydrogenase contained 14.3 ± 0.4 labile sulphur, 13.6 ± 1.1 iron and 3.8 ± 0.1 nickel atoms and approximately 1 FMN molecule per enzyme molecule. The oxidized hydrogenase showed an absorption spectrum with maxima (shoulders) at 380 nm and 420 nm and an electron spin resonance (ESR) spectrum with a signal at g= 2.01. The midpoint redox potential of the Fe‐S cluster giving rise to this signal was + 25 m V. In the reduced state, hydrogenase gave characteristic low‐temperature (10 – 20 K) and high‐temperature (>40 K) ESR spectra which were interpreted as due to [4Fe–4S] and [2Fe–2S] clusters, respectively. The midpoint redox potentials of these clusters were determined to be ‐420 mV and ‐285 mV, respectively.

The large hydrogenase dimer, consisting of subunits with relative molecular masses Mr of 64000 and 31000. contained 9.9 ± 0.4 S2− and 9.3 ± 0.5 iron atoms per protein molecule. This dimer contained the FMN molecule, but no nickel. The absorption and ESR spectra of the large dimer were qualitatively similar to the spectra of the whole enzyme. This dimer did not show any hydrogenase activity, but reduced several electron acceptors with NADH as electron donor (diaphorase activity).

The small hydrogenase dimer, consisting of subunits with Mr of 56000 and 27000, was demonstrated to have substantially different properties. For iron and labile sulphur average values of 3.9 and 4.3 atoms/dimer molecule have been determined, respectively. The dimer contained, in addition, about 2 atoms of nickel and was free of flavins. In the oxidized state this dimer showed an absorption spectrum with a broad band in the 400‐nm region and a characteristic ESR signal at g= 2.01. The reduced form of the dimer was ESR‐silent. The small dimer alone was diaphorase‐inactive and did not reduce NAD with H2, but it displayed high H2‐uptake activities with viologen dyes, methylene blue and FMN, and H2‐evolving activity with reduced methyl viologen. Hydrogen‐dependent NAD reduction was fully restored by recombining both subunit dimers, although the reconstituted enzyme differed from the original in its activity towards artificial acceptors and the, ESR spectrum in the oxidized state.
Isolement et étude structurale de glucanes de Phytophthora parasitica
Authors: FABRE Isabelle , BRUNETEAU Maud , RICCI Pierre , MICHEL et Georges
Published: March 2005

Several polysaccharides have been isolated from the cell walls of Phytophthora parasitica, a phytopathogenic fungus of carnation. The crude polysaccharides were fractionated by successive chromatographies on DEAE‐cellulose, Sephadex G‐25, concanavalin‐A‐Sepharose and Sephadex G‐200 columns. The neutral polysaccharides consist of a mixture of β(1 →3,1 →6)‐D‐glucans whose relative molecular masses varied from 9000 to about 200000. All these polysaccharides have a main chain of β(1 →3)‐linked D‐glucose residues. They differ by the presence of 1 →6 branched chains consisting of D‐glucose and D‐Glc‐(1 →3)‐D‐Glc, for the lowest molecular mass polysaccharides or D‐Glc‐(1 →3)‐D‐Glc‐(1 →3)‐D‐Glc for the highest molecular mass polysaccharides.
A potassium‐39 NMR study of potassium binding to double‐helical DNA
Authors: BRAUNLIN William H. , NORDENSKIÖLD Lars
Published: March 2005

The binding of K+ to double‐helical DNA has been examined by 39K NMR. The results obtained are in substantial agreement with previous measurements of 23Na+ binding to DNA. Thus, in both cases the fractional neutralization of DNA phosphate charge appears independent of the total univalent cation concentration. Further, the dependences of the 23Na+ and 39K + linewidths on Mg2+ and on temperature are strikingly similar. The implications of these results are discussed in terms of current theories of counterion binding to cylindrical polyelectrolytes.
Two different aldolase A mRNA species in rat tissues
Authors: TSUTSUMI Reiko , TSUTSUMI Ken‐ichi , NUMAZAKI Masayoshi , ISHIKAWA Kiichi
Published: March 2005

Double‐stranded DNA was synthesized with reverse transcriptase from size‐fractionated poly(A)‐containing RNA from rat ascites hepatoma cells. The cDNA was introduced into Escherichia coli HB101 using pBR 322 DNA as a cloning vector. Several plasmids containing aldolase A cDNA were identified by colony hybridization with 32P‐labeled cDNA prepared from immunologically purified aldolase A mRNA. The partial amino acid sequence of the cDNA sequence was determined, and found to coincide with that of rabbit aldolase A.

Using aldolase A cDNA as a hybridization probe, the aldolase A mRNA concentrations in various rat tissues were analysed, and two aldolase A mRNA species differing in nucleotide length were found; the smaller mRNA (about 1550 nucleotides) in muscle, and the larger one (about 1650 nucleotides) in brain and hepatoma cells.
Spontaneous modification of the oxoglutarate translocator in vivo
Authors: DUYCKAERTS Claire , SLUSE‐GOFFART Claudine M. , SLUSE Francis E. , GOSSELIN‐REY Cécile , LIÉBECQ Claude
Published: March 2005

In studying the oxoglutarate translocator of rat‐heart mitochondria over many years, we have observed an unexpected decrease in its efficiency. It has been divided by 2.48 ± 0.07 (S.E.M.) for the exchange of external oxoglutarate for internal malate at 2 °C when the internal‐malate concentration is 4 mM and is accompanied by an increase in its concentration (multiplied by 1.61 ± 0.02, S.E.M.).

The affinity of the external sites of the translocator for the external oxoglutarate is unchanged as well as the binding and kinetic cooperativities of the external oxoglutarate. This shows that the external side of the translocator has not been modified and suggests that its central part has not been modified either. The apparent Michaelis constant of the internal malate is increased (multiplied by 1.74 ± 0.23, S.E.M.) suggesting that the translocator has been modified on its matricial side.

Some control experiments show that a change in the diet of the rats, despite its effect on the fatty‐acid content of the mitoplasts, is probably not responsible for the observed modification. As it is nevertheless very likely that changes of the oxoglutarate translocator have occurred in vivo, it is proposed that the observed modification has a genetic origin.

The existence of two antagonist changes which are not directly related suggests that one of them is a response of the organism against the other; thus the oxoglutarate translocator may play a regulatory rôle in certain physiological conditions.
Complex structure of human bronchial mucus glycoprotein
Authors: SLAYTER Henry S. , LAMBLIN Geneviève , TREUT André LE , GALABERT Claude , HOUDRET Nicole , DEGAND Pierre , ROUSSEL Philippe
Published: March 2005

Human bronchial mucus glycoproteins or mucins were isolated from the sputum of two patients by a method avoiding reducing agents and involving water extraction and gel filtration on Sepharose CL‐2B in 6 M guanidinium chloride. The chemical analysis indicated approximately 25–40% lipid. The amino acid and carbohydrate analysis differ quantitatively from that of mucins purified after prior reduction of mucus. These fractions also have a higher proportion of aspartic and glutamic acids than that of the mucins from reduced sputum. These mucins are still contaminated by small amounts of peptides but do not seem to contain disulfide‐attached cross‐linking protein. Human bronchial mucins have a strong tendency to form aggregates except in 6 M guanidinium chloride.

Electron microscopy performed with various procedures indicates the presence of both micelles and flexible threads measuring 200–1000nm. Delipidation removes most of the micellar forms. Thereafter mucins appear mainly as polydisperse flexible extended threads and also as aggregates.