bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibitors of metabolic reactions
Authors: LILL Ute , KOLLMANN‐KOCH Angelika , BIBINGER Angelika , EGGERER Hermann
Published: March 2005

Substrate and intermediate analogue inhibitors of enzymes were prepared in which the thioester oxygen of acyl‐CoA substrates is replaced by hydrogen with formation of CoA‐thioethers.

Experiments performed with ATP citrate lyase and S‐(3,4‐dicarboxy‐3‐hydroxybutyl)‐CoA are consistent with citryl‐CoA but not with citryl‐enzyme being the direct precursor of the products acetyl‐CoA and oxaloacetate. Consistent with these results, a previously described isotopic exchange between acetyl‐CoA and [3H]CoASH, indicating the formation of an acetyl‐enzyme in the reaction pathway, could not be confirmed.

Substrate analogue CoA‐thioethers of malate synthase are inhibitors endowed with the affinity of the substrates. Acetyl carboxylase and fatty acid synthetase are not inhibited by the substrate analogue S‐ethyl‐CoA; S‐carboxyethyl‐CoA, which could substitute for malonyl‐CoA, is likewise not inhibitory. An explanation is proposed.

Previously suggested roles of S‐carboxymethyl‐CoA, an acetyl‐CoA‐related inhibitor of citrate synthase, are discussed in the light of new experimental data. S‐Acetyl, S‐propionyl and S‐carboxymethyl derivatives of 1,N6‐etheno‐CoA loose the high affinity of their CoA‐counterparts to citrate synthase, probably because the ethylene group prevents proper binding to the enzyme.
Rat liver dimethylglycine dehydrogenase
Authors: LANG Helmut , POLSTER Martin , BRANDSCH Roderich
Published: March 2005

Dimethylglycine dehydrogenase (Me2GlyDH), an enzyme of choline catabolism specifically expressed in the mammalian liver, was analyzed in rat hepatocytes in culture. This mitochondrial enzyme carries the FAD cofactor covalently attached to the polypeptide chain by its riboflavin 8α position to Nπ of histidine [Cook, R., Misono, K.S. & Wagner, C. (1980) J. Biol. Chem. 259, 12475–12480]. Subcellular fractionation of [14C]riboflavin‐labelled hepatocytes and immunoprecipitation with Me2GlyDH‐specific antiserum identified a [14C]riboflavin‐labelled polypeptide of the size of mature Me2GlyDH only in the mitochondrial fraction. Immunoprecipitation of extracts from [35S]Met‐labelled hepatocytes revealed a putative precursor protein to the mature Me2GlyDH in the cytoplasmic fraction. These Me2GlyDH polypeptides were not expressed in cells of the rat hepatoma cell line FAO. A Me2GlyDH cDNA clone of apparent full length was isolated from a rat liver cDNA bank constructed in the plasmid vector pcD‐X [Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T. & Arai, K. (1987) Methods Enzymol. 154, 3–28]. The nucleotide sequence of the cDNA contains an open reading frame encoding a protein of 96059 Da. This molecular mass agrees well with the migration on SDS/PAGE of the assumed Me2GlyDH precursor immunoprecipitated from the cytoplasm of [35S]Met‐labelled cells. Proteolytic cleavage at the putative mitochondrial processing protease‐recognition site Arg(‐2)‐Ala(‐1)‐Glu(+1) would lead to the formation of a protein of 91391 Da, which is in good agreement with the estimated 90 kDa of mature Me2GlyDH [Wittwer, A. J. & Wagner, C. (1981) J. Biol. Chem. 256, 4102–4108], and a 43‐amino‐acid leader peptide. The N‐terminus of Me2GlyDH contains a conserved amino acid sequence which forms the dinucleotide‐binding site in many enzymes with noncovalently bound FAD. Close to the modified histidine there is an amino acid sequence resembling a sequence conserved in thymidylate synthases and shown in these enzymes to be involved in the binding of the pteroyl polyglutamate cofactor.
Structural heterogeneity of membrane receptors and GTP‐binding proteins and its functional consequences for signal transduction
Authors: BOEGE Fritz , NEUMANN Eberhard , HELMREICH Ernst J. M.
Published: March 2005

Recent information obtained, mainly by recombinant cDNA technology, on structural heterogeneity of hormone and transmitter receptors, of GTP‐binding proteins (G‐proteins) and, especially, of G‐protein‐linked receptors is reviewed and the implications of structural heterogeneity for diversity of hormone and transmitter actions is discussed. For the future, three‐dimensional structural analysis of membrane proteins participating in signal transmission and transduction pathways is needed in order to understand the molecular basis of allosteric regulatory mechanisms governing the interactions between these proteins including hysteretic properties and cell‐cybernetic aspects.
Constitutive expression of heat‐shock protein 70 in mammalian cells confers thermoresistance
Authors: ANGELIDIS Charalampos E. , LAZARIDIS Ioannis , PAGOULATOS Gerassimos N.
Published: March 2005

A 70‐kDa heat‐shock‐protein (hsp70) expression vector which contains the human hsp70 gene linked to the human β‐actin promoter, was constructed and used to transfect CV1 monkey cells. Stably transfected CV1 clones were isolated which constitutively synthesized increased amounts of hsp70 at normal temperature. It is shown that these clones are resistant to elevated temperature. This finding indicates that hsp70 is involved in the protection of the cells against a lethal heat treatment and maybe responsible for the phenomenon of thermotolerance.
Ligand binding at membrane mimetic interfaces
Authors: DESFOSSES Bernard , CITTANOVA Nicole , URBACH Wladimir , WAKS Marcel
Published: March 2005

The behaviour of human serum albumin in the presence of three chemically distinct ligands: oxyphenyl‐butazone, dansylsarcosine and hemin, has been compared in buffer and in reverse micelles of isooctane, water, and either sodium bis(2‐ethylhexyl)sulfosuccinate or hexadecyl trimethylammonium bromide, systems selected to mimic the membrane‐water interface. Upon micellar incorporation, the dansylsarcosine‐albumin complex dissociated, as evidenced by fluorescence emission spectroscopy (red shift from 485 nm to 570 nm) and by fluorescence polarization measurements. In contrast, the hemin‐albumin complex remained stable in reverse micelles, as judged from the Soret absorption band at 408 nm and the molar absorption coefficient of 8.4 × 104 M−1 cm−1. The oxyphenylbutazone to albumin binding curves reveal that while the association constant remained unchanged (Kaη 1.0 × 105 M−1), only a fraction of the albumin molecules present reacted with the ligand. The results were unaffected by the nature and the concentration of the surfactant.

These findings can be interpreted in the light of conformational changes induced in human serum albumin by the large micellar inner surface area. The blue shift of the fluorescence emission maximum from 344 nm in buffer to 327 nm in sodium bis(2‐ethylhexyl)sulfosuccinate micelles and the lesser reactivity/accessibility of the fluorophore to oxidation by N‐bromosuccinimide, indicate perturbations of the sole tryptophan‐214 microenvironment. However, the distance between the indole residue and tyrosine‐411 does not seem substantially modified by the 15% decrease affecting the α helices of the albumin molecule. It is proposed that the results reported herein reflect the interactions of albumin with a membrane‐like interface which generates two protein subpopulations differing in their membrane‐surface and ligand affinities. Overall and local conformational changes, originating from this surface‐induced effect, may thus constitute a ligand‐release facilitating mechanism acting at cellular membrane levels.
Purple acid phosphatase from bovine spleen
Authors: DIETRICH Markus , MÜNSTERMANN Dieter , SUERBAUM Hildegard , WITZEL Herbert
Published: March 2005

Oxidation of the reduced (pink) phosphate‐free bovine spleen acid phosphatase with 1.5 mol H2O2 or sodium peroxodisulfate/mol, in the presence of Mes or Bistris pH 5, leads to a species with an absorption maximum at 558 nm. Addition of acetate or oxidation in the presence of acetate buffer engenders a species with a maximum at 550 nm. Addition of phosphate to both species shifts the maximum immediately to 540 nm; this is the species also found after preparation from the spleen. The assumption that these species represent strongly bidentate‐binding hydroxo, acetato and phosphato complexes of the Fe(III)‐Fe(III) system is supported by replacement reactions with other ligating oxoanions followed by their typical spectral shifts. These oxoanion complexes cannot be dissociated by gel filtration; this is possible only after reduction to the Fe(II)‐Fe(III) system. The oxidized species without EPR signals below g values of 2 still reveals 5% activity which cannot be reduced to zero even in the presence of higher concentrations of peroxodisulfate. The pH optimum of the reaction with α‐naphthyl phosphate shifts from 5.9 to 5.3 in the oxidized species.

The apparent pK values around 4.5 as derived from the pH dependence of activity, of the EPR spectra, and the spectral shifts of the phosphate‐saturated reduced and oxidized species are assigned to an aquo/hydroxo equilibrium at the Fe(III) or an equilibrium, where the phosphato ligand is replaced by a hydroxo ligand. A reaction mechanism is proposed in which a hydroxo ligand at the chromophoric Fe(III) attacks the phosphoric acid ester group only when that is monoprotonated and pre‐oriented by electrostatic interaction with the nonchromophoric metal ion. Binding and inhibition studies with the oxoanions indicate that they compete with the catalytically active hydroxo group of the reduced and oxidized enzyme with nearly the same inhibition constants. Catalysis is not affected by the oxoanions which replace the additional μ‐hydroxo ligand in the 558‐nm‐absorbing Fe(III)‐Fe(III) species. In contrast to hemerythrin and ribonucleotide reductase, a binuclear iron center is proposed for the purple acid phosphatase, which is bridged by a carboxylato and two aquo/hydroxo groups, but without a μ‐oxo bridge.
Structural, spectroscopic and catalytic activity studies on glutathione reductase reconstituted with FAD analogues
Authors: ERMLER Ulrich , GHISLA Sandro , MASSEY Vincent , SCHULZ Georg E.
Published: March 2005

FAD‐modified human glutathione reductases were reconstituted from apoenzyme using the FAD analogues 6‐SH‐FAD, 6‐SCN‐FAD, 6‐OH‐FAD, 6‐NH2‐FAD and 8‐OH‐FAD. The catalytic activities of the modified enzymes were substantially lower than for the native enzyme. All five species could be crystallized, but only those containing 6‐SH‐FAD, 6‐OH‐FAD and 6‐NH2‐FAD yielded crystals that could be analyzed. X‐ray analyses and structural refinements were performed at 0.27 nm and 0.30 nm resolution resulting in R factors around 13.5%. The crystal structures showed the additional non‐hydrogen atoms and small conformational changes of the polypeptide that were obviously induced by the substituents of the FAD analogues. The observed changes together with spectroscopic and activity data permit some conclusions about the chemical nature of the substituents.
ATP‐citrate lyase from rat liver
Authors: WELLS Timothy N. C.
Published: March 2005

The mechanism of ATP‐citrate lyase has been proposed to involve a citryl‐enzyme intermediate. When the enzyme is incubated with its substrates ATP and [14C]citrate, but in the absence of the final acceptor, two distinct types of citrate‐containing complex can be isolated. At early time points, a highly unstable complex can be isolated by gel filtration which has a half‐life of 36 s at 25°C. This complex reacts rapidly with CoA, but cannot be acid‐precipitated; behaviour consistent with its identification as enzyme‐citryl phosphate. However, ATP‐citrate lyase is also capable of undergoing a slow time‐dependent covalent incorporation of radiolabel from [14C]citrate. This modification is acid‐stable, non‐specific, and cannot be reversed by the addition of CoA. When cytochrome c is included in the reaction mixture as a heterologous acceptor, it is also citrylated. These reactions require that when ATP‐citrate lyase is incubated with all its substrates except for CoA, a freely diffusible citrylating species is generated within the active site. This evidence suggests that there is no requirement for the mechanism of ATP‐citrate lyase to proceed via a covalent citryl‐enzyme intermediate. By analogy with succinyl‐CoA synthetase, an enzyme which has a high degree of sequence similarity with ATP‐citrate lyase, a simple mechanism is proposed for the enzyme in which citryl‐CoA is produced by direct nucleophilic attack on citryl phosphate.
A family of bombinin‐related peptides from the skin of Bombina variegata
Authors: SIMMACO Maurizio , BARRA Donatella , CHIARINI Fernanda , NOVIELLO Lia , MELCHIORRI Pietro , KREIL Günther , RICHTER Klaus
Published: March 2005

A peptide fraction was isolated from the skin of Bombina variegata that showed antimicrobial activity. This fraction contained several molecular species, all of them consisting of 27 amino acid residues, with a constant C‐terminal region (from residues 14–27), including an amidated carboxyl end and a variable N‐terminal segment. These peptides are related but not identical to bombinin [Csordas, A. & Michl, H. (1970) Monatsh. Chem. 101, 182–189].

By using synthetic oligonucleotides corresponding to the C‐terminal region of the peptides, a cDNA library from the skin of B. variegata was screened and several positive clones coding for the corresponding peptide precursors were isolated and sequenced. Each clone contained the genetic information for a different bombinin‐like peptide. The antimicrobial activity towards different bacterial species of a synthetic peptide corresponding to one of the variants deduced from cDNA sequences was tested. This peptide was found to be mainly active against different isolates of Staphylococci and Escherichia coli.
Transient kinetics of flavin‐photosensitized oxidation of reduced redox proteins
Authors: NAVARRO José A. , De la ROSA Miguel A. , TOLLIN Gordon
Published: March 2005

We have used laser flash photolysis to investigate the kinetics of oxidation of reduced plastocyanins obtained from spinach and the green alga Monoraphidium braunii by the triplet states of lumiflavin, riboflavin and FMN. We have compared the results of these experiments with the kinetics of reduction of the oxidized forms of these proteins by the corresponding flavin semiquinones, as well as with the kinetics of flavin oxidation and reduction of cytochrome c552 (Class I c‐type cytochrome, generic name c553) from Monoraphidium. In all cases, the rate constants for oxidation were one or two orders of magnitude larger than for reduction, consistent with the greater thermodynamic driving force for the oxidation reaction. Similar steric and electrostatic effects were observed for both reactions with all proteins, suggesting that the same (or closely adjacent) sites were being utilized for electron removal and entry. The two algal proteins were quite similar to one another in their redox properties, consistent with their physiological role of being able to substitute for one another in photosynthetic electron transport. In contrast, the algal plastocyanin was more reactive than the spinach protein in both oxidation and reduction, suggesting differences in their steric properties at the site of electron transfer.
Modification of gelsolin with 4‐fluoro‐7‐nitrobenz‐2‐oxa‐1,3‐diazole
Authors: DOI Yukio , HASHIMOTO Tomoko , YAMAGUCHI Hisako , VERTUT‐DOÏ Aline
Published: March 2005

Pig plasma gelsolin was modified with the fluorescent reagent 4‐fluoro‐7‐nitrobenz‐2‐oxa‐1,3‐diazole (NBD‐F) for lysyl residues. The relationship between the gelsolin activity and the degree of NBD labeling suggested that a single lysyl residue, which reacted five times slower than the other reactive lysyl residues, was essential for the activity. Taking advantage of the slow reactivity of the essential residue, active NBD‐gelsolin was prepared. Limited cleavage of NBD‐gelsolin by chymotrypsin indicated that the fluorescent reagents were randomly incorporated into all fragments observed. When NBD‐gelsolin formed a gelsolin/actin (1:2) complex in the presence of micromolar Ca2+, the fluorescence spectra of NBD‐gelsolin were red‐shifted by 5 nm and the intensity decreased by 30%. However, on binding to phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2), the fluorescence spectra were blue‐shifted by 5 nm with a concomitant increase in intensity by 20%. The addition of PtdIns(4,5)P2 to the NBD‐gelsolin/actin (1:2) complex restored the fluorescence spectra to that obtained in the presence of PtdIns(4,5)P2 alone. These results indicated that NBD‐gelsolin, selectively labeled on lysyl residues not essential for activity, can be a useful probe to monitor the binding of PtdIns(4,5)P2 and actin.
Two‐dimensional proton‐NMR studies on a hybrid peptide between cecropin A and melittin
Authors: SIPOS David , CHANDRASEKHAR Kasibhatla , ARVIDSSON Klas , ENGSTRÖM Åke , EHRENBERG Anders
Published: March 2005

A hybrid peptide of cecropin A and melittin was investigated by two‐dimensional 1H‐NMR at pH 5.8 in aqueous solution with 30% (by vol.) hexafluoroisopropanol. The peptide contains 26 amino acids, is a combination of the first 13 residues of each of the two parent peptides, CA(1–13)M(1–13) ≡ CAM(1–26) and has an amidated C terminal. This peptide was recently synthesized [Boman, H. G., Wade, D., Boman, I. A., Wåhlin, B. & Merrifield, R. B. (1989) FEBS Lett. 259, 103–106] and shown to have strong antibacterial activity but to be harmless towards erythrocytes. All resonances of the main chain and side chain β‐protons are assigned except for those of the N‐terminal lysine. Several medium range NOE connectivities were observed showing two separated α‐helices, involving residues 4–12 and 16–26. The JNHα‐coupling constants in these sections support the conclusion. From the exchange rates of the NH protons it is concluded that the α‐helix of residues 16–26 is much more stable than the other helix. The circular dichroism data indicates about 30% less α‐helix character than the NMR data. A reduced contribution to the ellipticity from the unstable helix is suggested. The chemical‐shift differences between the two parts of the hybrid and the respective parent peptides are larger for the cecropin part than for the melittin part. For the latter, residues 17–26 of the hybrid are proposed to have a secondary structure very similar to that of residues 4–13 of melittin. The total results suggest that a hydrophobic α‐helix capable of spanning half a lipid bilayer combined with an amphiphilic α‐helix of two to three turns with a flexible hinge section in between are features of importance for the lytic antibacterial activity.
Primary structure and conformational analysis of peptide methionine—tyrosine, a peptide related to neuropeptide Y and peptide YY isolated from lamprey intestine
Authors: CONLON J. Michael , BJØRNHOLM Berith , JØRGENSEN Flemming S. , YOUSON John H. , SCHWARTZ Thue W.
Published: March 2005

A peptide belonging to the pancreatic‐polypeptide‐fold family of regulatory peptides has been isolated from the intestine of an Agnathan, the sea lamprey (Petromyzon marinus). The primary structure of the peptide (termed peptide methionine – tyrosine) was established as Met‐Pro‐Pro‐Lys‐Pro‐Asp‐Asn‐Pro‐Ser‐Pro10‐Asp‐Ala‐Ser‐Pro‐Glu‐Glu‐Leu‐Ser‐Lys‐Tyr20‐Met‐Leu‐Ala‐Val‐Arg‐Asn‐Tyr‐Ile‐Asn‐Leu30‐Ile‐Thr‐Arg‐Gln‐Arg‐Tyr CONH2. This sequence shows stronger structural similarity with pig neuropeptide Y (64%), particularly in the COOH‐terminal region, than with pig peptide tyrosine – tyrosine (61%) or with pig pancreatic polypeptide (42%). Molecular modelling and dynamic simulation, based upon sequence similarity with turkey pancreatic polypeptide, indicates that the conformations of the polyproline‐helix‐like region (residues 1–8) and the α‐helical region (residues 15–30) in turkey pancreatic polypeptide are conserved in peptide methionine – tyrosine, and that non‐bonded interactions between these domains have preserved the overall polypeptide fold in the molecule. The substitution of the otherwise totally conserved Gly9 residue by serine in lamprey peptide methionine – tyrosine, however, results in a preferred structure in which the conformation of the β‐turn between the two helical domains (residues 9–14) is appreciably different.
Effect of the cobalt‐N coordination on the cobamide recognition by the human vitamin B12 binding proteins intrinsic factor, transcobalamin and haptocorrin
Authors: STUPPERICH Erhard , NEXØ Ebba
Published: March 2005

The binding of several corrinoids to the binding site of human intrinsic factor, transcobalamin or haptocorrin was investigated. p‐Cresolyl cobamide and 2‐amino‐vitamin B12 are complete corrinoids, whose nucleotide at the lower face of the corrin ring is not coordinated to the cobalt. These corrinoids were ≥ 103 times less efficiently recognized by intrinsic factor or transcobalamin than vitamin B12, which contains a Co‐coordinated nucleotide. Pseudovitamin B12, with a weak Co‐N coordination bond, revealed only moderate affinity to intrinsic factor. From these findings it is concluded that the cobamide binding to intrinsic factor and transcobalamin is strongly affected by the Co‐N coordination bonds of their lower cobalt nucleotide ligands. We suggest that the Co‐N coordination bond positions the nucleotide at a critical distance to the corrin ring, which is recognized by the binding proteins.

Human haptocorrin, however, disclosed to distinctive selectivity regarding the different corrinoid structures. The protein bound all corrinoids with similar efficiency, independent of the strength of their Co‐N coordinations, or the structures of their lower Coα ligands. Hence, the corrin ring, rather than a structural feature induced by the Co‐N coordination, has to be considered responsible for the corrinoid binding to haptocorrin.
Membrane‐bound cytochromes in Chloroflexus aurantiacus studied by EPR
Authors: van VLIET Pieter , ZANNONI Davide , NITSCHKE Wolfgang , RUTHERFORD A. William
Published: March 2005

The heme components of chlorosome‐depleted membranes of the green‐gliding bacterium Chloroflexus aurantiacus were studied by EPR spectroscopy. The four major species, which are present in approximately equimolar quantities, are characterized by the following gz values, redox midpoint potentials and orientations of heme planes with respect to the plane of the membrane: gz= 3.40, Em=+ 280 mV, 30°; gz= 3.33, Em= 0 mV, 45°gz= 3.03, Em=+ 95 mV, 40–50° and gz= 2.95, Em=+ 150 mV, 90°. These four hemes were attributed to cytochrome c554, the membrane‐bound immediate electron donor to the photosynthetic reaction centre in Chloroflexus. All hemes except that with the highest potential were able to undergo photooxidation at 4 K. The photooxidation of the lowest potential heme was stable, whereas that of the + 95 mV and the + 150 mV hemes reversed on increasing the temperature to 100 K in darkness, due to charge recombination. The ability to photooxidize these hemes at 4 K was lost upon aging of samples. The results demonstrate that a reaction‐centre‐associated tetraheme cytochrome subunit, analogous to that of purple bacteria, is also present in C. aurantiacus.
Determination of amino acid sequences involved in the processing of the ARG5/ARG6 precursor in Saccharomyces cerevisiae
Authors: BOONCHIRD Chuenchit , MESSENGUY Francine , DUBOIS Evelyne
Published: March 2005

In Saccharomyces cerevisiae, the ARG5/ARG6 locus encodes both acetylglutamate kinase and acetylglutamyl‐phosphate reductase, localized in the mitochondria. Genetic analysis, determination of the nucleotide sequence of the ARG5/ARG6 gene and identification of the transcript indicate that it encodes a single translation product containing two enzyme activities. However, analysis of cellular extracts revealed that the activities are completely separable. In this work, we define different domains in the ARG5/ARG6 polypeptide; a mitochondrial target sequence and the acetylglutamate‐kinase and acetylglutamyl‐phosphate‐reductase domains. We show that deletions in the N‐terminal end of the protein and point mutations in the junction region between the acetylglutamate‐kinase and acetylglutamyl‐phosphate‐reductase domains lead to the accumulation of a large precursor. Our data support the idea that import of the ARG5/ARG6 precursor into the mitochondria is required for its processing into two mature enzymes.
Autoprocessing of the HIV‐1 protease using purified wild‐type and mutated fusion proteins expressed at high levels in Escherichia coli
Authors: LOUIS John M. , McDONALD Richard A. , NASHED Nashaat T. , WONDRAK Ewald M. , JERINA Donald M. , OROSZLAN Stephen , MORA Peter T.
Published: March 2005

Various constructs of the human immunodeficiency virus, type 1 (HIV‐1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose‐binding protein of the malE gene of Escherichia coli. The full‐length fusion proteins did not exhibit self‐processing in E. coli, thereby allowing rapid purification by affinity chromatography on cross‐linked amylose columns. Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site‐specific autoprocessing to release the 11‐kDa protease. Rapid purification involving two column steps gave an HIV‐1 protease preparation of > 95% purity (specific activity ∼ 8500 pmol · min−1·μg protease−1) with an overall yield of about 1 mg/l culture. Incubation of an inactive mutant protease fusion protein with the purified wild‐type protease resulted in specific trans cleavage and release of the mutant protease. Analysis of products of the HIV‐1 fusion proteins containing mutations at either the N‐or the C‐terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non‐mutated site. Such mutated full‐length and truncated protease fusion proteins possess very low levels of proteolytic activity (∼ 5 pmol · min−1·μg protein−1).
Isolation of chick tenascin variants and fragments
Authors: CHIQUET Matthias , VRUČINIĆ‐FILIPI Nevenka , SCHENK Susanne , BECK Konrad , CHIQUET‐EHRISMANN Ruth
Published: March 2005

The extracellular‐matrix glycoprotein, tenascin, consists of disulfide‐linked subunits of 190, 200 and 230 kDa (the three splicing variants reported in chicken) and usually exists as a six‐armed structure under the electron microscope. We used monoclonal antibodies to isolate and characterize different splicing variants and proteolytic fragments obtained from the native protein. Purified monomeric tenascin has a native molecular mass of 216 kDa and is structured as single arms. Tenascin fragments obtained by pepsin digestion bind to monoclonal antibody (mAb) TnM1 which is directed against epidermal‐growth‐factor‐like repeats in the N‐terminal half of all subunits. These fragments represent the thin proximal part of the tenascin arms and they are still partially linked to dimers and trimers via disulfide bridges. Using mAb Tn68, that reacts with a fibronectin‐type‐III repeat towards the C‐terminus, a tenascin fragment, generated by treatment with pronase, can be isolated. Ultrastructurally, this fragment looks like the thicker distal part of the tenascin arms. Only the 230‐kDa variant of tenascin gives rise to this distal fragment after cleavage within the alternatively spliced fibronectin‐type‐III repeats. Native tenascin and all fragments containing the distal part of its arms bind to heparin‐agarose, whereas the proximal fragments do not. Oligomeric and monomeric tenascin inhibit fibronectin‐mediated fibroblast spreading with comparable efficiency when added to the culture medium, while the proximal fragment has no effect. The distal fragment as well as reduced and alkylated tenascin are active in this assay, but only at higher molar concentrations when compared to the native protein.
Prepeptide sequence of cinnamycin (Ro 09–0198): the first structural gene of a duramycin‐type lantibiotic
Authors: KALETTA Cortina , ENTIAN Karl‐Dieter , JUNG Günther
Published: March 2005

The tetracyclic polypeptide antibiotic cinnamycin (Ro 90–0198) belongs to the duramycin‐type lantibiotics and contains the unusual amino acids threo‐3‐methyl‐lanthionine, meso‐lanthionine, lysinoalanine and 3‐hydroxy‐aspartic acid. Its structural gene, referred to as cinA, has been identified on isolated chromosomal DNA of the Ro 09–0198‐producing strain Streptoverticillium griseoverticillatum via a 39‐residue oligonucleotide probe derived from fragment 7–19 of the hypothetical prolantibiotic sequence CRQSCSFGPFTFVCDGNTK. This propeptide part was then found within an open reading frame of 77 amino acids. In contrast to the nisin‐type prelantibiotics, this first duramycin‐type prelantibiotic has an unusually long leader sequence of 58 amino acids. It also differs in the processing site and the direction of the formation of the threo‐3‐methyl‐lanthionine bridges is from N‐terminal cysteine to C‐terminal dehydrated threonine residues, whereas the meso‐lanthionine and lysinoalanine bridges are formed by addition reactions from C‐terminal cysteine or lysine to N‐terminal dehyrated serine residues.
The promotion of self‐association of horse‐heart cytochrome c by hexametaphosphate anions
Authors: WHITFORD David , CONCAR David W. , WILLIAMS Robert J. P.
Published: March 2005

In the presence of the highly charged hexametaphosphate anion, horse heart cytochrome c aggregates to form stable protein complexes. The formation of protein aggregates has been detected by high‐resolution 1H‐NMR spectroscopy from an increase in the linewidth of resolved ferricytochrome c resonances with hexametaphosphate concentration. Alternatively, analytical ultracentrifugation reveals protein association from the increase in apparent sedimentation coefficients of cytochrome c in the presence of equimolar hexametaphosphate. Protein aggregation is dependent on the concentration of background electrolyte since in the range 10–150 mM sodium cacodylate alternative stabilisation of dimeric and trimeric complexes was observed by both NMR and analytical ultracentrifugation. A model is proposed for the mechanism of protein aggregation caused by polyphosphate binding to the surface of cytochrome c.
Differential interactions of estrogens and antiestrogens at the 17β‐hydroxy or counterpart function with the estrogen receptor
Authors: BORGNA Jean‐Louis , SCALI Jacqueline
Published: March 2005

The action of diethylpyrocarbonate on lamb uterine estrogen receptor produced an homogeneous population of the receptor (∼ 55%) which still bound triarylethylene antiestrogens such as 4‐hydroxytamoxifen with a high affinity but bound classical potent estrogens such as estradiol or diethylstilbestrol with a very low affinity. To specify the structural features of the ligands involved in the decrease of ligand affinity upon modification of the estrogen receptor, we determined the relative affinity constants of 17 steroidal estrogens or antiestrogens (deriving from estradiol by a 7α‐ or 11β‐substitution) and 14 nonsteroidal estrogens or antiestrogens (all including the 1,2‐trans‐diphenylethylene structure of diethylstilbestrol) for native and diethylpyrocarbonate‐modified estrogen receptors. Then the ratio of the relative affinity constant for the native receptor to that for the modified receptor (δ) was calculated for each ligand, to compare the variation in the affinity of the ligand upon modification of the receptor to that of 4‐hydroxytamoxifen (δ= 1). The results showed that the strong decrease of ligand affinity upon modification of the receptor displayed by classical estrogens (δ≥ 200) is strictly dependent on the presence of the 17β‐hydroxyl group in steroidal compounds or its α‐4‐ and β‐4‐counterparts in diethylstilbestrol‐related compounds. However, for the 7α‐ or 11β‐derivatives of estradiol displaying potent antiestrogenic properties, the relative decrease in affinity was much more limited (δ≤ 19). For 11β‐derivatives displaying a relative estrogenic activity weaker than that of estradiol itself, an average decrease in affinity was observed (23 ≤δ≤ 62). With the diethylstilbestrol‐related compounds, bearing or not the α‐4‐hydroxyl and/or the β‐4‐hydroxy functions and showing either weak relative estrogenic or antiestrogenic properties, the relative variation in affinity was weak (0.6 ≤δ≤ 24). These results indicate that the interaction of 7α‐ or 11β‐substituted steroidal antiestrogens and of 1,2‐trans‐diphenylethylene or triphenylethylene derivatives, displaying either weak relative estrogenic or antiestrogenic properties with the receptor, differs at the 17β‐hydroxy or at the α‐4‐/β‐4‐hydroxy functions from that of potent estrogens. They suggest that the strong decrease in the relative affinity of ligands upon receptor modification may reflect the high efficiency of the ligands to activate the receptor properly. Consequently, the screening of 17β‐hydroxylated steroids or diethylstilbestrol‐related compounds with native and diethylpyrocarbonate‐modified receptors could be a useful method for distinguishing between potent estrogens (δ > 200) and weak estrogens or antiestrogens (δ < 20).
Primary structure of porin from Rhodobacter capsulatus
Authors: SCHILTZ Emile , KREUSCH Andreas , NESTEL Uwe , SCHULZ Georg E.
Published: March 2005

The primary structure of the integral membrane protein porin from the purple bacterium Rhodobacter capsulatus was determined. The protein was cleaved with trypsin, CNBr and Asp‐N protease. The peptides were isolated, sequenced and aligned to a total length of 301 residues with an Mr of 31536. The low isoelectric point of 3.9 is confirmed by the high excess of 34 Asp and 17 Glu (16.9%) over 10 Lys, 7 Arg and 2 His (6.3%). Overall sequence similarity to other porins is not evident when using sequence alignment programs. However, a partial relationship to Neisseria porins seems to exist. The established sequence has been used as the basis for a three‐dimensional structure determination by X‐ray diffraction at 0.18‐nm resolution. The arrangement of the sequence in the 16‐stranded β‐barrel of porin is given. Some sequence‐structure correlations are discussed.
On the importance of the support material for enzymatic synthesis in organic media
Authors: ADLERCREUTZ Patrick
Published: March 2005

Enzymes were deposited on different porous support materials and these preparations were used to catalyze reactions in organic media. Reactions were carried out at specific water activities, achieved by equilibrating both the enzyme preparation and the substrate solution at the desired water activity before mixing them and thereby starting the reactions. The reaction rates obtained at the same water activity with different supports differed greatly, indicating a direct influence of the support on the enzyme. For horse liver alcohol dehydrogenase, Celite was the best support, and the reaction rate increased with increasing water activity. In the α‐chymotrypsin‐catalyzed alcoholysis of N‐acetyl‐l‐phenylalanine ethyl ester with 1‐butanol, high rates were again obtained with Celite, but with this support only about one third of the ethyl ester was converted to butyl ester, the rest was hydrolyzed. With the polyamide support, Accurel PA6, alcoholysis was the dominating reaction, and by using a low water activity (0.33), hydrolysis was completely suppressed while still maintaining a high alcoholysis activity. Controlled pore glass (CPG), derivatized with either hexyl or glucosyl groups, had quite different properties as enzyme supports. For horse liver alcohol dehydrogenase, glucose‐CPG was a much better support than hexyl‐CPG, and in the α‐chymotrypsin‐catalyzed reactions, glucose‐CPG favored hydrolysis, and hexyl‐CPG alcoholysis, at water activities exceeding 0.8. The results are discussed considering the absorption of water on the enzymes, on the supports and the solubility of water in the reaction media; all these parameters were measured separately.
Peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F cannot release glycans with fucose attached α1 → 3 to the asparagine‐linked N‐acetylglucosamine residue
Authors: TRETTER Verena , ALTMANN Friedrich , MÄRZ Leopold
Published: March 2005

The ability of peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum and PNGase A from sweet almonds to deglycosylate N‐glycopeptides and N‐glycoproteins from plants was compared. Bromelain glycopeptide and horseradish peroxidase‐C glycoprotein, which contain xylose linked β1 → 2 to β‐mannose and fucose linked α1 → 3 to the innermost N‐acetylglucosamine, were used as substrates.

In contrast to PNGase A, the enzyme from F. meningosepticum did not act upon these substrates even at concentrations 100‐fold higher than required for complete deglycosylation of commonly used standard substrates. After removal of α1 → 3‐linked fucose from the plant glycopeptide and glycoprotein by mild acid hydrolysis, they were readily degraded by PNGase F at moderate enzyme concentrations.

Hence we conclude that α1 → 3 fucosylation of the inner N‐acetylglucosamine impedes the enzymatic action of PNGase F. Knowledge of this limitation of the deglycosylation potential of PNGase F may turn it from a pitfall into a useful experimental tool.
Acid‐base properties of nucleosides and nucleotides as a function of concentration
Authors: CORFÙ Nicolas A. , SIGEL Helmut
Published: March 2005

The acid‐base properties of the nucleic base residues of ITP, GTP, and ATP, and for comparison also as far as possible of the corresponding nucleosides, were studied in dependence on their concentration, i. e. on the effect of self‐association. From the dependence between the 1H‐NMR chemical shifts of H‐2 (where applicable), H‐8, and H‐1′, and the pD of the solutions, the acidity constants for the deprotonation of the D+ (N‐7) site in D2(ITP)2−, D2(GTP)2−, D(Ino)+, and D(Guo)+, and of the D+ (N‐1) site in D2(ATP)2− and D(Ado)+ were calculated. Chemical shift/pD profiles for a whole series of varying concentrations of the nucleic base derivatives (= N) were constructed, including those for infinite dilution (δo), which give the acidity constant for the monomeric N, and for infinitely concentrated solutions (δ∞), which give the acidity constant of an N in an infinitely long stack. The acidity constants determined from the δo/pD plots are in excellent agreement with the pKa values measured by potentiometric pH titrations of highly diluted solutions of N. The effects of self‐association are striking: e.g. the pKa value of the D+ (N‐7) site in D2(GTP)2− is lowered by about 1 (as calculated from the δo/pD and δ∞/pD profiles), while the pKa value of the D+ (N‐1) site in D2(ATP)2− is increased by approximately 0.3; i.e. in the first case deprotonation is facilitated and in the second it is inhibited. The increasing inhibition of the H+ (N‐1) deprotonation with an increasing ATP concentration is due to the high stability of the dimeric [H2(ATP)]24− stack for which the intermolecular H+(N‐1)/γ‐P(OH)(O)2− ion pairs between the two ATP molecules are crucial. In those cases where no other significant interaction but aromatic‐ring stacking in the self‐association process occurs, the release of protons from protonated nitrogen‐ring sites is facilitated with increasing stacking; this holds not only for D2(GTP)2− as indicated above, but also for D2(ITP)2−, D(Ino)+, and D(Ado)+. The latter example especially suggests that the situation for the D2(ATP)2− system is exceptional. Some consequences of the considered acid‐base properties for biological sytems are indicated.
Regulation of smooth‐muscle myosin‐light‐chain kinase
Authors: SOBIESZEK Apolinary
Published: March 2005

The kinetic mechanism of turkey gizzard smooth muscle myosin‐light‐chain kinase was investigated using the isolated 20‐kDa light chain of myosin as substrate. The kinetic and product inhibition patterns of the forward reaction indicated an ordered sequential mechanism in which MgATP bound first, ADP was released last. The order of substrate binding and product release was confirmed independently by competitive, dead‐end inhibition patterns obtained using the non‐hydrolizable ATP analog adenosine 5′‐[β,γ‐imido]triphosphate. The mechanism was also characterized by a relatively strong product inhibition by ADP and a weak one by phosphorylated 20‐kDa light‐chain myosin, in addition to a significant inhibition by the latter product via a formation of a dead‐end complex. [γ‐32P]ATP⇋[32P]phosphorylated light chain isotope‐exchange data were consistent with the deduced mechanism and with the presence of the latter dead‐end complex.
GDP‐fucose: β‐N‐acetylglucosamine (Fuc to (Fucα1 → 6GlcNAc)‐Asn‐peptide) α1 → 3‐fucosyltransferase activity in honeybee (Apis mellifica) venom glands
Authors: STAUDACHER Erika , ALTMANN Friedrich , GLÖSSL Josef , MÄRZ Leopold , SCHACHTER Harry , KAMERLING Johannis P. , HÅRD Karl , VLIEGENTHART Johannes F. G.
Published: March 2005

Incubation of honeybee (Apis mellifica) venom‐gland extracts with GDP‐[14C]fucose and GlcNAcβ1 → 2Manα1 → 6(GlcNAcβ1 → 2Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(Fucα1 → 6)GlcNAcβ1 → N‐Asn‐peptide(NAc) gave a labeled product in 40% yield. Analysis by 500‐MHz 1H‐NMR spectroscopy indicated the transferred fucose (Fuc) residue to be α1 → 3‐linked to the Asn‐bound GlcNAc. Further proof was provided by one‐dimensional and two‐dimensional 1H‐NMR analysis of the incubation mixture, after incubation with β‐N‐acetylhexosaminidase. The established carbohydrate structure

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proves the existence of a novel α1 → 3‐fucosyltransferase with the ability to effect difucosylation of the Asn‐bound GlcNAc in N‐glycans.
Structure of the β‐glucosidase gene bglA of Clostridium thermocellum
Authors: GRÄBNITZ Folke , SEISS Monika , RÜCKNAGEL Karl P. , STAUDENBAUER Walter L.
Published: March 2005

The nucleotide sequence of the Clostridium thermocellum gene bglA, coding for the thermostable β‐glucosidase A, has been determined. The coding region of 1344 bp was identified by comparison with the N‐terminal amino acid squence of recombinant β‐glucosidase A purified from Escherichia coli. The deduced amino acid sequence corresponds to a protein of 51482 Da. The coding region is flanked by putative promoter and transcription terminator sequences. The protein is unrelated to β‐glucosidase B of C. thermocellum, but has a high level of similarity with other bacterial β‐glucosidases and phospho‐β‐glucosidases. Similarity is also observed with the β‐galactosidase of the archaebacterium Sulfolobus solfataricus. Unexpectedly, it was found that human lactasephlorizin hydrolase contains three copies of a sequence closely related to C. thermocellumβ‐glucosidase A (up to 40% sequence identity). These diverse β‐glucosidases can therefore be grouped into an enzyme family (BGA) of common structural design. Sequence comparison by hydrophobic cluster analysis revealed that all BGA enzymes share a well conserved region which is homologous to the catalytic domain of the widely distributed cellulase family A. A distinctive feature of this domain is the sequence motif His – Ans‐Glu‐Pro in which the catalytic residues His and Glu are separated by 35–55 amino acid residues. The cellulase family A and the β‐glucosidase family BGA might thus be considered as members of a protein super‐family comprising β‐glucanases and β‐glycosidases from all three primary kingdoms of living organisms.
Association of ribosomal subunits
Authors: HERRERA Flor , CORREIA Heriberto , TRIANA Ledia , FRAILE Germán
Published: March 2005

A yeast ribosomal subunit association factor (AF) has been purified from a high‐salt ribosomal wash. The purified enzyme is a thermostable protein that associates ribosomal subunits at low Mg2+ concentration without requiring energy. It appears to be an aggregate of trimers or dimers (molecular mass 125 or 79 kDa) which on sodium dodecyl sulfate gels shows the presence of a major protein band whose estimated molecular mass is 43 kDa. Evidence also indicates the existence of a 50‐kDa polypeptide which seems to be unstable since with freezing and thawing it gives rise to the 43‐kDa polypeptide. It was shown that the labelled factor interacts with 80S ribosomes and with 40S ribosomal subunits. The purified polypeptide reacts with antibodies directed against EF‐1α, this last protein recognizing the antibodies raised against AF. Likewise, both EF‐1α and AF associate ribosomal subunits in the same way. When EF‐1 is heated, it not only maintains its association activity, but also behaves like a 43‐kDa polypeptide in an SDS electrophoresis run. These observations strongly suggest that AF originates from EF‐1α, which implies that the well‐known elongation factor may also play a role in the initiation step of protein synthesis.
Cytoplasmic aspartyl‐tRNA synthetase from Saccharomyces cerevisiae
Authors: ERIANI Gilbert , PREVOST Gilles , KERN Daniel , VINCENDON Pascale , DIRHEIMER Guy , GANGLOFF Jean
Published: March 2005

Aspartyl‐tRNA synthetase (AspRS) from yeast, a homodimer of 125 kDa, was shortened by several residues from the C‐ and N‐termini, via site‐directed mutagenesis, to examine the contribution of the removed peptides to the enzyme properties. This study showed that the N‐terminal sequence up to amino acid 70 (which confers peculiar ionic properties to the protein) is dispensable for activity. Domains located beyond amino acid 70 appeared to have increasing catalytic importance; the removal of 80 or 90 residues affected the Km values for ATP and deletions of 101 or 140 amino acids profoundly modified the physicochemical properties of AspRS, and by consequence, its structural organisation (extraction of the mutated proteins out of the cells required the presence of SDS). On the C‐terminal side, very limited modifications readily affected the enzyme properties. Deletion of as few as three residues increased the Km for ATP and reduced the aminoacylation kcat as well as the thermostability of the adenylate synthesis activity; the kcat of this step was impaired after deletion of two further residues. Finally, shortening the C‐terminal decapeptide completely inactivated AspRS, whilst affecting neither its affinity for tRNAAsp nor its dimerisation capacity. These data reveal the role of the C‐terminal decapeptide as a determinant in both reactions catalysed by AspRS. This peptide is involved in ATP binding, stabilising the functional conformation of the amino‐acid‐activating domain and probably maintaining the tRNA‐acceptor end in a reactive position with regard to the activated amino acid.
Isolation and characterization of hyodeoxycholic‐acid: UDP‐glucuronosyltransferase from human liver
Authors: MATERN Heidrun , LAPPAS Norbert , MATERN Siegfried
Published: March 2005

The enzyme hyodeoxycholic‐acid: UDP‐glucuronosyltransferase was purified about 230‐fold from a solubilized human liver microsomal preparation utilizing anion‐exchange chromatography, ampholyte‐displacement chromatography and UDP‐hexanolamine—Sepharose affinity chromatography. The homogeneity of the final enzyme preparation was judged by two criteria: the appearance of a single band of Mr 52000 in SDS/PAGE; the elution of a single peak in reversed‐phase FPLC. The isolated enzyme catalyzed the glucuronidation of the 6α‐hydroxy bile acids hyodeoxycholic and hyocholic acids, and of the steroid hormone estriol, with a ratio of relative reaction rates of 13:1:2.7. UDP‐glucuronosyltransferase activities toward the 3α‐hydroxy bile acid lithocholic acid, androsterone, testosterone, bilirubin and P‐nitrophenol were not detectable in the pure enzyme preparation and were shown to be separated from enzyme activity toward hyodeoxycholic acid during ampholyte‐displacement chromatography and/or UDP‐hexanolamine—Sepharose affinity chromatography. Two‐substrate kinetic analysis of hyodeoxycholic‐acid‐conjugating activity gave a sequential mechanism with apparent Km values of 12 μM and 4μM for hyodeoxycholic acid and UDP‐glucuronic acid, respectively. Phospholipids were required for reconstitution of maximal activity toward hyodeoxycholic acid. Phosphatidylcholine was the most effective activator of enzyme activity.