bluuebunny/crossref_metadata_2025 huggingface dataset:
12α‐Hydroxysteroid dehydrogenase from Clostridium group P, strain C 48–50
Authors: BRAUN Manfred , LÜNSDORF Heinrich , BÜCKMANN Andreas F.
Published: March 2005

NADP(H)‐dependent 12α‐hydroxysteroid dehydrogenase (HSDH) from Clostridium group P, strain C 48–50, is still expressed at unusual high level (approximately 1% of total protein) under cultivation conditions where the usual expensive brain/heart infusion complex medium is replaced by inexpensive technical grade yeast autolysate. An inexpensive anaerobic bioprocess for the production of HSDH was developed provisionally up to 900‐1 scale (9000 U/l, 7 g HSDH, specific activity 1.0 U/mg crude protein, 55 U/g wet cells). By a simple two‐step affinity chromatography procedure, easily adaptable to a large‐scale operation, using columns of small dimensions of Sephacryl‐S‐400–Procion‐orange‐P‐2R (5 cm × 28 cm) and Sephacryl‐S‐400—Procion‐red‐HE‐7B (2.6 cm × 14 cm) approximately 140 mg (1.8 × 104 U), HSDH was purified to apparent homogeneity and concentrated directly from a crude cell extract (overall yield 53%, specific activity 128 U/mg). As confirmed by fast native and SDS/PAGE, isoelectric focussing and electron microscopy, HSDH has a molecular mass of approximately 105 kDa and consists of four flattened tetrahedrically arranged identical subunits (26 kDa). The enzyme exhibits a rather low isoelectric point of 4.6, a pH optimum of 8.5–9.5 and a temperature optimum of approximately 55°C for the oxidation of cholic acid. Inhibition by SH reagents and pyridoxal 5′‐phosphate has been observed. Chelating agents have no inhibitory effect. The presence of NADP increases considerably the thermostability (t1/2 4—10 d, 25°C; 2—5 d, 37°C). Steady‐state kinetic analysis for both reaction directions indicated that the reaction proceeds through an ordered bi bi mechanism with NADP(H) binding first to the free enzyme. Km, Vmax [forward (Vf) and reverse reactions (Vr)] and the dissociation constants Kd for the binary complexes with NADP and NADPH were as follows. NADP, Km= 35 μm, Kd= 35 μm; cholic acid, Km= 72 μm; deoxycholic acid, Km= 45 μm, Vf= 160 U/mg; NADPH, Km= 8.5 μm, Kd= 16 μm; 12‐oxochenodeoxycholic acid, Km= 12 μm, V=r= 66 U/mg (conditions, 0.1 M potassium phosphate, pH 8.0, 25°C). N6‐functionalized NADP derivatives, e.g. N6‐(2‐aminoethyl)NADP (Km= 4.5 mM) are poorly accepted as coenzyme by HSDH.
Methanol esterification reactions catalyzed by snake venom and bovine intestinal 5′‐nucleotide phosphodiesterases
Authors: GARCIA‐DIAZ Miguel , AVALOS Martin , CAMESELLE José Carlos
Published: March 2005

It is not known whether the enzymes 5′‐nucleotide phosphodiesterase/nucleotide pyrophosphatase (EC 3.1.4.1/EC 3.6.1.9) catalyze the transfer of nucleotides to acceptors other than water. We have investigated the action of snake venom and bovine intestinal mucosa phosphodiesterases on nucleoside 5′‐polyphosphates in the presence of methanol. In those conditions, GTP was converted by snake venom phosphodiesterase to a mixture of GMP and another compound with a different retention time in reverse‐phase high‐performance liquid chromatography. That compound, by ultraviolet, 1H‐ and 13C‐nuclear magnetic resonance spectroscopic analysis, and by enzyme analysis, was characterized as the methyl ester of GMP (GMP‐OMe). The molar fraction [GMP‐OMe]/ [GMP + GMP‐OMe] formed was higher than the molar fraction of methanol as a solvent in reaction mixtures. Similar reactions took place at comparable rates with snake venom and bovine intestinal mucosa phosphodiesterases using several nucleoside 5′‐polyphosphates as substrates. The ability of 5′‐nucleotide phosphodiesterases to catalyze transfer reactions to a non‐water acceptor is relevant to the mechanism of the enzymes, to their use as analytical tools, and to their possible use/role in the preparative/in vivo synthesis of nucleotide esters.
Glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus
Authors: CONSALVI Valerio , CHIARALUCE Roberta , POLITI Laura , GAMBACORTA Agata , De ROSA Mario , SCANDURRA Roberto
Published: March 2005

An NAD(P)‐dependent glutamate dehydrogenase was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme is a hexamer (subunit mass 45 kDa) which dissociates into lower states of association when submitted to gel filtration. Isoelectric focusing analysis of the purified enzyme showed a pI of 5.7 and occasionally revealed microheterogeneity. The enzyme is strictly specific for the natural substrates 2‐oxoglutarate and l‐glutamate, but is active with both NADH and NADPH. S. solfataricus glutamate dehydrogenase revealed a high degree of thermal stability (at 80°C the half‐life was 15 h) which was strictly dependent on the protein concentration. Very high levels of glutamate dehydrogenase were found in this archaebacterium which suggests that the conversion of 2‐oxoglutarate and ammonia to glutamate is of central importance to the nitrogen metabolism in this bacterium.
Isolation and characterization of catalytic and calmodulin‐binding domains of Bordetella pertussis adenylate cyclase
Authors: MUNIER Hélène , GILLES Anne‐Marie , GLASER Philippe , KRIN Evelyne , DANCHIN Antoine , SARFATI Robert , BÂRZU Octavian
Published: March 2005

A truncated Bordetella pertussis cya gene product was expressed in Escherichia coli and purified by affinity chromatography on calmodulin‐agarose. Trypsin cleavage of the 432‐residue recombinant protein (Mr= 46 659) generated two fragments of 28 kDa and 19 kDa. These fragments, each containing a single Trp residue, were purified and analyzed for their catalytic and calmodulin‐binding properties. The 28‐kDa peptide, corresponding to the N‐terminal domain of the recombinant adenylate cyclase, exhibited very low catalytic activity, and was still able to bind calmodulin weakly, as evidenced by using a fluorescent derivative of the activator protein. The 19‐kDa peptide, corresponding to the C‐terminal domain of the recombinant adenylate cyclase, interacted only with calmodulin as indicated by a shift in its intrinsic fluorescence emission spectrum or by the enhancement of fluorescence of dansyl‐calmodulin. T28 and T19 fragments exhibited an increased sensitivity to denaturation by urea as compared to uncleaved adenylate cyclase, suggesting that interactive contacts between ordered portions of T28 and T19 in the intact protein participate both in their own stabilization and in stabilization of the whole tertiary structure. The two fragments reassociated into a highly active calmodulin‐dependent species. Reassociation was enhanced by calmodulin itself, which ‘trapped’ the two complementary peptides into a stable, native‐like, ternary complex, which shows similar catalytic properties to intact adenylate cyclase.
Comparison of the membrane transport of anthracycline derivatives in drug‐resistant and drug‐sensitive K562 cells
Authors: FREZARD Frédéric , GARNIER‐SUILLEROT Arlette
Published: March 2005

One of the phenotypes of multidrug resistance is characterized by a decrease in the intracellular concentration of drug in resistant cells as compared to sensitive cells. This is correlated with the presence in the membrane of resistant cells of a 150–180‐kDa glycoprotein, P‐glycoprotein, responsible for an active efflux of the drug. The fluorescence emission spectra from anthracycline‐treated cells suspended in buffer have been used to compare the membrane transport of five anthracycline derivatives: adriamycin, daunorubucin, 4′‐o‐tetrahydropyranyladriamycin, carminomycin and aclacinomycin in drug‐sensitive and drug‐resistant K562 cells. The initial rate of uptake of these five drugs has been measured as a function of the extracellular pH, pHe. The data show that the uptake occurs through free permeation of the neutral form of the drug. For each drug an influx coefficient kpHe, characteristic of the drug and of the cell type has been defined and calculated: k+7.2=V+/[D]e·n where V+ and [D]e are the initial rate of uptake and the concentration of drug in the medium at pHe= 7.2 respectively and n is the number of cells. This coefficient is characteristic of a passive diffusion of the neutral form of the drug through the lipid bilayer. Efflux coefficients k−7.2 at pHi= 7.2 (the intracellular pH value) have also been calculated. In the case of sensitive cells, k+7.2 and k−7.2 are equal. For resistant cells, the efflux coefficient is composed of two terms: (a) (k−)p corresponding to the passive diffusion of the neutral form of the drug and (k−)p) =k+; (b) (k−)a corresponding to an active efflux mediated by the P‐glycoprotein. Our data suggest that the anthracycline derivatives efflux actively in the neutral form.
Effect of dietary fish oil and corn oil on lipid metabolism and apolipoprotein gene expression by rat liver
Authors: RIBEIRO Agnès , MANGENEY Marise , CARDOT Philippe , LORIETTE Claude , RAYSSIGUIER Yves , CHAMBAZ Jean , BEREZIAT Gilbert
Published: March 2005

A 3‐week fish oil diet induced in weanling rats a decrease in plasma lipids and liver triacylglycerol, and an increase in insulinemia, compared to a corn oil diet. At the same time, plasma apolipoprotein (apo) A‐I was slightly lower and plasma heavy apo B/light apo B ratio was higher in fish‐oil‐fed than in corn‐oil‐fed rats. Hepatocytes obtained from fish‐oil‐fed and corn‐oil‐fed rats were used to examine how fish oil affects lipid and apolipoprotein synthesis and secretion. Primary culture of hepatocytes from fish‐oil‐fed rats displayed a lower ability to synthesize and secrete triacylglycerol than hepatocytes from corn‐oil‐fed rats, as measured by mass determination or [U‐14C]glycerol incorporation. Hepatocytes from fish‐oil‐fed rats exhibited a lower synthesis of cholesterol, measured by [14C]acetate incorporation, than hepatocytes from corn‐oil‐fed rats. This impairment was associated with an increase in β‐oxidation, a higher channeling of oleic acid into phospholipids, and a lower triacylglycerol/diacylglycerol ratio in hepatocytes from fish‐oil‐fed rats than in hepatocytes from corn‐oil‐fed rats. Incorporation of [35S]methionine into secreted apoB was reduced in hepatocytes from fish‐oil‐fed rats, but was not paralleled by a decrease in apo B mRNA. The appearance of degradative forms of apo B suggest an increase in apo B degradation in hepatocytes from fish‐oil‐fed rats. Incorporation of [35S]methionine into cellular and secreted apo A‐I was lower in hepatocytes from fish‐oil‐fed rats than in hepatocytes from corn‐oil‐fed rats, and was not paralleled by any difference in the apo A‐I mRNA level. Finally, [35S]methionine incorporation into cellular and secreted forms of apo E and apo A‐I mRNA were reduced in hepatocytes from fish‐oil‐fed rats, compared with hepatocytes from corn‐oil‐fed rats. These combined data show that fish oil diet reduces triacylglycerol synthesis and secretion and affects apo B synthesis at a post‐transcriptional level, and reduces cholesterol synthesis and affects apo E and apo A‐I synthesis at a transcriptional and a post‐transcriptional level.
Differences in primary structure among five phospholipases A2 from Heloderma suspectum
Authors: VANDERMEERS André , VANDERMEERS‐PIRET Marie‐Claire , VIGNERON Laurence , RATHE Jean , STIEVENART Michel , CHRISTOPHE Jean
Published: March 2005

Five increasingly anionic phospholipases A2 (Pa1– Pa5) exist in the venom of the lizard Heloderma suspectum. We recently elucidated the sequence of Pa5, the most abundant and most active variant, towards emulsfied phosphatidylcholines. Here we present the primary structures of Pa2, Pa3 (subvariants a and b) and Pa4, based on Edman degradation of tryptic, endoproteinase Arg‐C and chymotryptic fragments of the reduced and S‐carboxymethylated proteins. Pa1– Pa5, considered collectively, belong to an original class of secretory phospholipases A2 with 141 – 143 residues, a short hydrophobic N‐terminus, 10 half‐cystine residues and an extended C‐terminus. The only known phospholipase A2 with characteristics close enough to be a member of the same class is that present in the venom from the insect Apis mellifera.

More specifically, the sequences of Pa3 and Pa5 are almost identical, and those of Pa2 and Pa4 are also quite similar. Both groups diverge enough to indicate the translation of two mRNA species in the venom gland. The primary structure of Pa3 reveals the existence of subvariants a and b, the sequence of which is identical to that previously defined for Pa5, except that the C‐terminal tripeptide GEG in Pa5 is replaced by the dipeptide GE in Pa3a and the tetrapeptide GEGR in Pa3b. Pa4, when compared to Pa5, shows 21 substitutions with a cluster of five modified amino acids in positions 40 – 44, immediately after the catalytic segment amino acids 30 – 39, and added changes scattered before the C‐terminus. Pa2 differs from Pa4 only by the absence of the Gly142 C‐terminal residue. The 15% difference in primary structure observed between the Pa3– Pa5 and Pa2– Pa4 subgroups might be largely responsible for their distinct biological properties.
Activation and induction by copper of Cu/Zn superoxide dismutase in Saccharomyces cerevisiae
Authors: GALIAZZO Francesca , CIRIOLO Maria Rosa , CARRÌ Maria Teresa , CIVITAREALE Patrizia , MARCOCCI Lucia , MARMOCCHI Franco , ROTILIO Giuseppe
Published: March 2005

The Cu/Zn superoxide dismutase activity of Saccharomyces cerevisiae was found to be strictly related to the extent of oxygen metabolism, since cells grown under anaerobic or repressed conditions were found to contain 10% and 40% the activity of derepressed cells, respectively. The dependence of Cu/Zn superoxide dismutase on oxygen was found to be related to the availability of copper to the cells since the enzyme activity and immunoreactive protein measured under the various conditions was roughly proportional to the copper content of cells and in anaerobic cells a large fraction of the enzyme was found to be in the form of an inactive proenzyme which was activated by the addition of copper to cell extracts. The Cu/Zn superoxide dismutase mRNA did not parallel the dependence of the enzyme concentration on oxygen metabolism, suggesting that the gene expression was affected by copper also at the post‐transcriptional level. However, under conditions of copper overloading, a more direct effect on transcription was observed and the presence of the inactive proenzyme in anaerobic cultures was associated with the over‐expression of metallothionein.
Isolation of human type X collagen and immunolocalization in fetal human cartilage
Authors: KIRSCH Thorsten , VON DER MARK Klaus
Published: March 2005

Type X Collagen was extracted with 1 M NaCl and 10 mM dithiothreitol at neutral pH from fetal human growth plate cartilage and purified to homogeneity by gel filtration and anion‐exchange chromatography. The purified protein migrates in SDS/polyacrylamide gels with an apparent Mr of 66000 under reducing conditions, and as a high‐Mr oligomer under non‐reducing conditions. Purified collagenase digests most of the molecule; pepsin digestion at 4°C decreases the Mr of the monomer to 53000. A rabbit antiserum was raised against purified human type X collagen; the IgG fraction was specific for this collagen by criteria of ELISA and immunoblotting after absorption with collagen types I, II, VI, IX and XI. Immunohistological studies localized type X collagen exclusively in the zone of hypertrophic and calcifying cartilage.
Characterization of a new cobalt‐containing nitrile hydratase purified from urea‐induced cells of Rhodococcus rhodochrous J1
Authors: NAGASAWA Toru , TAKEUCHI Koji , YAMADA Hideaki
Published: March 2005

A new cobalt‐containing nitrile hydratase was purified from extracts of urea‐induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500–530‐kDa protein composed of two different subunits (α subunit 26kDa, β subunit 29kDa). The enzyme contained approximately 11–12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5–6.8. The enzyme was more stable than ferric nitrile hydratases. The amino‐terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the β subunit. However, the amino acid sequence of the α subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes.
Characterization of the 12S globulin complex of Brassica napus
Authors: SJÖDAHL Staffan , RÖDIN Joakim , RASK Lars
Published: March 2005

Cruciferin (125 globulin) is the major seed protein in Brassica napus (oil seed rape). It is synthesized during seed development and consists of six subunit pairs. Each of these pairs is synthesized as a precursor containing one α and one β chain. Atleast three different precursors exist (P1–3), giving rise to four different mature subunits (cru1–4). Several cruciferin clones were isolated from a seed mRNA cDNA library. Comparison of the deduced amino acid sequences of these clones to amino acid sequences of purified cruciferin chains and peptides identified them as coding for cru2/3 and cru4 subunits. From the amino acid sequences deduced from two overlapping cDNA clones, the precursor of the cru4 subunit was shown to consist of 465 amino acid residues. Comparison of cruciferin and cruciferin‐related sequences from B. napus and Arabidopsis thaliana, respectively, suggested that early during evolution the Brassicaceae family only possessed two types of 11–12S globulin genes, like the present‐day Fabaceae.
Production and purification of recombinant human interleukin‐5 from yeast and baculovirus expression systems
Authors: INGLEY Evan , CUTLER Robert L. , FUNG Ming‐Chiu , SANDERSON Colin J. , YOUNG Ian G.
Published: March 2005

A cDNA for human interleukin‐5 (hIL‐5) was created from the hIL‐5 gene using site‐directed mutagenesis to splice out the introns in vitro. This cDNA was expressed in yeast and baculovirus systems, utilizing in both cases an in‐frame fusion to the pre sequence of the α‐mating‐type factor to direct secretion. The highest level of production was achieved from Sf9 cells using a baculovirus vector in serum‐containing medium (2.7 mg/l), whereas in serum‐free medium ten times less hIL‐5 was produced. In the yeast system much lower levels of hIL‐5 were produced (12.5 μg/l). Recombinant hIL‐5 was purified to homogeneity from serum‐free baculovirus cultures. The rhIL‐5 consisted of a 30‐kDa homodimer linked by disulfide bridging. The purified recombinant protein had a specific activity on murine BCL1 cells of 1.5x104 U/mg, of 3x105 U/mg in the murine eosinophil differentiation factor assay, and 2.4x107 U/mg in a human peripheral eosinophil maintenance assay.
The amino acid sequence of peanut agglutinin
Authors: YOUNG N. Martin , JOHNSTON Rosemary A. Z. , WATSON David C.
Published: March 2005

The amino acid sequence of peanut (Arachis hypogaea) agglutinin was determined from three major fragments obtained by mild acid cleavage at Asp‐Pro peptide bonds. The sequence of 236 amino acids has residues identical to those that form the metal‐binding site and the hydrophobic pocket in concanavlin A and other lectins, although the overall similarity is only 42%. In the segments of peanut agglutinin that correspond to the four loops that form the carbohydrate‐binding site in concanavalin A and favin, several central residues are homologous, while others show changes to smaller side chains, such as Tyr → Gly. The carbohydrate‐binding site of peanut agglutinin may therefore have a similar peptide‐backbone architecture, but form a considerably more open cleft.
Binding and toxicity of apamin
Authors: LABBÉ‐JULLIÉ Catherine , GRANIER Claude , ALBERICIO Fernando , DEFENDINI Maria‐Léria , CEARD Brigitte , ROCHAT Hervé , VAN RIETSCHOTEN Jurphaas
Published: March 2005

The structural features of apamin, a natural octadecapeptide from bee venom, enabling binding to its receptor and the expression of toxicity in mice, have been delineated by studying the effects on binding and toxicity of chemical modifications and amino acid substitutions in synthetic analogues. The results obtained indicate that the only hydrophobic residue, leucine at position 10, can be changed to alanine without a significant decrease in the specific activity. The need for a correct conformation has been established and also the importance of Gln‐17 and the side chains of Arg‐13 and Arg‐14 (besides the charge effects). The interaction of apamin with its receptor, a calcium‐activated potassium channel, is thus mediated by a precise topology around these three residues. Due to the ability to detect very low specific activities for some of the analogues, it has been shown that, individually, none of these interactions constitute an essential criteria for binding per se, but that their presence is necessary for the high specific activity of the toxin.
A spectroscopic analysis of the Pro35 → Ala mutant of Rhodobacter capsulatus cytochrome c2
Authors: GOOLEY Paul R. , CAFFREY Michael S. , CUSANOVICH Michael A. , MACKENZIE Neil E.
Published: March 2005

Visible, near‐ultraviolet circular dichroic, near‐infrared and nuclear magnetic resonance spectroscopies show that the secondary and tertiary structures of the mutant Pro35 → Ala Rhodobacter capsulatus ferrocytochrome c2 are similar to the wild‐type protein. The near‐infrared spectrum shows that the methionine‐S – Fe‐heme bond is intact; however, a small red shift in the heme M transition of the near‐ultraviolet circular dichroic spectrum of the mutant indicates that the heme environment may differ slightly between the two proteins. This difference may be a consequence of changes in the ligand and hydrogen bonds of His17 [Gooley, P. R. & MacKenzie, N. E. (1990) FEBS Lett. 260, 225–228]. 1H and 15N chemical shift differences suggest that the microenvironment of pyrrole rings III and IV of the heme prosthetic group differs between the two proteins. As the rings of the Phe51 and Tyr53 flip faster in the mutant protein than the wild type, these chemical shift differences may reflect changes in the time‐average ring‐current effects and not structural alterations.
15N‐ and 13C‐NMR investigations of glucose oxidase from Aspergillus niger
Authors: SANNER Christoph , MACHEROUX Peter , RÜTERJANS Heinz , MÜLLER Franz , BACHER Adalbert
Published: March 2005

The apoprotein of glucose oxidase from Aspergillus niger was reconstituted with specifically 15N‐ and 13C‐enriched FAD derivatives and investigated by 15N‐ and 13C‐NMR spectroscopy. On the basis of the 15N‐NMR results it is suggested that, in the oxidized state of glucose oxidase, hydrogen bonds are formed to the N(3) and N(5) positions of the isoalloxazine system. The hydrogen bond to N(3) is more pronounced than that to N(5) as compared with the respective hydrogen bonds formed between FMN and water. The resonance position of N(10) indicates a small decrease in sp2 hybridization compared to free flavin in water. Apparently the isoalloxazine ring is not planar at this position in glucose oxidase.

Additional hydrogen bonds at the carbonyl groups of the oxidized enzyme‐bound FAD were derived from the 13C_NMR results. A strong downfield shift observed for the C(4a) resonance may be ascribed in part to the decrease in sp2 hybridization at the N(10) position and to the polarization of the carbonyl groups at C(2) and C(4). The polarization of the isoalloxazine ring in glucose oxidase is more similar to FMN in water than to that of tetraacetyl‐riboflavin in apolar solvents.

In the reduced enzyme the N(1) position is anionic at pH 5.6. The pKa is shifted to lower pH values by at least 1 owing to the interaction of the FAD with the apoprotein.

As in the oxidized state of the enzyme, a hydrogen bond is also formed at the N(3) position of the reduced flavin. The N(5) and N(10) resonances of the enzymes‐bound reduced FAD indicate a decrease in the sp2 character of these atoms as compared with that of reduced FMN in aqueous solution.

Some of the 15N‐ and 13C‐resonance positions of the enzyme‐bound reduced cofactor are markedly pH‐dependent. The pH dependence of the N(5) and C(10a) resonances indicates a decrease in sp2 hybridization of the N(5) atom with increasing pH of the enzyme solution.
Monoclonal antibodies that inhibit binding of propolypeptide of von Willebrand factor to collagen
Authors: FUJISAWA Tomoyuki , TAKAGI Junichi , SEKIYA Fujio , GOTO Akira , MIAKE Fumio , SAITO Yuji
Published: March 2005

We reported previously that bovine propolypeptide of von Willebrand factor (pp‐vWF) binds to type I collagen. To determine the collagen‐binding sites of pp‐vWF we generated monoclonal antibodies (mAbs) against bovine pp‐vWF. One mAb, designated TC8, very strongly inhibited the binding of pp‐vWF to type I collagen; three other mAbs, designated TC2, TC6 and TC7, exhibited moderate inhibition. Competition between the mAbs for binding to intact pp‐vWF revealed that the epitope for TC8 was structurally independent of that for TC6 and TC7. To determine directly the location of the epitope for each mAb on the bovine pp‐vWF molecule, we tested the reactivity of mAbs by immunoblotting toward peptide fragments obtained by digestion with lysylendopeptidase. TC2 and TC8 recognized a fragment of mass 21 kDa, while Tc6 and TC7 recognized a distinct fragment of 18 kDa. These two fragments were purified to homogeneity and their N‐terminal amino acid sequences were determined. Comparing these sequences with the sequence of human pp‐vWF, the locations of these fragments in the primary structure were estimated to be Phe570–Lys682 for the 21‐kDa fragment and Glu281–Lys375 for the 18‐kDa fragment. These data suggest that pp‐vWF contains at least two collagen‐binding sites which lie within or close to the regions between Phe570–Lys682 and Glu281–Lys375.
Enzyme‐mediated peptide synthesis using acylpeptide hydrolase
Authors: FARRIES Timothy C. , AUFFRET Anthony D. , AITKEN Alastair
Published: March 2005

Acylpeptide hydrolase is shown to catalyse the specific addition of a single amino acid to the N‐terminus of a peptide. The stabilised Sepharose‐coupled form of the enzyme is used to couple a carboxy‐methylated N‐formyl (or N‐acetyl) amino acid to a short pre‐existing peptide. The yield is improved by optimal timing of the reaction and the presence of moderate concentrations (5%) of N, N‐dimethylformamide. Two tripeptides, Ac‐Ala‐Ala‐Ala and fMet‐Leu‐Phe (f, formyl) were synthesized by this technique (in yields of 2% and 0.064% respectively). The products were characterised by HPLC, amino acid analysis, mass spectroscopy and protein sequencing. The synthetic fMet‐Leu‐Phe also had biological activity, in that it stimulated superoxide generation by granulocytes. Acylpeptide hydrolase could therefore be a very useful tool for the synthesis and modification of peptides.
Some structural features of the iron‐uptake regulation protein
Authors: SAITO Takeshi , WORMALD Mark R. , WILLIAMS Robert J. P.
Published: March 2005

An extensive proton nuclear magnetic resonance study of the iron‐uptake regulation protein (Fur) from Escherichia coli has been made. Considerable difficulties were experienced in the NMR experiments in 1H2O which may be due unfavourable proton exchange rates in the pH range > 6.2, where the protein is soluble. Even in 2H2O, the two‐dimensional NMR spectra were not easily interpreted due to widely differing line widths, as a result of the protein side‐chains having very differing mobilities. Despite these problems, virtually all the 20 aromatic amino acids have been assigned. Small regions of the protein core were assigned by taking advantage of the approximately 20 non‐exchanging peptide‐NH resonances in 2H2O. Using two‐dimensional J‐correlated, homonuclear Hartmann‐Hahn and NOE spectroscopies, we have been able to give some assignments in which there is considerable confidence for about one third of the amino acids. Taking advantage of two series of probe experiments, using Mn(II) and a spin label, together with longer range NOE data and results from structure predictions and CD data, we have put forward a tentative fold for the protein which is seen to have a relatively rigid series of interior strands and more flexible exterior strands, many of which are likely to be helical. The Mn(II) probe experiments have also allowed us to define the Fe(II) binding site.
Monoclonal antibodies specific for the core protein of the β‐subunit of the gastric proton pump (H+/K+ ATPase)
Authors: JONES Claerwen M. , TOH Ban‐Hock , PETTITT John M. , MARTINELLI Teresa M. , HUMPHRIS Danielle C. , CALLAGHAN Judy M. , GOLDKORN Isabela , MU Fi‐Tjen , GLEESON Paul A.
Published: March 2005

The gastric H+/K+ ‐transporting adenosine triphosphatase (H+/K+ ATPase) (proton pump) consists of a catalytic α‐subunit and a recently proposed 60–90‐kDa glycoprotein β‐subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60–90‐kDa glycoprotein.

The monoclonal antibodies (1) specifically stained the cytoplasm of unfixed and formalin‐fixed dog gastric parietal cells; (2) specifically reacted by ELISA with gastric tubulovesicular membranes; (3) recognised epitopes located on the luminal face of parietal cell tubulovesicular membranes, the site of the proton pump, by immunogold electron microscopy; (4) immunoblotted a 60–90‐kDa molecule from tubulovesicular membranes and a 35‐kDa component from peptide N‐glycosidase‐F‐treated membrane extracts; (5) immunoblotted the 60–90‐kDa parietal cell autoantibody‐ or tomato‐lectin–Sepharose 4B affinity columns, and the 35‐kDa protein core of this autoantigen; this autoantigen has amino acid sequence similarity to the β‐subunit of the related NH+/K+ ‐transporting adenosine triphosphatase (Na+/K+ ATPase) [Toh et al. (1990) Proc. Natl Acad. Sci. 87, 6418–6422]; (6) co‐precipitated a molecule of 95kDa with the 60–90‐kDa molecule from 125I‐labelled detergent extracts of dog tubulovesicular membranes; and (7) co‐purified the catalytic α‐subunit of the H+/K+ ATPase with the 60–90‐kDa molecule by immunoaffinity chromatography of tubulovesicular membrane extracts on a monoclonal antibody 3A6‐Sepharose 4B column, indicating a physical association between the two molecules.

These results provide further evidence that the 60–90‐kDa glycoprotein is the β‐subunit of the gastric H+/K+ ATPase. We conclude that the monoclonal antibodies specifically recognise luminal epitopes on the 35‐kDa core protein of the 60–90‐kDa β‐subunit of the gastric proton pump, a major target molecule in autoimmune gastritis and pernicious anaemia. These monoclonal antibodies will be valuable probes to study the structure and function of this associated β‐subunit, as well as the ontogeny of the gastric proton pump.
Intracellular modifications of rat α1inhibitor3
Authors: SJÖBERG Mathilda , ESNARD Frédéric , FRIES Erik
Published: March 2005

α1 Inhibitor3 (α1I3) is a monomeric protease inhibitor of about 190 kDa which is secreted by rodent hepatocytes. We have studied intracellular modifications of this protein in [35S]methionine‐labelled rat hepatocytes by pulse/chase experiments followed by immunoprecipitation and gel electrophoresis under reducing and nonreducing conditions. Directly after the pulse, most of the unreduced α1I3 migrated faster than the reduced form, indicating that disulphide bridges are formed during or shortly after synthesis yielding a compact structure. With increasing chase time however, an increasing portion of the unreduced α1I3 migrated with a mobility lower than that of the reduced protein, half‐maximal conversion occurring after about 10 min. This finding suggests that α1I3 undergoes a conformational change in the endoplasmic reticulum, possibly becoming more elongated. During 10–30 min of chase, the protein acquired the capacity to undergo autolytic cleavage upon heating, a property due to the existence of an internal thiolester bond [Howard, J. B., Vermeulen, M. & Swenson, R. P. (1980) J. Biol. Chem. 255, 3820–3823; Esnard, F., Gutman, N., El Moujahed, A. & Gauthier, F. (1985) FEBS Lett. 182, 125–129]. Analysis by subcellular fractionation indicated that this bond is formed in the endoplasmic reticulum. Finally, we show that secreted α1I3 is sulphated, presumably at Tyr618.
Characterisation of glutamine uptake in rat liver mitochondria
Authors: SOBOLL Sibylle , LENZEN Christoph , RETTICH Dirk , GRÜNDEL Sonja , ZIEGLER Brigitte
Published: March 2005

Glutamine is taken up by rat liver mitochondria in an electroneutral manner with a Km of 3.3 mM and a Vmax of 33 nmol × min−1× mg−1 at 10°C. The uptake is driven by the mitochondrial pH/cytosolic pH difference in isolated mitochondria, as well as in the intact rat liver. The rate of uptake is stimulated at a more alkaline matrix pH due to a stimulation of mitochondrial glutaminase. Our data confirm the notion that glutamine metabolism is regulated by pH, not only at the site of its metabolism but also through regulation of its transport systems.
Characterization of fMet‐Leu‐Phe‐stimulated phospholipase C in streptolysin‐O‐permeabilised cells
Authors: STUTCHFIELD Jane , COCKCROFT Shamshad
Published: March 2005

Phospholipase C (specific for inositol lipids) is known to be present both in membranes and cytosol. Receptor‐mediated activation of this enzyme occurs via a guanine nucleotide regulatory protein (G‐protein), designated Gp. We have compared the stimulation of this enzyme by fMet‐Leu‐Phe via the G‐protein in HL60 membranes and in permeabilised cells. fMet‐Leu‐Phe stimulated phospholipase C in membranes at 2 min and the response was dependent on exogenously added GTP. GTP alone also stimulated phospholipase C activity such that at 10 min the response to fMet‐Leu‐Phe was minimal. In comparison, the response to fMet‐Leu‐Phe in permeabilised cells was greater in extent but did not require added GTP. However, it was antagonized by GDP analogues (GDP[βS] > GDP > dGDP) and by pertussis toxin pretreatment, indicating that fMet‐Leu‐Phe‐stimulated phospholipase C activity was also mediated via Gp. GTP and its analogue GTP[γS] also stimulated phospholipase C and their effects were strictly additive to the stimulation obtained with fMet‐Leu‐Phe. Such additivity was also observed when two receptor‐directed agonists, fMet‐Leu‐Phe and ATP, were used to stimulate intact cells. It is concluded that (a) the size of the response with fMet‐Leu‐Phe in membranes is limited by the loss of a component, possibly phospholipase C, and (b) stoichiometry and physical organisation of multiple species of G‐proteins and/or phospholipases C may explain the independent nature of phospholipase C activation by fMet‐Leu‐Phe, ATP and guanine nucleotides.
3‐Aminobenzamide inhibits cytotoxicity and adhesion of phorbol‐ester‐stimulated granulocytes to fibroblast monolayer cultures
Authors: MEYER Thomas , LENGYEL Helgard , FANICK Werner , HILZ Helmuth
Published: March 2005

Damage of 3T3 fibroblasts as induced by short‐term co‐cultivation with O2−‐producing granulocytes, stimulated by 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), was compared with that induced by treatment with enzymically generated O2− and with the alkylating agent dimethyl sulfate. The action of stimulated granulocytes was different in several aspects: (a) DNA fragmented by the products of TPA‐stimulated granulocytes showed a biphasic alkaline elution pattern while fragmentation induced by alkylation or by enzymically produced O2− was monophasic. (b) Poly(ADP‐ribosyl)ation of nuclear proteins after treatment with TPA‐stimulated granulocytes exhibited a lag phase and was, in most experiments, less pronounced than after equitoxic dimethyl sulfate treatment. (c) 3‐Aminobenzamide, the most widely used inhibitor of ADP‐ribosylation, partially protected target cells from the cytotoxic effects of TPA‐stimulated granulocytes, while it enhanced alkylation‐induced and O2−‐induced cytotoxicity. Protection by 3‐aminobenzamide in the granulocyte system was apparently not mediated by an inhibition of nuclear poly(ADP‐ribosyl)ation. Other inhibitors, like benzamide and nicotinamide, augmented cytotoxicity of TPA‐stimulated granulocytes. The unique effect of 3‐aminobenzamide in this system appeared to relate to TPA‐induced adhesion of the neutrophils to surfaces. In the presence of 1 mM 3‐aminobenzamide, but not of benzamide, the adhesion of stimulated granulocytes to 3T3 monolayer cultures was markedly reduced or even abolished. This effect was also seen in granulocyte preparations depleted of monocytes. Since 3‐aminobenzamide at the doses applied does not inhibit TPA‐induced superoxide production in isolated granulocytes, its specific anticytotoxic effect appears to result from a ‘dilution’ of granulocyte‐derived damaging agents into the medium. Our data suggest that prevention of granulocyte adhesion is likely to reduce tissue damage and carcinogenesis in areas of chronic inflammation.
Sequence requirements for prohormone processing in mouse pituitary AtT‐20 cells
Authors: NAGAHAMA Masami , NAKAYAMA Kazuhisa , MURAKAMI Kazuo
Published: March 2005

Although cleavage of peptides at sites marked by paired basic amino acids is a common feature of prohormone processing, little is known about the properties of endoprotease(s) responsible for cleavage of the precursor. To examine the cleavage specificity of a processing endoprotease, we have altered the Lys‐Arg cleavage site of human prorenin to Arg‐Arg, Lys‐Lys and Arg‐Lys by site‐directed mutagenesis, and expressed the native and mutated precursors in mouse pituitary AtT‐20 cells which are known to process foreign prohormones, including prorenin, at paired basic sites during the regulated secretory process. All native and mutated human prorenins were sorted into the regulated secretory pathway. The mutated precursor with Arg‐Arg instead of the Lys‐Arg native pair was processed at about half the efficiency of the native one, while the Lys‐Lys and Arg‐Lys mutants were not processed. Rat prorenin, which naturally has a Lys‐Lys pair, was not processed in the cells. In addition, mouse Ren2 prorenin, which has a Ser residue next to the Lys‐Arg pair, but not mouse Ren1 prorenin, which has a Pro residue next to the pair, was processed. These results suggest that the Arg residue at the COOH side of the basic pair is essential for cleavage of prorenins by a processing enzyme during the regulated secretory process in AtT‐20 cells, although the NH2‐side Lys residue also plays a role. The results also demonstrate that the processing enzyme cannot cleave the Arg‐Pro peptide bond.
Cell/cell channel formation involves disulfide exchange
Authors: DAHL Gerhard , LEVINE Eric , RABADAN‐DIEHL Cristina , WERNER Rudolf
Published: March 2005

The oocyte cell/cell‐channel assay was used to identify amino acids involved in the process of cell/cell‐channel formation. The expression of the rat liver gap‐junction protein, connexin 32, in single oocytes, results in the accumulation of a pool of channel precursors. Upon pairing of such oocytes, cell/cell channels form rapidly from this pool. The rate of formation is affected by thiol‐specific reagents and the pH. This suggests the involvement of extracellular cysteine residues in the channel formation process. Two connexin‐32 mutants were generated by site‐directed mutagenesis in which cysteine residues were replaced by serine. Both mutant connexins were unable to form cell/cell channels. Thus, the cysteine residues appear to play an important role in the channel formation process.
Complete structure of the chicken α2(VI) collagen gene
Authors: HAYMAN Alison R. , KÖPPEL Judith , TRUEB Beat
Published: March 2005

Type VI collagen is a hybrid molecule consisting of a short triple helix flanked by two large globular domains. These globular domains are composed of several homologous repeats which show a striking similarity to the collagen‐binding motifs found in von Willebrand factor. The α2(VI) subunit contains three of these homologous repeats termed D1, D2 and D3. We have isolated and characterized the entire gene for chicken α2(VI) collagen. This gene, which is present as a single copy in the chicken genome, is 26 kbp long and comprises 28 exons. All exons can be classified in three groups. (a) The triple‐helical domain is encoded by 19 short exons (27–90 bp) separated by introns of phase class 0. These exons are multiples of 9 bp and encode an integral number of collagenous Gly‐Xaa‐Yaa triplets. (b) The homologous repeats D1–D3 are encoded by one or two very long exons each (153–1578 bp). These exons are separated by introns of phase class 1. (c) The homologous repeats and the collagen sequence are linked to each other by three short adapter segments which are each encoded by a single exon (21–46 bp). The modular nature of the polypeptide is thus clearly reflected by the mosaic structure of its gene. The size of the exons and the phase class of the introns suggest that the α2(VI) gene evolved by duplication and shuffling of two different primordial exons, one of 9 bp encoding a collagen Gly‐Xaa‐Yaa triplet and one of 600 bp encoding the precursor of the homologous repeats.
Release of proteinases from stimulated polymorphonuclear leukocytes
Authors: SCHETTLER Almut , THORN Hans , JOCKUSCH Brigitte M. , TSCHESCHE Harald
Published: March 2005

The secretion of matrix‐degrading proteinases and protein components involved in the production of cytotoxic metabolites is an important step in the sequence of defense reactions executed by polymorphonuclear leukocytes (PMNL) in response to stimulation. In the present report, we have analyzed degranulation of PMNL stimulated either with soluble synthetic peptides fLeu‐Phe (fMet, formylmethionyl), or fAhx‐Leu‐Phe‐Ahx‐Tyr‐Phe (Ahx, aminohexyl) which trigger chemotaxis and degranulation, or with opsonized zymosan which induces phagocytosis.

The release of elastase, myeloperoxidase and lactoferrin‐containing granules was not at all or only slightly (< 6%) induced either by fAhx‐Leu‐Phe‐Ahx‐Tyr‐Leu or by zymosan particles. In contrast, type‐I collagenase and gelatinase were secreted in significant amounts after treatment with these agents. The disruption of microfilaments by cytochalasin B and subsequent stimulation of PMNL with the formyl‐peptide led to the secretion of elastase, myeloperoxidase and lactoferrin, and enhanced the release of gelatinase. Disruption of microtubules by incubation with colcemid resulted in inhibition of fAhx‐Leu‐Phe‐Ahx‐Thyr‐Leu and fAhx‐Leu‐Phe‐Ahx‐Tyr‐Leu/cytochalasin‐B‐induced granule release. Furthermore, we found different patterns of enzyme distribution after fractionation by centrifugation: most (> 90%) type‐I collagenase and gelastinase was measured in the supernatant whereas 60–90% of elastase, myeloperoxidase and lactoferrin had partitioned into the cytoskeleton‐containing pellet. Our results suggest that the two main types of secretory vesicles identified in PMNL (specific and azurophilic granules) consist of subpopulations. The differential association of the various types of granules with cytoskeletal elements may serve to control their sequential discharge.
Purification and characterization of a novel broad‐specificity (α1 → 2, α1 → 3 and α1 → 6) mannosidase from rat liver
Authors: BONAY Pedro , HUGHES R. Colin
Published: March 2005

We have identified a mannosidase in rat liver that releases α1 → 2, α1 → 3 and α1 → 6 linked manose residues from oligosaccharide substrates, MannGlcNAc where n= 4–9. The end product of the reaction is Manα1 → 3[Manα1 → 6]Manβ1 → 4GlcNAc. The mannosidase has been purified to homogeneity from a rat liver microsomal fraction, after solubilization into the aqueous phase of Triton X‐114, by anion‐exchange, hydrophobic and hydroxyapatite chromatography followed by chromatofocusing. The purified enzyme is a dimer of a 110‐kDa subunit, has a pH optimum between 6.1 and 6.5 and a Km of 65 μM and 110 μM for the Man5GlcNAc‐oligosaccharide or Man9GlcNAc‐oligosaccharide substrates, respectively. Enzyme activity is inhibited by EDTA, by Zn2+ and Cu2+, and to lesser extent by Fe2+ and is stabilized by Co2+. The pattern of release of mannose residues from a Man6GlcNAc substrate shows an ordered hydrolysis of the α1 → 2 linked residue followed by hydrolysis of α1 → 3 and α1 → 6 linked residues. The purified enzyme shows no activity against p‐nitrophenyl‐α‐mannoside nor the hybrid GlcNAc Man5GlcNAc oligosaccharide. The enzyme activity is inhibited by swainsonine and 1‐deoxymannojirimycin at concentrations 50–500‐fold higher than required for complete inhibition of Golgimannosidase II and mannosidase I, respectively. The data indicate strongly that the enzyme has novel activity and is distinct from previously described mannosidases.
Effects of amphotericin B on the glucose metabolism inSaccharomyces cerevisiaecells
Authors: TRAN‐DINH Son , HERVÉ Martine , LEBOURGUAIS Odette , JEROME Muriel , WIETZERBIN Jaime
Published: March 2005

A new approach is proposed to investigate the metabolic perturbation induced by drugs in cells. The effects of various concentrations of amphotericin B on the aerobic [1‐13C]glucose metabolism in glucose‐grown repressedSaccharomyces cerevisiaecells were studied as a function of time using13C‐,1H‐NMR and biochemical methods. The13C enrichment of different compounds such as ethanol, glycerol and trehalose were determined by1H‐NMR spectroscopy. In the absence of amphotericin B, glycerol diffuses slowly from the internal to the external medium, whereas in its presence this diffusion is greatly facilitated by the formation of pores in the cell membrane. Amphotericin B has been found to exert a marked influence on the glucose consumption and the production of all metabolites; for example, at 1 μM, the glucose consumption and the production of ethanol decrease while the production of glycerol and trehalose increases. The13C relative enrichments of ethanol, glycerol and trehalose are almost the same with and without the drug. Thus it can be concluded that amphotericin B induces a large effect on the production of these compounds in the cytosol but shows no significant influence on the mechanism of their formation. Upon addition of glucose, all the amino acid concentrations decrease continuously with time; this effect is more pronounced in the presence of the drug. The ratio of the integrated resonances of glutamate (C2 + C3)/C4 reflects the activity of pyruvate carboxylase relative to citrate synthase rather than to pyruvate dehydrogenase. Without amphotericin B, this ratio (∼ 1.0) is pratically constant upon addition of glucose which suggests that the activities of pyruvate carboxylase and citrate synthase are equivalent. By contrast, upon coaddition of 25 mM glucose and 1 μM amphotericin B, the glutamate C4 resonance remains virtually unchanged while that of glutamate C2 is much smaller than in its absence and continuously decreases with time. It seems likely that amphotericin B induces a reduction in the activity of pyruvate carboxylase in the mitochondria.