bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant laminin B1 chains exhibit intact short‐arm domains but do not form oligomeric molecules
Authors: PIKKARAINEN Timo , SCHULTHESS Therese , ENGEL Jürgen , TRYGGVASON Karl
Published: March 2005

The human laminin B1 chain has been produced in the baculovirus expression system in sufficient amounts for biochemical and functional studies. A full‐length cDNA, which was constructed of four partially overlapping clones and verified by in vitro transcription and translation to be functional, was cloned into the transfer vector pVL1392 behind the polyhedrin promoter. The recombinant construct was incorporated by in vivo homologous recombination into the genome of the wild‐type baculovirus, Autographa californica nuclear polyhedrosis virus. Infection of Spodoptera frugiperda cells (Sf9) with the recombinant virus resulted in the expression of the recombinant B1 chain (recB1) in these insect cells. The recB1 was found to be synthesized in two forms with apparent molecular masses of 220 kDa and 200 kDa. The 220‐kDa form is an N‐glycosylated form of recB1, because it was not present in cultures containing tunicamycin, an inhibitor of N‐linked glycosylation. The recB1 accumulated inside the cell and only a small portion of it was secreted into the culture medium. Thus purification had to be started from the cell extract in order to obtain reasonable amounts of the protein. About 500 μg was obtained from a 500‐ml culture with three steps of chromatography, concavalin A, DEAE‐Sepharose and Mono Q anion‐exchange chromatography. Only the glycosylated form was purified. The recB1 was found to be sensitive to degradation during the purification, because two proteolytic forms of about 180 kDa were present in every preparation. The accumulation of recB1 inside the cell was possibly due to the lack of correct assembly. Electron microscopy studies showed that the short arm part had a native or near‐native structure, but the C‐terminal heptad repeat domain had not foided correctly and did not exist in an α‐helical structure, as it does in native laminin. Electron microscopy and cross‐linking studies further revealed that recB1 was a monomeric protein. It was also shown to be unable to oligomerize in vitro, suggesting that the B1 chain is not designed to form homo‐oligomers. Finally, cell attachment assays were carried out, but the native recB1 appeared to be inactive in these assays.
Ferrisiderophore reductases of Pseudomonas
Authors: HALLÉ Félix , MEYER Jean‐Marie
Published: March 2005

Purification of the ferripyoverdine reductase from Pseudomonas aeruginosa, strain PAO1, lead to the isolation of a soluble protein of M 27000–28000, as determined by HPLC sieving filtration and by denaturating gel electrophoresis. In the presence of NADH as the reductant, ferripyoverdine as the iron substrate, ferrozine as an iron(II)‐trapping agent and FMN, this protein displayed an ironreductase activity which resulted in the formation of ferrozine‐iron(II) complex, providing that the enzymic assay was run under strict anaerobiosis. FMN was absolutely required for the activity to occur, but the lack of a visible spectrum and the lack of fluorescence for the protein in solution suggested that ferripyoverdine reductase is not a flavin‐containing protein and that covalently bound FMN is not a prerequisite for the enzymatic reaction. A search of ferripyoverdine reductase by immunological detection amongst the different cellular compartments of P. aeruginosa lead to the conclusion that the soluble enzyme, which represented more than 95% of the total cellular enzyme, is not located in the periplasm but specifically in the cytoplasm. A strongly immunoreacting material, corresponding to a protein with identical Mr as the ferripyoverdine reductase of P. aeruginosa PAO1, was detected in all the eighteen fluorescent pseudomonad strains belonging to the P. aeruginosa, P. fluorescens, P. putida and P. chlororaphis species, as well as in P. stutzeri, a non‐fluorescent species, suggesting that the enzyme acting as a ferripyoverdine reductase in P. aeruginosa PAO1 is ubiquitous among the Pseudomonas.
Use of serine‐protease inhibitors as probes for the different proteolytic activities of the rat liver multicatalytic proteinase complex
Authors: DJABALLAH Hakim , HARNESS Jane A. , SAVORY Peter J. , RIVETT A. Jennifer
Published: March 2005

The multicatalytic proteinase (MCP) complex catalyses cleavage of bonds on the carboxy‐group side of basic, hydrophobic or acidic amino acid residues. Orginally, it was proposed that the complex contained three distinct types of catalytic component. MCP from rat liver has been assayed for so‐called trypsin‐like activity with Boc‐Leu‐Ser‐Thr‐Arg‐NH‐Mec (Mec, 4‐methylcoumarin; Boc, t‐butoxycarbonyl), for chymotrypsin‐like activity with Ala‐Ala‐Phe‐NH‐Mec and Suc‐Leu‐Leu‐Val‐Tyr‐NH‐MEc (Suc, succinyl), and peptidyl‐glutamylpeptide hydrolase activity with Cbz‐Leu‐Leu‐Glu‐Nap (Nap, naphthylamide; Cbz, benzyloxycarbonyl). Results of these studies suggest that as many as five distinct components can be distinguished, one for the trypsin‐like activity and two for each of the others. The activities were tested with a variety of serine‐protease inhibitors, and other novel effectors have also been identified. The two most effective inhibitors were 4‐(2‐amino‐ethyl)benzenesulphonyl fluoride, which selectivity inactivates the trypsin‐like activity, and 3,4‐dichloroisocoumarin which inhibits chymotrypsin‐like activity and the second, cooperative component [Djaballah, H. & Rivett, A. J. (1992) Biochemistry 31, 4133–4141] of peptidylglutamylpeptide hydrolase activity. The three activities inhibited by 3,4‐dichloroisocoumarin can easily be distinguished by the effects of chymostatin analogues, diisopropylfluorophosphate, guanidine/HCl and casein. The results support the view that the enzyme is a novel type of serine protease and suggest that it may contain at least five distinct catalytic components. Marked differences in the reactivities of the different catalytic sites with different reagents can be used to distinguish between them.
Biochemical characterization of thioredoxin 1 from Dictyostelium discoideum
Authors: WETTERAUER Birgit , VÉRON Michel , MIGINIAC‐MASLOW Myroslawa , DECOTTIGNIES Paulette , JACQUOT Jean‐Pierre
Published: March 2005

Multiple genes for thioredoxins (TRX) have been demonstrated in Dictyostelium discoideum. We expressed the cDNA for one of these genes (DdTrxl) in E. coli and purified the protein to homogeneity. The interaction with classic substrates as well as TRX reductases was analysed. It reacted with every tested substrate: insulin, NADP‐dependent malate dehydrogenase and fructose‐1,6‐bisphosphatase. With a S0.5 of 20 uM, the reactivity with the fructose‐1,6‐bisphosphatase is the highest ever found for a heterologous TRX. DdTRX1 itself is accepted as a substrate by the chloroplast ferredoxin‐dependent TRX reductase, as well as by the E. coli NADPH‐dependent TRX reductase. Thus, the Dictyostelium TRX is functionally promiscuous. Its reactivity with insulin, chloroplast NADP‐dependent malate dehydrogenase and ferredoxin‐dependent TRX reductase resemble those of other TRX. It is, however, clearly different in its good interaction with chloroplast fructose‐1,6‐bisphos‐phatase and in its poor interaction with E. coli NADP‐dependent TRX reductase.
Secondary and tertiary structures involving chondroitin and chondroitin sulphates in solution, investigated by rotary shadowing/electron microscopy and computer simulation
Authors: SCOTT John E. , CHEN Yuan , BRASS Andy
Published: March 2005

Rotary shadowing/electron microscopy of chondroitin 6‐sulphate (CS6) and 4‐sulphate (CS4) showed that the former, but not the latter, aggregated to mesh works. Preparations made from salt (ammonium acetate) solutions showed enhanced aggregation. Computer modelling, using molecular mechanics and dynamics, was applied to secondary structures (twofold helices) derived from NMR studies, to determine geometric and energetic constraints on duplex and higher‐aggregate formation. The calculations suggested that chondroitin, CS6 and undersulphated CS4 could form duplexes, while CS4 could not, thus bridging the gap between atomic dimensions (NMR) and high polymer scale (electron microscopy). Calculations suggested that water structure helped to stabilise the twofold helix. It is proposed that the twofold helical, flat, tape‐like molecules aggregate via hydrophobic bonding between the very extensive hydrophobic patches (9 CH units) repeated on alternating sides of the polymers. The negative charge of the polyanions opposes aggregate formation. Calculations showed that duplexes were formed with decreasing stability as the charge density increased, and as the charge was concentrated towards the centre line of the polymer (i.e. in CS4). The unsulphated polymer chondroitin could form duplexes and higher aggregates as readily as hyaluronan. Hyaluronan was calculated to form stable heteroduplexes with CS6 and CS4. The frequency and positioning of the sulphate‐ester group within the polymer thus determines whether the molecule participates in duplex formation.
Proteolytic cleavage of atrial natriuretic factor receptor in bovine adrenal membranes by endogenous metalloendopeptidase
Authors: ABE Tetsuaki , MISONO Kunio S.
Published: March 2005

Atrial natriuretic factor (ANF) is a peptide hormone from the heart atrium with potent natriuretic and vasorelaxant activities. The natriuretic activity of ANF is, in part, mediated through the adrenal gland, where binding of ANF to the 130‐kDa ANF receptor causes suppression of aldosterone secretion. Incubation of bovine adrenal membranes at pH <5.6 caused a rapid and spontaneous cleavage of the 130‐kDa ANF receptor, yielding a 65‐kDa polypeptide that could be detected by photoaffinity labeling by 125I‐labeled Nα4‐azidobenzoyl‐ANF(4–28) followed by SDS/PAGE under reducing conditions. Within 20 min of incubation at pH 4.0, essentially all the 130‐kDa receptor was converted to a 65‐kDa ANF binding protein. This cleavage reaction was completely inhibited by inclusion of 5 mM EDTA. When SDS/PAGE was carried out under non‐reducing conditions, the apparent size of the ANF receptor remained unchanged at 130 kDa, indicating that the 65‐kDa ANF‐binding fragment was still linked to the remaining part(s) of the receptor polypeptide through a disulfide bond(s). The disappearance of the 130‐kDa receptor was accompanied by a parallel decrease in guanylate cyclase activity in the membranes. Inclusion of EDTA in the incubation not only prevented cleavage of the 130‐kDa receptor, but also protected guanylate cyclase activity, indicating that proteolysis, but not the physical effects of the acidic pH, causes inactivation of guanylate cyclase. The 130‐kDa ANF receptor in adrenal membranes was competitively protected from photoaffinity labeling by ANF(1–28) or ANF(4–28), but not by atriopeptin I [ANF(5–25)] or C‐ANF [des‐(18–22)‐ANF(4–23)‐NH2]. On the contrary, the 65‐kDa ANF‐binding fragment generated after incubation at pH 4.0 was protected from labeling by any of the above peptides, indicating broader binding specificity. After incubation in the presence of EDTA, the 130‐kDa ANF receptor, which was protected from proteolysis, retained binding specificity identical to that of the 130‐kDa receptor in untreated membranes. The results indicate that the broadening of selectivity is caused by cleavage, but not by the physical effect of acidic pH. Spontaneous proteolysis of ANF receptor by an endogenous metalloendopeptidase, occurring with concomitant inactivation of guanylate cyclase activity and broadening of ligand‐binding selectivity, may be responsible for the generation of low‐molecular‐mass receptors found in the adrenal gland and other target organs of ANF. The proteolytic process may play a role in desensitization or down‐regulation of the ANF receptor.
Isolation and characterization of alliinase cDNA clones from garlic (Allium sativum L.) and related species
Authors: VAN DAMME Els J. M. , SMEETS Koen , TORREKENS Sophie , VAN LEUVEN Fred , PEUMANS Willy J.
Published: March 2005

cDNA libraries constructed from poly(A)‐rich RNA isolated from Allium sativum (garlic), Allium cepa (onion) and Allium ascalonicum (shallot) were screened for cDNA clones encoding the alliinase using colony hybridization. Sequence analysis of the alliinase cDNA clones from different Alliaceae species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level.

Apparently, the alliinases are translated from mRNA species of approximately 2200 nucleotides. The primary translation products are preproproteins which are converted into the mature alliinases following post‐translational modifications. In the case of the garlic alliinase, the mRNA encodes a 486‐amino‐acid polypeptide with a molecular mass of 55623 Da. Cleavage of the signal peptide (28 amino acids) results in a preprotein which extends 10 amino acids before the first amino acid of the mature protein of 51451 Da. Southern‐blot analysis of genomic DNA has shown that the alliinases are most probably encoded by a family of closely related genes, which is in good agreement with the sequence heterogeneity found between different alliinase cDNA clones of one species.
Solution conformation of human neuropeptide Y by 1H nuclear magnetic resonance and restrained molecular dynamics
Authors: DARBON Hervé , BERNASSAU Jean‐Marie , DELEUZE Colette , CHENU Jacques , ROUSSEL Alain , CAMBILLAU Christian
Published: March 2005

The solution structure of human neuropeptide Y has been solved by conventional two‐dimensional NMR techniques followed by distance‐geometry and molecular‐dynamics methods. The conformation obtained is composed of two short contiguous α‐helices comprising residues 15–26 and 28–35, linked by a hinge inducing a 100° angle. The first helix (15–26) is connected to a polyproline stretch (residues 1–10) by a tight hairpin (residues 11–14). The helices and the polyproline stretch are packed together by hydrophobic interactions. This structure is related to that of the homologous avian pancreatic polypeptide and bovine pancreatic polypeptide. The C‐ and N‐terminii, known to be involved in the biological activity for respectively the receptor binding and activation, are close together in space. The side chains of residues Arg33, Arg35 and Tyr36 on the one hand, and Tyr1 and Pro2 on the other, form a continuous solvent‐exposed surface of 4.9 nm2 which is supposed to interact with the receptor for neuropeptide Y.
Identification of functionally active fragments of staphylococcal enterotoxin B
Authors: ALAKHOV Valery Yu. , KLINSKY Eugenii Yu. , KOLOSOV Mikhail I. , MAURER‐FOGY Ingrid , MOSKALEVA Elizaveta Yu. , SVESHNIKOV Peter G. , POZDNYAKOVA Lyubov P. , SHEMCHUKOVA Olga B. , SEVERIN Eugenii S.
Published: March 2005

It has been found that staphylococcal enterotoxin B contains a proteolysis‐sensitive sequence in the cysteine loop formed by two half‐cystines located in the middle of the toxin polypeptide chain. Fragments of the enterotoxin formed as a result of its digestion in this region have been isolated, their N‐terminal sequences have been determined and sites of proteolysis have been identified. It has been demonstrated that the N‐terminal fragment of staphylococcal enterotoxin B is capable of activating T cell proliferation in the culture of human mononuclear cells practically to the same degree as the intact enterotoxin. The toxin's C‐terminal fragment possesses an ability to activate calmodulin‐dependent enzymes and is probably the toxicogenic part of the enterotoxin.
Recombinant chicken egg white cystatin variants of the QLVSG region
Authors: AUERSWALD Ennes A. , GENENGER Gabriele , ASSFALG‐MACHLEIDT Irmgard , MACHLEIDT Werner , ENGH Richard A. , FRITZ Hans
Published: March 2005

Using recombinant DNA methods, seven cystatin variants were produced by cassette mutagenesis of a chicken egg white cystatin variant which already contains the mutations Ala3, Glu2, Phe1, Ser1 → Met, Met29 → and Met 89 → Leu. When characterized by structural and functional studies, they were all found to harbour mutations in the first hairpin loop, the so‐called ‘QXVXG’ region, which is highly conserved within the cystatin superfamily and thought to be important for its inhibitory activity towards cysteine proteinases. They were purified to more than 90% homogeneity and analysed by SDS/PAGE, HPLC, tryptic peptide mapping, N‐terminal amino acid sequencing and ELISA. Structural model building of the variants and their complexex with papain was performed using computer graphics based on the crystallographic coordinates of chicken egg white cystatin and the papain‐stefin complex. Only minor conformational changes were required for modelling the mutants or complexes. Equilibrium dissociation constants and rate constants of complex formation of the variants with papain, actinidin as well as cathepsin B and L were determined by kinetic measurements using fluorogenic substrates. The single exchanges Gln53 → Glu, Gln53 → Asn, Val44 → Asp, Gly57 → Ala and the double exchanges Arg52 → Leu, Gln53 → Glu, Gln53 → Asn, Ser56 → Ala, Leu54 → Met, Gly57 → Ala reduced the inhibition of papain, actinidin and cathespin B significantly by 10‐1000‐fold. With the exception of the Val55 → Asp variant, the differences in the Ki values are mainly due to larger koff values, whereas the kon values seem to be more or less unaffected by the selected mutations. The effect on the inhibition of papain is generally smaller than the effects on actinidin and cathepsin B inhibition. Cathepsin L inhibition is strikingly insensitive to all mutations. These distinct effects of the inhibitor variants indicate differences in proteinase – inhibitor‐protein interactions between closely related cysteine proteinases. In addition, the results verify the prediction, made earlier from sequence alignment studies and from a docking model of the chicken cystatin‐papain complex, that the first hairpin loop of cystatins is essential for effective inhibition.
Chemical and structural characterisation of iron cores of haemosiderins isolated from different sources
Authors: WARD Roberta J. , RAMSEY Michael H. , DICKSON Dominic P. E. , FLORENCE Anne , CRICHTON Robert R. , PETERS Timothy J. , MANN Stephen
Published: March 2005

The elemental content of the iron cores of haemosiderins isolated from animal and human tissues has been determined to ascertain whether changes in composition are correlated with structural differences previously identified in these mineralisation products.

Significant differences were observed in the elemental composition of haemosiderins isolated from patients subjected to desferrioxamine‐chelation therapy compared to patients who had been venesected. The P/Fe molar ratio was considerably higher in haemosiderin isolated from treated primary haemochromatosis (0.83), compared to untreated primary haemochromatosis (0.10) and treated secondary haemochromatosis (0.25), and this could account for the amorphous nature of these iron cores. The levels of M/Fe (M = Ca, Cu, Zn) were reduced in the haemosiderins derived from treated secondary haemochromatosis patients, possibly due to the chelation of these ions by desferrioxamine therapy. In an experimentally iron‐loaded rat, receiving either desferrioxamine or 1,2‐diethyl‐3‐hydroxypyrid‐4‐one, selective decreases in these three elements were also observed after two weeks of desferrioxamine therapy. Such changes may be important determinants in the modification of biomineralisation of the iron cores.
Structural relationship between the hexameric and tetrameric family of glutamate dehydrogenases
Authors: BRITTON K. Linda , BAKER Patrick J. , RICE David W. , STILLMAN Timothy J.
Published: March 2005

The family of glutamate dehydrogenases include a group of hexameric oligomers with a subunit Mr of around 50000, which are closely related in amino acid sequence and a smaller group of tetrameric oligomers based on a much larger subunit with Mr 115000. Sequence comparisons have indicated a low level of similarity between the C‐terminal portion of the tetrameric enzymes and a substantial region of the polypeptide chain for the more widespread hexameric glutamate dehydrogenases. In the light of the solution of the three dimensional structure of the hexameric NAD+‐linked glutamate dehydrogenase from Clostridium symbiosum, we have undertaken a detailed examination of the alignment of the sequence for the C‐terminal domain of the tetrameric Neurospora crassa glutamate dehydrogenase against the sequence and the molecular structure of that from C. symbiosum. This analysis reveals that the residues conserved between these two families are clustered in the three‐dimensional structure and points to a remarkably similar layout of the glutamate‐binding site and the active‐site pocket, though with some differences in the mode of recognition of the nucleotide cofactor.
A comparative EPR investigation of the multicopper proteins nitrous‐oxide reductase and cytochrome c oxidase
Authors: ANTHOLINE William E. , KASTRAU Dieter H. W. , STEFFENS Guy C. M. , BUSE Gerhard , ZUMFT Walter G. , KRONECK Peter M. H.
Published: March 2005

The multicopper proteins, nitrous‐oxide reductase (N2OR) and cytochrome c oxidase (COX), were investigated by EPR spectroscopy at microwave frequencies 2.4–35 GHz. Our results support a Cu‐Cu interaction in COX and N2OR. At least 10 lines in the 2.7‐GHz, 12 lines in the 4.6‐GHz and 14 lines in the 9.2 GHz spectra were resolved for N2OR. Eight copper lines at 2.7 GHz, about nine lines at 4.6 GHz and about six lines at 9.2 GHz were resolved for COX. Simulations of the EPR spectra were consistent with most of the resonances of the multiline spectra, including regions in the center of the spectra where overlap of the three seven‐line patterns is proposed. These simulations indicated that Cu‐Cu interaction, in a mixed‐valence [Cu(1.5)…Cu(1.5)], S=1/2 site is consistent with, if not proof of, the unusual spectral features observed for N2OR and COX.
Evidence for a protein kinase cascade in higher plants
Authors: MACKINTOSH Robert W. , DAVIES Stephen P. , CLARKE Paul R. , WEEKES John , GILLESPIE John G. , GIBB Barry J. , HARDIE D. Grahame
Published: March 2005

Protein phosphorylation is well established as a regulatory mechanism in higher plants, but only a handful of plant enzymes are known to be regulated in this manner, and relatively few plant protein kinases have been characterized. AMP‐activated protein kinase regulates key enzymes of mammalian fatty acid, sterol and isoprenoid metabolism, including 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductase. We now show that there is an activity in higher plants which, by functional criteria, is a homologue of the AMP‐activated protein kinase, although it is not regulated by AMP. The plant kinase inactivates mammalian HMG‐CoA reductase and acetyl‐CoA carboxylase, and peptide mapping suggests that it phosphorylates the same sites on these proteins as the mammalian kinase. However, with the target enzymes purified from plant sources, it inactivates HMG‐CoA reductase but not acetyl‐CoA carboxylase. The kinase is located in the soluble, and not the chloroplast, fraction of leaf cells, consistent with the idea that it regulates HMG‐CoA reductase, and hence isoprenoid biosynthesis, in vivo. The plant kinase also appears to be part of a protein kinase cascade which has been highly conserved during evolution, since the kinase is inactivated and reactivated by mammalian protein phosphatases (2A or 2C) and mammalian kinase kinase, respectively. This contrasts with the situation for many other mammalian protein kinases involved in signal transduction, which appear to have no close homologue in higher plants. To our knowledge, this represents the first direct evidence for a protein kinase cascade in higher plants.
Characterization of the 56‐kDa subunit of yeast trehalose‐6‐phosphate synthase and cloning of its gene reveal its identity with the product of CIF1, a regulator of carbon catabolite inactivation
Authors: BELL Walter , KLAASSEN Paul , OHNACKER Martin , BOLLER Thomas , HERWEIJER Marga , SCHOPPINK Peter , VANDERZEE Peter , WIEMKEN Andres
Published: March 2005

Trehalose‐6‐phosphate synthase is the key enzyme for biosynthesis of trehalose, the major soluble carbohydrate in resting cells of yeast. This enzyme was purified from a strain of Saccharomyces cerevisiae lacking vacuolar proteases. It was found to be a multimeric protein of 630 kDa. Monoclonal antibodies were raised against its smallest subunit (56 kDa) and used for screening a yeast cDNA library. This yielded an immunopositive cDNA clone of 1.7 kb, containing an open reading frame of 1485 base pairs. Its sequence, called TPS1 (for trehalose‐6‐phosphate synthase), was represented by a single gene in the yeast genome and was found to be almost identical with the recently sequenced CIF1, a gene important for carbon catabolite inactivation, believed to be allelic with FDP1. A mutant obtained by disruption of TPS1 had a very low activity of trehalose‐6‐phosphate synthase, indicating that TPS1 is an important component of the enzyme. The mutant also showed a growth defect when transferred from glycerol to glucose, a phenotype similar to that of the cif1 and fdp1 mutants deficient in carbon catabolite inactivation. Thus, the smallest subunit of the biosynthetic enzyme trehalose‐6‐phosphate synthase appears to have, in addition, a central regulatory role in the carbohydrate metabolism of yeast.
A substrate‐like form of plasminogen‐activator‐inhibitor type 1
Authors: URANO Tetsumei , STRANDBERG Leif , JOHANSSON Lennart B.‐Å. , NY Tor
Published: March 2005

Recombinant plasminogen‐activator‐inhibitor type 1 (PAI‐1) purified in an active form from Escherichia coli and eucaryotic cells was found to contain a mixture of three functionally distinct forms: an active form that forms complexes with plasminogen activators (PAs), an inactive (latent) form that remains intact after incubation with PAs, and a substrate‐like form which is easily cleaved by PAs. Since active PAI‐1 purified from bacteria (rpPAI‐1) contains only trace amounts of the inactive latent and the substrate‐like forms, this material was used to study the effect of sodium dodecyl sulphate (SDS) on the structure and function of active PAI‐1. After treatment with 0.01% SDS, active rpPAI‐1 was converted to an inactive form that did not form complexes with PAs, but exhibited characteristics similar to those of latent PAI‐1. After treatment with 0.1% SDS, PAI‐1 lost its inhibitory activity and was cleaved as a substrate in the reactive center. Circular dichroism spectral analysis reveals that SDS changed the conformation of PAI‐1 dramatically, mainly by increasing its α‐helical content.
The incorporation of divalent metal ions into recombinant human tyrosine hydroxylase apoenzymes studied by intrinsic fluorescence and 1H‐NMR spectroscopy
Authors: HAAVIK Jan , MARTÍNEZ Aurora , OLAFSDOTTIR Sigridur , MALLET Jacques , FLATMARK Torgeir
Published: March 2005

Three isoforms of human tyrosine hydroxylase were expressed in Escherichia coli and purified to homogeneity as the apoenzymes (metal‐free). The apoenzymes exhibit typical tryptophan fluorescence emission spectra when excited at 250–300 nm. The emission maximum (342 nm) was not shifted by the addition of metal ions, but reconstitution of the apoenzymes with Fe(II) at pH 7–9 reduced the fluorescence intensity by about 35%, with and end point at 1.0 iron atom/enzyme subunit. The fluorescence intensity of purified bovine adrenal tyrosine hydroxylase, containing 0.78 mol tightly bound iron/mol subunit, was reduced by only 6% on addition of an excess amount of Fe(II). Other divalent metal ions [Zn(II), Co(II), Mn(II), Cu(II) and Ni(II)] also reduced the fluorescence intensity of the human enzyme by 12–30% when added in stoichiometric amounts. The binding of Co(II) at pH 7.2 was also found to affect its 1H‐NMR spectrum and this effect was reversed by lowering the pH to 6.1. The quenching of the intrinsic fluorescence of the human isoenzymes by Fe(II) was reversed by the addition of metal chelators. However, the addition of stoichiometric amounts of catecholamines, which are potent feedback inhibitors of tyrosine hydroxylase, to the iron‐reconstituted enzyme, prevented the release of iron by the metal chelators. Fluorescence quenching, nuclear magnetic relaxation measurements and EPR spectroscopy all indicate that the reconstitution of an active holoenzyme from the isolated apoenzyme, with stoichiometric amounts of Fe(II) at neutral pH, occurs without a measurable change in the redox state of the metal. However, on addition of dopamine or suprastoichiometric amounts of iron, the enzyme‐bound iron is oxidized to a high‐spin Fe(III) (S= 5/2) form in an environment of nearly axial symmetry, thus providing an explanation for the inhibitory action of the catecholamines.
Purification and characterization of rat brain cytosolic 3,5,3′‐triiodo‐l‐thyronine‐binding protein
Authors: LENNON Ana Maria
Published: March 2005

A rat brain cytosolic 3,5,3′‐triiodo‐l‐thyronine‐(T3)‐binding protein (CTBP) was purified using, successively, carboxymethyl‐Sephadex, DEAE‐Spherodex, T3–Sepharose‐4B affinity chromatography and Sephacryl S‐200. The molecular mass determined by SDS/PAGE wa 58 kDa. The binding characteristics determined by Scatchard analysis revealed a singla class of binding sites with a Ka of 1.56 nM−1 and a maximal binding capacity of 7500 nmol T3/g protein. The relative binding affinities of iodothyronine analogues were D‐T3 > L‐T3 > L‐T4 > 3,3′‐5‐triiodothyroacetic acid > reverse T3. The optimum pH for binding was 7.5. Purified brain CTBP was reversibly inactivated by charcoal. NADPH, NADP and thioredoxin restored binding activity to a level higher than that of the control; this effect was concentration dependent. Maximal activation was observed at 25 nM NADPH. NADP was effective only in the presence of 1 mM dithiothreitol; maximal activity was obtained at 10 nM NADP. At concentrations higher than 50 nM NADP, the binding gradually decreased. Thioredoxin in the presence of 1 mM dithiothreitol activated CTBP; maximal binding was obtained with 4 μM thioredoxin. In the presence of NADPH, NADP or thioredoxin the maximal binding capacity increased 2–4 times and the Ka was 2.6 nM−1. These results show that the activity of purified cytosolic brain T3‐binding protein may be modulated by NADPH, NADP or thioredoxin.
Coordination and internal exchange of two DNA molecules in a RecA filament in the presence of hydrolysing ATP
Authors: TAKAHASHI Masayuki , NORDÉN Bengt
Published: March 2005

Solution structure of complexes between DNA and recombinase RecA from Escherchia coli, in the presence of the physiological cofactor ATP, is probed by flow linear dichroism (LD) spectroscopy. A problem of ADP accumulation which promotes dissociation of DNA‐RecA is circumvented by using an ATP‐regenerating system. The LD features indicate that the local structure of the complex is very similar to that found in the presence of the non‐hydrolysable analog of ATP, adenosine‐5′‐O‐[γ‐thio]triphosphate (ATP[γS]); the DNA bases are oriented with their planes preferentially perpendicular to the long axis of the filament, while the indole chromophores of the two tryptophan residues of RecA are rather parallel to this reference direction. A much smaller overall amplitude of the LD spectrum, compared to ATP[γS], is interpreted as a result of fast dissociation of RecA due to hydrolysis of ATP, producing transiently naked DNA regions which act like flexible joints, diminishing the macroscopic orientation of the RecA filaments. However, the ATP hydrolysis is not found to prevent simultaneous accommodation of two non‐complementary DNA molecules in the RecA complex, as judged from the LD behaviour upon successive addition of two different polynucleotides or modified DNA strands. A notable differnce from corresponding complexes formed with ATP[γS] is that, in the presence of ATP hydrolysis, the order in which the two DNA molecules have been added is insignificant as judged from virtually identical resulting structures; this observation indicates that exchange of DNA occurs between the two DNA accommodation sites within the RecA filament.
Investigation of the mechanism of non‐turnover‐dependent inactivation of purified human 5‐lipoxygenase
Authors: PERCIVAL M. David , DENIS Danielle , RIENDEAU Denis , GRESSER Michael J.
Published: March 2005

Human 5‐lipoxygenase is a non‐heme iron protein which is reported to be highly unstable in the presence of oxygen. The results of this investigation demonstrate that H2O2 generated during air oxidation of thiols is the main factor in non‐turnover‐dependent inactivation of purified recombinant human 5‐lipoxygenase for the following reasons: catalase protects against oxygen‐dependent inactivation of the enzyme in the presence of dithiothreitol; the active, stable enzyme can be prepared under aerobic conditions with the exclusion of dithiothreitol and contaminating metal ions; 10 μM H2O2 causes the rapid inactivation of the enzyme. The native (ferrous) enzyme is approximately seven times more sensitive to inactivation by H2O2 than the ferric enzyme, suggesting that the mechanism of inactivation involves a Fenton‐type reaction of the ferrous enzyme with H2O2, resulting in the formation of an of an activated oxygen species. Purification of 5‐lipoxygenase under aerobic conditions (no dithiothreitol) results in an increase in both the specific activity of the purified protein [up to 70 μmol 5(S)‐hydroperoxy‐6‐trans‐8, 11, 14‐cis‐icosatetraenoic acid (5‐HPETE)/mg protein] and in the ratio of specific activity to enzyme iron content compared to enzyme purified under anaerobic conditions in the presence of dithiothreitol. The reaction of the highly active 5‐lipoxygenase enzyme shows a dependence on physiological intracellular calcium concentrations, half‐maximal product formation being obtained at 0.9 μM free Ca2+. The maximal enzyme activity is also dependent on EDTA and dithiothreitol and low amounts of carrier protein, as well as the known activators PtdCho and ATP. Ca2+ can be substituted by Mn2+, Ba2+ and Sr2+, although lower levels of stimulation are obtained. 5‐Lipoxygenase is strongly inhibited by low concentrations (≤ 10 μM) of Zn2+ and Cu2+. The inhibition by Cu2+ is apparently irreversible, whereas that by Zn2+ is slowly reversed (t1/2= 2 min) in the presence of excess EDTA. These observations on the mechanism of non‐turnover‐dependent inactivation of 5‐lipoxygenase, and the optimisation of assay conditions, have facilitated the purification of large quantities of relatively stable enzyme that will be useful for further kinetic and physical studies.
Protection by glutathione and other thiol compounds against the loss of protein thiols and tocopherol homologs during microsomal lipid peroxidation
Authors: MURPHY Michael E. , SCHOLICH Heiner , SIES Helmut
Published: March 2005

Microsomes from rat liver were used to investigate the mechanisms by which thiol compounds protect cellular membranes against damage from oxidants. Glutathione (GSH), dihydrolipoate and dithioerythritol, but not cysteine, ameliorated the loss of thiol groups of microsomal proteins attacked by Fe/ADP/NADPH or Fe/ADP/ascorbate prooxidant systems. The protection by GSH, but not dihydrolipoate or dithioerythritol, appeared to be enzymic since it was lost after microsomes were heated or treated with trypsin. The blocking of microsomal protein thiols with N‐ethylmaleimide also diminished the protective effect of GSH. Lipid peroxidation, as assessed by chemiluminescence and vitamin‐E loss, was inhibited in parallel with the protection of protein thiols. In microsomes lacking vitamin E, the protection of protein thiols by exogenous thiols was diminished. However, the GSH‐dependent protection of vitamin E showed no preference for α‐tocopherol over other tocopherol homologs. It is suggested that a GSH‐dependent enzyme maintains protein thiols in the face of oxidative damage during microsomal peroxidation. A maintenance of protein thiols might not only protect important metabolic functions, but may also afford an antioxidant capacity to membranes, and account for one facet of the GSH‐dependent inhibition of lipid peroxidation.
Probing the active site of the reconstituted aspartate/glutamate carrier from mitochondria
Authors: STAPPEN Reiner , DIERKS Thomas , BRÖER Angelika , KRÄMER Reinhard
Published: March 2005

Treatment of the reconstituted aspartate/glutamate carrier from mitochondria with 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole (Nbd‐Cl) led to complete inactivation of carrier function. Inhibition could be attributed to chemical modification of one single cysteine in the active site. This residue was specifically protected in the presence of aspartate or glutamate, 50% substrate protection being observed at half‐saturation of the external binding site. The bifunctional reagent 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonate (DIDS) also modified the same cysteine and, in addition, an active‐site lysine identified previously [Dierks, T., Stappen, R., Salentin, A. & Krämer, R. (1992) Biochim. Biophys. Acta 1103, 13–24]. The proximity of the cysteine [Cys(a)] and the lysine residue was confirmed by a mutual exclusion of the respective reagents when added consecutively. By using a variety of reagents a further cysteine [Cys(b)] and probably a histidine residue could be discriminated from Cys(a) and the lysine. The applied reagents were classified according to functional and structural criteria. Class A reagents, like Nbd‐Cl, modified the active‐site Cys(a) thereby inhibiting the antiport function. Class B reagents, like HgCl2, reacted with both Cys(a) and Cys(b) leading to a conversion of the carrier from antiport to uniport function [Dierks, T., Salentin, A., Heberger, C. & Krämer, R. (1990) Biochim. Biophys. Acta 1028, 268–280]. DIDS at relatively high concentration (60 μM) also acted as a uniport inducer. Class C reagents finally, like pyridoxal phosphate or diethyl pyrocarbonate, modified the active‐site lysine or histidine, respectively, and blocked antiport and uniport activity. By testing the accessibility of the mentioned residues to the various reagents, when applied in different order, topological relationships could be elaborated indicating the location of these amino acids with respect to the exofacial active site of the carrier protein.
Kinetics of ATP synthesis catalyzed by the H+‐ATPase from chloroplasts (CF0F1) reconstituted into liposomes and coreconstituted with bacteriorhodopsin
Authors: RICHARD Peter , GRÄBER Peter
Published: March 2005

The H+‐ATPase from chloroplasts (CF0F1) was isolated, purified and reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. A transmembrane pH difference, pH, and a transmembrane electric potential difference, Ψ, were generate by an acid/base transition. The rate of ATP synthesis was measured at constant pH and constant Ψ as a function of temperature between 5°C and 45°C. The activation energy was 55 kJ mol−1.

CF0F1 was coreconstituted with bacteriohodopsin at a molar ratio of approximately 1:170 in the same type of liposomes. Illumination of the proteoliposomes leads to proton transport into the vesicles generating a constant pH = 1.8. The dependence of the rate of ATP synthesis on ADP concentration was measured with CF0F1 in the oxidized state, Eox, and in the reduced state, Ered. The results can be described by Michaelis‐Menten kinetics with the following parameters: Vmax= 0.5 s−1, Km= 8 μLM for Eox and Vmax= 2.0 s−1, Km= 8 μLM for Ered.
1H‐NMR study on the chitotrisaccharide binding to hen egg white lysozyme
Authors: FUKAMIZO Tamo , IKEDA Yasuo , OHKAWA Takeshi , GOTO Sachio
Published: March 2005

Interaction between hen egg white lysozyme and chitotrisaccharide was investigated by 1H‐NMR spectroscopy using partially acetylated chitotrisaccharides and chemically modified lysozyme. Monoacetyl (GlcN‐GlcN‐GlcNAc), diacetyl (GlcN‐GlcNAc‐GlcNAc), or triacetyl chitotrisaccharide [(GlcNAc)3] was added to the lyozyme solution, and the changes in the 1H‐NMR signals of the lysozyme were analyzed. Although many of the resonances were affected by addition of the saccharide, the most remarkable effect was seen on the signal of Trp28 C5H which is in a hydrophobic box adjacent to the saccharide‐binding site. The signal shifted upfield by 0.2 ppm upon (GlcNAc)3 binding, whereas the chemical shift change of the signal resulting from binding of GlcN‐GlcNAc‐GlcNAc or GlcN‐GlcN‐GlcNAc was smaller than that resulting from (GlcNAc)3 binding. When the Asp101‐modified lysozyme was used instead of the native lysozyme, the chemical shift change of the Trp28 C5H signal resulting from (GlcNAc)3 binding was also smaller than that for the native lysozyme. The chemical shift change of the signal reflects the conformational change of the hydrophobic box region which should synchronize with the movement of the binding site resulting from the saccharide binding. Therefore, the conformational change resulting from the saccharide binding might be reduced when the sugar residues located at binding subsites A and B of the lysozyme are deacetylated, as well as when Asp101 interacting with the sugar residues at the same subsites is modified.
Insulin signalling and regulation of glucokinase gene expression in cultured hepatocytes
Authors: NOUSPIKEL Thierry , IYNEDJIAN Patrick B.
Published: March 2005

In cultured rat hepatocytes, transcription of the glucokinase‐gene is turned on by insulin and turned off by glucagon/cAMP, the latter being the dominant effector system. It is thus possible that in the absence of hormones the gene is maintained in a repressed state by the basal level of cAMP and that insulin turns on transcription by relieving cAMP repression, for instance via activation of a cyclic‐nucleotide phosphodiesterase. Three inhibitors of this class of enzymes were tested for their effect on the insulin‐dependent induction of the glucokinase gene in hepatocytes. Isobutyl methylxanthine, the prototype inhibitor, abrogated the gene response to insulin, as shown by run‐on transcription assay. Among the drugs investigated, Ly186126, a preferential inhibitor of type‐III phosphodiesterase, proved the most potent in inhibiting insulin‐induced accumulation of glucokinase mRNA. Type‐III phosphodiesterase is inhibited by cGMP. Induction of glucokinase mRNA was prevented in hepatocytes challenged with insulin in presence of 8‐bromoguanosine‐3′,5′‐phosphate. These results are consistent with the involvement of type‐III phosphodiesterase in transduction of the insulin signal to the glucokinase gene. However, we were unable to detect significant decreases in total cellular cAMP level or cAMP‐dependent‐protein‐kinase ratio after the addition of insulin to hepatocytes. Many effects of glucagon are mediated via cAMP‐dependent protein‐kinase phos‐phorylation of regulatory proteins and, conversely, insulin effects are often accompanied by protein dephosphorylation. A specific inhibitor of protein phosphatases PP1 and PP2A, okadaic acid, was shown to abolish the transcriptional response of the glucokinase gene to insulin. Thus, interference of insulin with the cAMP signal transduction pathway at several steps may be a critical aspect of insulin action on hepatic glucokinase gene expression. In addition, insulin induction of glucokinase mRNA was suppressed by inhibitors of protein synthesis. The underlying mechanism was a severe inhibition of the transcriptional effect of insulin, rather than mRNA destabilization, as demonstrated by run‐on transcription assays with nuclei from cycloheximide‐treated or pactamycin‐treated cells. Transcription of the glucokinase gene may therefore depend on de novo synthesis of the product of an early‐response gene induced by insulin, or may require a short‐lived trans‐acting or accessory factor of transcription. Alternatively, insulin signalling may be compromised in hepatocytes by a mechanism indirectly related to the arrest of protein synthesis.
The effect of driving force on intramolecular electron transfer in proteins
Authors: FARVER Ole , SKOV Lars K. , VAN DE KAMP Mart , CANTERS Gerard W. , PECHT Israel
Published: March 2005

An intramolecular electron‐transfer process has previously been shown to take place between the Cys3‐Cys26 radical‐ion (RSSR−) produced pulse radiolytically and the Cu(II) ion in the blue single‐copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad. Sci. USA 86, 6868–6972]. To further investigate the nature of this long‐range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single‐site mutation of the wild‐type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu‐coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of the copper site and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site.

The rate of intramolecular electron transfer from RSSR− to Cu(II) in the wild‐type P. aeruginosa azurin (G°=−68.9 kJ/mol) has previously been determined to be 44 ± 7 s−1 at 298 K, pH 7.0. The [M44K]azurin mutant (G°=− 75.3 kJ/mol) was now found to react considerably faster (k= 134 ± 12 s−1 at 298 K, pH 7.0) while the [H35Q]azurin mutant (G°=− 65.4 kJ/mol) exhibits, within experimental error, the same specific rate (k= 52 ± 11 s−1, 298 K, pH 7.0) as that of the wild‐type azurin. From the temperature dependence of these LRET rates the following activation parameters were calculated: H≠= 37.9 ± 1.3 kJ/mol and 47.2 ± 0.7 kJ/mol and S≠=−86.5 ± 5.8 J/mol · K and ‐46.4 ± 4.4 J/mol · K for [H35Q]azurin and [M44K]azurin, respectively. Using the Marcus relation for intramolecular electron transfer and the above parameters we have determined the reorganization energy, λ and electronic coupling factor, β. The calculated values fit very well with a through‐bond LRET mechanism.
Characterization of peptides related to neuropeptide tyrosine and peptide tyrosine‐tyrosine from the brain and gastrointestinal tract of teleost fish
Authors: JENSEN Jörgen , CONLON J. Michael
Published: March 2005

Neuropeptide Y was isolated from the brain of the Atlantic cod, Gadus morhua and its primary structure established as Tyr‐Pro‐Ile*‐Lys‐Pro‐Glu*‐Asn‐Pro‐Gly‐Glu10‐Asp‐Ala‐Pro‐Ala‐Ala‐Asp*‐Glu*‐Leu*‐Ala‐Lys*‐Tyr20‐Tyr‐Ser‐Ala‐Leu‐Arg‐His‐Tyr‐Ile‐Asn‐Leu30‐Ile‐Thr‐Arg‐Gln‐Arg‐Tyr‐CONH2. Residues denoted by an asterisk are different from the corresponding sequence of human neuropeptide Y. A structurally similar peptide was isolated from the brain of the trout, Oncorhynchus mykiss. Trout neuropeptide Y contains four substitutions (Ile3→Val, Ala14→Thr, Asp15→Glu and Ser22→Thr) compared with cod neuropeptide Y. A second peptide of the neuropeptide Y family was identified in the trout brain and this component was structurally similar to peptide tyrosine‐tyrosine previously isolated from frog intestine (six amino acid substitutions) and identical to a peptide isolated from the pancreas of the closely related species, Oncorhynchus kisutch (Coho salmon). Peptide tyrosine‐tyrosine, with the same primary structure as the brain peptide, was also isolated from an extract of the trout stomach. The data indicate that a peptide analogous to mammalian neuropeptide Y is present in the brain of teleost fish and a peptide analogous to mammalian peptide tyrosine‐tyrosine is present in brain, gastrointestinal tissue and pancreas. We speculate, therefore, that the putative gene duplication that led to pancreatic polypeptide in the higher vertebrates took place after the time of divergence of fish and tetrapods.