bluuebunny/crossref_metadata_2025 huggingface dataset:
Photoaffinity labeling of Torpedo acetylcholine receptor by physostigmine
Authors: SCHRATTENHOLZ Andre , GODOVAC‐ZIMMERMANN Jasminka , SCHÄFER Hans‐Jochen , ALBUQUERQUE Edson X. , MAELICKE Alfred
Published: March 2005

The plant alkaloid physostigmine, an established anti‐cholinesterase agent of the carbamate type, has recently been shown to bind to the nicotinic acetylcholine receptor from Torpedo marmorata electrocytes [Okonjo, K. O., Kuhlmann, J. & Maelicke, A. (1991) Eur. J. Biochem. 200, 671–677]. Pharmacological studies of physostigmine‐induced ion flux into nicotinic‐acetylcholine‐receptor‐rich membrane vesicles, indicated distinct binding sites for physostigmine and acetylcholine. As shown in this study by photoaffinity labeling with [phenyl‐(n)‐3H](‐)physostigmine, the physostigmine‐binding site is located within the same subunit (α polypeptide) of the receptor as the acetylcholine‐binding site. Using a variety of proteolytic cleavage conditions for the purified α polypeptide, several [3H]physostigmine‐labeled peptides were isolated and sequenced. From the radioactivity released in the course of the Edman degradations of the labeled peptides, it was found that the label was associated in all cases with Lys125. These results identify a novel ligand‐binding site for the Torpedo nicotinic acetylcholine receptor that is different in location from binding sites identified previously for acetylcholine, its established agonists and antagonists, and direct channel blockers.
Molecular cloning and functional expression of different molecular forms of rat amiloride‐binding proteins
Authors: LINGUEGLIA Eric , RENARD Stephane , VOILLEY Nicolas , WALDMANN Rainer , CHASSANDE Olivier , LAZDUNSKI Michel , BARBRY Pascal
Published: March 2005

The colon and lung amiloride‐binding proteins were cloned from rat tissues. Two sizes of transcripts were identified. The 2.7‐kb transcript codes for an 85‐kDa protein, whereas the 1.2‐kb transcript codes for a 25‐kDa polypeptide. The 2.7‐kb transcript was detected in the proximal and distal colon and in duodenum, liver, placenta and thymus. The 1.2‐kb transcript was the only form present in lung and spleen, and it was also detected in placenta and colon. The short form corresponds to the 3′ terminus of the longer one. It is formed by alternative transcription under the control of an internal promoter.

Cells stably transfected with cDNAs encoding these two proteins were used for binding studies using [3H]phenamil, a potent blocker of the epithelial Na+ channel, derived from amiloride. Both the long and short forms of the protein bind amiloride and some of its derivatives, but they have distinct pharmacologies. The order of potency of the different amiloride derivatives to inhibit [3H]phenamil binding was phenamil (K0.5= 10 nM) > benzamil (K0.5= 43 nM) > amiloride (K0.5= 1.4 μM) ∼ ethylisopropylamiloride (K0.5= 1.6 μM) for the long form, whereas it was phenamil (K0.5= 68 nM) > amiloride (K0.5= 3.2 μM) = ethylisopropylamiloride (K0.5= 4 μM) ∼ benzamil (K0.5= 6.3 μM) for the short form. Although the binding proteins described here are distinct from the pore‐forming protein of the epithelial Na+ channel, the pharmacological profile of the long form of the ABP is identical to that described previously in pig and human kidney, and similar to that expected for an epithelial Na+ channel. The pharmacological profile of the short form resembles that previously described for an amiloride‐binding protein in pneumocytes. Results presented in this paper suggest that previously purified preparations showing Na+ channel activity contain different forms of the amiloride‐binding protein, possibly associated with other proteins.

The similarity between amiloride‐binding proteins and a protein identified in seminal vesicles suggests that amiloride‐binding proteins are the first members of a new family of epithelia‐specific proteins.
Rabbit procathepsin E and cathepsin E
Authors: KAGEYAMA Takashi
Published: March 2005

The structure of rabbit procathepsin E was determined by molecular cloning of its cDNA. The proenzyme consisted of 379 amino acids and had structural features common to human and guineapig procathepsin E species. The highly conserved tripeptide sequence at the active site of aspartic proteinases, Asp‐Thr(Ser)‐Gly, is, however, replaced by Asp‐Thr‐Val in rabbit procathepsin E. To our knowledge, this is the first case of such a variation in aspartic proteinases. The processed form, cathepsin E, hydrolyzed various biologically active peptides maximally at around pH 5 Tachykinins, such as substance P and neurokinin A, were hydrolyzed most rapidly, with specific cleavge of sequences essential for their activity. The rates of hydrolysis were several hundred‐fold higher than those of cathepsin D. Furthermore, cathepsin E was able to inactivate a functional‐domain peptide of fibroblast growth factor, the sequence of which resembles those of tachykinins, and it was active in the generation of functional peptides, such as endothelin and angiotensin I, from their respective precursors. Procathepsin E was detected at high levels in various fetal tissues, such as the liver, stomach and blood cells. At the adult stage, the proenzyme was detectable only in specific tissues, such as the urinary bladder, duodenum and colon. Northern‐blot analysis showed similar stage‐specific and tissue‐specific expression of the mRNA for procathepsin E. Since tachykinins and other suited peptide substrates of cathepsin E have been shown to have mitogenic activity, (pro)cathepsin E may regulate the growth and differention of embryonic and fetal tissues by degrading or processing these peptides. The enzyme may also regulate the physiological activities of adult tissues which are mediated by substance P and related tachykinins.
GTPase activity of small GTP‐binding proteins in HL‐60 membranes is stimulated by arachidonic acid
Authors: LIGETI Erzsébet , PIZON Veronique , WITTINGHOFER Alfred , GIERSCHIK Peter , JAKOBS Karl H.
Published: March 2005

The GTPase activity of membranes isolated from differentiated HL‐60 cells was investigated to obtain information about the possible involvement of membrane‐bound GTP‐binding proteins in the regulation of the NADPH oxidase. A more than tenfold increase in the rate of hydrolysis of membrane‐bound GTP was observed when cytosol and arachidonic acid were added simultanously, i.e. under the same conditions where NADPH oxidase becomes activated. There were parallel changes in GTPase and NADPH oxidase activities when the concentration of arachidonic acid or the species of the fatty acid was varied or different detergents were applied. Separation of the GTP‐binding proteins of the solubilized membrane by sucrose density gradient centrifugation, allowed us to ascribe the observed effect to the stimulation of the GTPase activity of small GTP‐binding proteins by cytosolic components(s). Indirect evidence suggests that, in contrast to the effect upon recombinant ras and ras‐GTPase‐activating protein, in intact HL‐60 membranes the interaction of rap1A with rap‐GTPase‐activating protein, is strongly enhanced by arachidonic acid.
Determinants for the enhanced thermostability of hybrid (1‐3,1‐4)‐β‐glucanases
Authors: POLITZ Oliver , SIMON Ortwin , OLSEN Ole , BORRISS Rainer
Published: March 2005

Hybrid (1–3,1–4)‐β‐glucanases which contain an N‐terminal region derived from the Bacillus amyloliquefaciens enzyme and a C‐terminal region of the closely related B, macerans enzyme may exhibit a thermostability superior to both parental enzymes. A systematic series of hybrid enzymes were constructed in order to delineate the amino acid residues that affect protein stability. Hybrid enzymes with between one and four of the N‐terminal residues for the mature B, amyloliquefaciens (1–3,1–4)‐β‐glucanase exhibit no significant changes in biochemical characteristics as compared with the parental B, macerans enzyme. However, significantly enhanced thermostability was observed in the hybrid enzyme. However, significantly enhanced thermostability was observed in the hybrid enzyme containing an N‐terminal segment of eight amino acid residues derived from the B. amyloliquefaciens enzyme. Site‐directed mutagenesis revealed that the combined effect of Gln1, Thr2, Ser5 and Phe7 confer enhanced stability on hybrid enzymes, probably by improving the hydrogen bonding that stabilizes the interactions between the two termini of the polypeptide chain. Furthermore, deletion of Tyr13 in the hybrid enzyme containing the 12 N‐terminal amino acids from the B. amyloliquefaciens (1–3,1–4)‐β‐glucanase results in a dramatic incrase in stability at 70°C with the half‐life of 6 min increased to around 4 h. This is twofold higher than the hitherto most stable hybrid enzyme in which the N‐terminal domain consisted of 16 residues of the B. amyloliquefaciens enzyme.
Identification of important bases in a single‐stranded region (SSrC) of the hepatitis delta (δ) virus ribozyme
Authors: KAWAKAMI Junji , KUMAR P. K. R. , SUH Young‐Ah , NISHIKAWA Fumiko , KAWAKAMI Keiko , TAIRA Kazunari , OHTSUKA Eiko , NISHIKAWA Satoshi
Published: March 2005

Models for the secondary structure of genomic and antigenomic self‐cleaving RNAs of human hepatitis delta (δ) virus (HDV) have been proposed by several groups. Our recent results support a pseudoknot structure and have allowed us to identify functionally important nucleotides in single‐stranded regions [nucleotides 726–731 (SSrA) and nucleotides 762–766 (SSrB)]. For the identification of the important residues in the remaining single‐stranded region, nucleotides 708–715 (SSrC), of the genomic HDV ribozyme, we made derivatives with a single‐base substitution in the SSrC region. To screen inactive mutants rapidly, we use a simplified in‐vitro selection method. Among the various base substitutions in mutants in the SSrC, U708A, C709(A/G/U) and G713C variants had less than 10% of the cleavage activity of the wild‐type SSrC (HDV86). By analyzing the self‐cleavage activities of various mutants, we determined the base requirements for SSrC as 5′‐(U/C/G)‐C‐N‐N‐(C/A/G)‐(G/A/U)‐N‐N‐3′.
Molecular cloning of a cDNA encoding an inducible calmodulin‐dependent nitric‐oxide synthase from rat liver and its expression in COS 1 cells
Authors: ADACHI Hiroko , IIDA Sachio , OGUCHI Shinobu , OHSHIMA Hiroshi , SUZUKI Hisanori , NAGASAKI Koichi , KAWASAKI Hiroshi , SUGIMURA Takashi , ESUMI Hiroyasu
Published: March 2005

Calmodulin‐dependent nitric‐oxide synthase, with an apparent molecular mass of 125 kDa, was induced in the liver of rats treated with Propionibacterium acnes and Escherichia coli lipopolysaccharide. Clones were isolated from a cDNA library obtained from induced rat liver using oligonucleotide probes which were synthesized based on the amino acid sequences of peptides of the purified enzyme. Four overlapping cDNA clones for a 3.8‐kbp region were isolated and the nucleotide sequences were determined. These clones encompassed an open‐reading frame of 3441 bases encoding 1147 amino acids. The deduced amino acid sequence of the cDNA suggested that the protein contains binding sites for NADPH, FAD and FMN. The structure of the possible calmodulin‐binding site, consisting of a strongly hydrophobic region surrounded by basic amino acids, is present. The full‐length cDNA was expressed in COS 1 cells under the control of a cytomegalovirus promoter and the expressed enzyme was found to be a calmodulin‐dependent nitric‐oxide synthase. A structural comparison suggested that the liver nitric‐oxide synthase is the same as the macrophage enzyme. Northern‐blot analysis showed that the mRNA in the liver is approximately 4.2 kb long and is induced transcriptionally by treatment with P. acnes and lipopolysaccharide.
Specific covalent modification of the tryptophan residues in murine interleukin‐6
Authors: ZHANG Jian‐Guo , REID Gavin E. , MORITZ Robert L. , WARD Larry D. , SIMPSON Richard J.
Published: March 2005

Modification of recombinant murine interleukin‐6 (mIL‐6) with the tryptophan‐specific reagent 2‐nitrophenylsulfenyl chloride under mild acidic conditions, 0.1 M sodium acetate, pH 3.5, yielded a derivative containing 2.02 mol 2‐nitrophenylsulfenyl tryptophan/mol protein. The sites of modification were identified as Trp36 and Trp160. No detectable side reactions occurred on other amino acids in the molecule, as indicated by the combination of endoproteinase Asp‐N peptide mapping, Edman degradation and electrospray mass spectrometry. Sulfenylation of the two tryptophan residues in mIL‐6 caused a 50% reduction in both the biological activity in the murine‐hybridoma‐growth‐factor assay using 7TD1 cells and receptor‐binding affinity to mIL‐6 receptors. Sulfenylation of mIL‐6 did not significantly affect the overall conformation of the protein as measured by farultraviolet circular dichroism and binding to the neutralizing anti‐mIL‐6 mAb 6B4. The sulfenylated protein was, however, significantly less stable [ΔΔG(H2O) = 3.98 kJ/mol] than unmodified mIL‐6 as measured by urea‐gradient gel electrophoresis.
Interactions between active‐site serine β‐lactamases and so‐called β‐lactamase‐stable antibiotics
Authors: MATAGNE André , LAMOTTE‐BRASSEUR Josette , FRÈRE Jean‐Marie
Published: March 2005

The interactions between imipenem and four monobactams and three class A β‐lactamases have been studied in detail. Despite their reputation as being β‐lactamase‐stable, some of these compounds were significantly hydrolysed by the enzymes. The results obtained with the Streptomyces albus G β‐lactamase have been analysed in the light of molecular modelling studies. The discussion is extended to include other so‐called β‐lactamase‐stable antibiotics to demonstrate that this appellation can often be misleading.
Nuclear magnetic resonance study of the codon‐anticodon interaction in Bombyx mori tRNAGCCGly
Authors: AMANO Mayumi , KYOGOKU Yoshimasa
Published: March 2005

NMR spectra of Bombyx mori tRNAGCCGly were recorded in the GCC absence and presence of the oligonucleotide, GGCUp, which contains the codon sequence, GGC. The difference between the spectra with and without the codon oligonucleotide indicates the appearance of five new imino proton peaks. For the assignment of these peaks, G*GCUp, in which the 5′‐terminal G was enriched with 95%15N, was prepared (G*, 15N‐labeled guanosine). In the imino proton spectrum of B. mori tRNAGCCGly on the addition of G*GCUp, the peak at 12 ppm became a doublet due to coupling with 15N nuclei. In the two‐dimensinal 1H‐15N heteronuclear multiple‐quantum correlation (HMQC) spectrum, only the peak at 12 ppm was observed, and thus it was assigned to the imino proton of the 5′‐terminal G of GGCUp interacting with tRNAGCCGly. Judging from the temperature effect and chemical shifts, the five new imino proton peaks are presumed to be due to three G · C base pairs, induced by the codon‐anticodon interaction, and one U · U base pair, induced by an interaction between the 3′ terminal U of GGCUp and U33 neighboring the anticodon. The binding of three trinucleotides (GGCp, GGUp and GCUp) to B. mori tRNAGCCGly was also investigated. Ultracentrifugation analysis showed that tRNAGCCGly underwent dimerization through the anticodon‐anticodon interaction, but the dimerization was broken on addition of GGCUp. On 1H‐NMR and ultracentrifugation analysis, it was found that GCUp not complementary to the anticodon also binds to the anticodon loop.
Amino acid sequence of the small cadmium‐binding protein (MP II) from Nereis diversicolor (annelida, polychaeta)
Authors: DEMUYNCK Sylvain , LI Ka Wan , Van der SCHORS Roel , DHAINAUT‐COURTOIS Nicole
Published: March 2005

The primary sequence of the low‐molecular‐mass cadmium‐binding protein metalloprotein II of Nereis diversicolor (Hediste diversicolor, recent denomination) has been determined. This protein is composed of 119 amino acids and has 80.8% identity with the N. diversicolor myohemerythrin [Takagi, T. & Cox, J. A. (1991) FEBS Lett. 285, 25–27]. The fact that iron, which normally binds to myohemerythrin, is not found to be associated with the cadmium‐binding protein metalloprotein II in cadmium‐exposed animals could be the result of the complete abolition of the iron‐binding capacity of the protein due to the binding of cadmium.
Side‐chain specificities of human and bovine cytochromes P‐450scc
Authors: TUCKEY Robert C. , CAMERON Kathryn J.
Published: March 2005

Cytochrome P‐450scc catalyses the conversion of cholesterol to pregnenolone by the sequential hydroxylation of the side chain of cholesterol. This occurs at a single active site and produces 22R‐hydroxycholesterol and 22R‐20α‐dihydroxycholesterol as intermediates. To further define the active site of human and bovine cytochromes P‐450scc, we have examined the kinetics of the conversion of structural analogues of cholesterol with modified side chains, to pregnenolone. Analysis of the side‐chain cleavage of analogues of cholesterol modified at C22 confirmed the high degree of structural specificity for the 22R position by cytochrome P‐450scc, the major effect being on the turnover number (kcat) rather than on binding. The analogues of cholesterol that had a polar group at C24, C25 or C26 had much lower Km values and generally lower kcat values than the non‐polar analogues which were tested. Km values of the polar analogues were 3–25‐times lower than the Km for cholesterol and kcat values were also much lower than the kcat values for cholesterol, particularly for the human enzyme. The data suggest that the tight binding of the analogues with a hydroxyl or ketone group at C24, C25 or C26 places C20 and C22 in a poor orientation relative to the heme group for hydroxylation to occur. Many of the polar analogues which were tested are postulated regulators of cellular cholesterol metabolism. Several of these analogues are good substrates for bovine and human cytochromes P‐450scc at low substrate concentration, as determined from their kcat/Km values. This study also indicates that the active site of cytochrome P‐450scc is well conserved between bovine and human cytochromes. However, small species differences are evident since lower kcat values relative to the kcat of cholesterol are observed for some polar side‐chain analogues of cholesterol with the human enzyme.
Abduction of iron(III) from the soluble methane monooxygenase hydroxylase and reconstitution of the binuclear site with iron and manganese
Authors: ATTA Mohamed , FONTECAVE Marc , WILKINS Patricia C. , DALTON Howard
Published: March 2005

The apo‐form of the soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath) was prepared via chelation of iron(III) with 3,4‐dihydroxybenzaldehyde. The apohydroxylase was reconstituted by the anaerobic addition of Fe(II) followed by air oxidation. The enzyme thus prepared regained 85–90% of its original catalytic activity. The incorporation of two manganese(II) ions/mol of apohydroxylase was monitored by EPR spectroscopy. The Mn(II) ions occupy the native diiron active site and remain in the +2 oxidation state. The EPR data suggest strong coupling between the two Mn(II) ions and retention of the μ‐hydroxo (alkoxo) bridge. The results of this study indicate that the M. capsulatus (Bath) hydroxylase contains a single diiron site.
Intracellular and extracellular processing of chromogranin A
Authors: METZ‐BOUTIGUE Marie‐Hélène , GARCIA‐SABLONE Pascale , HOGUE‐ANGELETTI Ruth , AUNIS Dominique
Published: March 2005

Chromogranins are a family of acidic soluble proteins which exhibit widespread distribution in endocrine cells and neurons. Chromogranin A (CGA), the major soluble component of the secretory granules in chromaffin cells of the adrenal medulla, is a single polypeptide chain of 431 residues with an apparent molecular mass of 70–75 kDa and a pI of 4.5–5. In mature bovine chromaffin granules about 50% of the CGA has been processed. In the present paper, the structural features of the proteolytic degradation mechanism have been characterized with regard to the possible function of CGA as a prohormone, as suggested by recent studies.

CGA‐derived components present in chromaffin granules were subjected to either two‐dimensional gel electrophoresis or HPLC and the N‐terminal of each fragment was sequenced. Immunoblotting with antisera to specific sequences within the CGA molecule were used to characterize these fragments further at their C‐terminal. In addition, a similar approach was performed to characterize CGA‐derived fragments released into the extracellular space from directly depolarized bovine cultured chromaffin cells.

Our results identified several proteolytic cleavage sites involved in CGA degradation. Intragranular processing occurs at 12 cleavage sites along the peptide chain located in both N‐ and C‐terminal moieties of the protein; a preferential proteolytic attack in the C‐terminal part was noted. We found that CGA processing also occurs in the extracellular space after release, generating new shorter fragments. The proteolytic cleavage sites identified in this study were compared with the cleavage points which are thought to be involved in generating CGA fragments with specific biological activity: pancreastatin, chromostatin and N‐terminal vasostatin fragments. In addition, a new 12‐amino‐acid CGA‐derived peptide corresponding to the sequence 65–76 was identified in the soluble core of purified chromaffin granules. This short peptide was released, together with catecholamines, after stimulation of cultured chromaffin cells suggesting its presence within the storage complex of chromaffin granules. The specific biological activity of this CGA‐derived fragment remains to be determined.
Regulation of neurosecretory habituation by cAMP
Authors: MARTIN Paul T. , CHUNG Ben Tsu‐Ping , KOSHLAND Daniel E.
Published: March 2005

The role of adaptation in receptors was studied with regard to the processing of neural information by cAMP in PC12 cells. Both secretin and adenosine A2 receptors elevated cAMP levels. During prolonged stimulation, secretin receptors demonstrated adaptation of cAMP levels, while adenosine A2 receptors did not. Incubation of cells with secretin increased the norepinephrine secretion resulting from stimulation by acetylcholine, but not from high potassium depolarization. The difference in the adaptation of cAMP by secretin and adenosine A2 receptors was responsible for the changes observed in norepinephrine secretion. This difference in secretory responses was caused by protein‐kinase‐C activation, which occurred during acetylcholine stimulation but not during depolarization by high potassium. Secretin receptors also demonstrated a reduced ability to elevate cAMP levels during repetitive stimulation, even for stimulation times where no adaptation of cAMP signals was observed, suggesting that secretin receptors possess the ability to habituate in addition to undergoing adaptation. These results are discussed with regard to the modulatory roles cAMP may play in neurosecretory habituation.
Probing the role of metal ions in the mechanism of inositol monophosphatase by site‐directed mutagenesis
Authors: POLLACK Scott J. , KNOWLES Michael R. , ATACK John R. , BROUGHTON Howard B. , RAGAN C. Ian , OSBORNE SallyAnn , McALLISTER George
Published: March 2005

Since inhibition of myo‐inositol monophosphatase (EC 3.1.3.25) by lithium ions and the resulting attenuation of phosphatidylinositol cycle activity may be the mechanism by which lithium exerts its therapeutic effect in the treatment of manic depression, it is of great interest to understand the mechanism of the enzyme and how lithium and other metals interact with it. Divalent magnesium is essential for enzyme activity, whereas Li+ and high concentrations of Mg2+ act as uncompetitive inhibitors with respect to substrate. From the recently solved crystal structure of the human enzyme, several amino acid residues in the active site were targeted for mutagenesis studies. Nine single‐residue substituted mutants were characterized with regard to catalytic parameters, Mg2+ dependence, and Li+ inhibition. In addition, a terbium fluorescence assay was developed to determine the metal binding properties of the wild‐type and mutant enzymes. Although none of these mutations affected Km for substrate substantially, the mutations Glu70→Gln, Glu70→Asp, Asp90→Asn and Thr95′Ala, in which residues within coordinating distance of the active site metal were modified, all resulted in large reductions in catalytic activity. The position of Glu70 in the crystal structure further suggests that this residue may be involved in activating water for nucleophilic attack on the substrate. The mutations Lys36→Ile, Asp90→Asn, Thr95′Ala, Thr95′Ser, His217→Gln, and Cys218→Ala all resulted in parallel reductions in both lithium and magnesium affinity, suggesting that Li+ and Mg2+ share a common binding site.
Complement fragment C3a stimulates Ca2+ influx in neutrophils via a pertussis‐toxin‐sensitive G protein
Authors: NORGAUER Johannes , DOBOS Gustav , KOWNATZKI Eckhard , DAHINDEN Clemens , BURGER Reinhard , KUPPER Roland , GIERSCHIK Peter
Published: March 2005

The signal pathways of neutrophils following stimulation with the complement fragment C3a (C3a) were studied in neutrophils and compared to the pathways activated by complement fragment C5a (C5a). Analysis of polyphosphoinositol lipid turnover showed that C5a, but not C3a, activated phosphatidylinositol‐bisphosphate‐3‐kinase (PtdInsP2 3‐kinase) indicating that different signal pathways are activated by the two anaphylatoxins. To examine whether C3a stimulated Ca2+ transients, cytosolic free Ca2+ levels were analyzed in Fluo‐3‐labelled neutrophils by flow cytometry. C3a stimulated a fast and concentration‐dependent increase of cytosolic free Ca2+. Comparison of the C3a response with that of C5a revealed a more pronounced C5a‐triggered Ca2+ rise. Addition of EGTA to the extracellular buffer prior to stimulation did not significantly alter the initial Ca2+ rise at low C5a concentrations, but reduced the time course of the Ca2+ transients at high concentrations. In marked contrast, EGTA completely blocked the Ca2+ response stimulated by C3a in neutrophils labeled with either Indo‐1/AM or Fluo‐3. Preincubation of neutrophils with pertussis toxin inhibited both C3a‐ and C5a‐stimulated Ca2+ transients, indicating the involvement of guanine‐nucleotide‐binding proteins (G proteins) in these processes. In order to examine whether the C3a receptor is coupled to G proteins, binding of guanosine 5′‐O‐(3‐[35S]thiotriphosphate) ([35S]GTP[S]) to purified neutrophil plasma membranes was studied. Both C3a and C5a stimulated high‐affinity binding of [35S]GTP[S] up to 1.5‐fold and 3‐fold, respectively. These data suggest that the two anaphylatoxins activate pertussis‐toxin‐sensitive G proteins, which then trigger different signal transduction pathways. C3a specifically stimulated Ca2+ influx from the extracellular medium, whereas C5a additionally activated the PtdInsP2 3‐kinase and stimulated Ca2+ mobilization from intracellular stores.
Determination of relative binding affinity of influenza virus N9 sialidases with the Fab fragment of monoclonal antibody NC41 using biosensor technology
Authors: GRUEN L. Clem , KORTT Alexander A. , NICE Edouard
Published: March 2005

The relative binding affinities of influenza virus N9 sialidase from tern and whale with the Fab fragment of monoclonal antibody NC41 were determined using biosensor technology (Pharmacia BIAcoreTM). The apparent association and dissociation rate constants were measured in real time for the interaction of the Fab with both sialidases, the Fab being immobilised on the sensor surface. Although three‐dimensional structural studies have shown that there are no apparent structural differences between the tern and whale N9 sialidase epitopes to which the NC41 Fab binds, the apparent binding constant for the interaction with tern N9 sialidase was ∼ 2.4‐fold higher than that with whale N9 sialidase. The kinetic analysis showed that the association rate constant for the binding of whale N9 sialidase was higher than that for tern N9 sialidase (12.0 × 104 M−1 s−1 compared to 4.3 × 104 M−1 s−1) and the dissociation rate constants for the whale N9‐sialidase–Fab complex were × 6‐fold higher than for the tern N9‐sialidase–Fab complex. Furthermore, kinetic analysis of the dissociation reaction showed that it was composed of two stages, an initial, faster rate followed by a late, slower rate. The values of the relative affinity constants calculated using the initial dissociation rate constant were similar to the values measured at equilibrium in the BIAcore and those determined in true solution equilibrium studies using sedimentation equilibrium. The late, slower, dissociation rate constant yielded affinity constants significantly higher than those obtained by true solution methods.
Post‐transcriptional and post‐translational regulatory steps are crucial in controlling the appearance and stability of thylakoid polypeptides during the transition of etiolated tobacco seedlings to white light
Authors: PALOMARES Rosario , HERRMANN Reinhold G. , OELMÜLLER Ralf
Published: March 2005

We have investigated the expression of nuclear‐encoded chloroplast proteins that are not associated with chlorophyll (the lumenal 33‐kDa and 23‐kDa polypeptides of the oxygen‐evolving system of photosystem II, plastocyanin and the Rieske Fe/S protein) by comparing mRNA‐accumulation rates with those of the corresponding proteins during illumination of etiolated tobacco seedlings. Using subcellular fractionation, pulse/chase, Northern and Western techniques, we found that the biogenesis and stability of these proteins are regulated both translationally, as well as post‐translationally, including the efficiency of mRNA uptake into polysomes, processes that operate between translation and assembly or monitor the status (soluble and membrane‐attached) of a terminally processed polypeptide. Polypeptide synthesis is generally not limited by mRNA amounts. For instance, steady‐state transcript levels may increase 10‐fold during illumination, while those associated with polysomes increase only 2–3‐fold without measurable influence on the rate of protein synthesis. The 23‐kDa and Rieske polypeptides are predominantely membrane associated, but plastocyanin and the 33‐kDa polypeptide are distributed among both soluble and membrane‐associated protein fractions. Plastocyanin appears to be comparably stable in both forms. However, for the 33‐kDa polypeptide, only the membrane‐attached form is stable (>8h) and only this pool increases upon illumination. Its soluble form is rapidly degraded with a half‐life of approximately 1 h under the chosen conditions. Our findings probably reflect part of a more general regulatory principle operating in the differentiation and maintenance of subcellular structure.
Stimulatory effect of NH4+ on the transport of leucine and glucose in an anaerobic alkaliphile
Authors: KOYAMA Noriyuki
Published: March 2005

An anaerobic alkaliphile, EP01, specifically requires NH4+ for the acceleration of amino acid and glucose transport [Koyama, N. (1988) FEBS Lett. 253, 187–189]. In this paper, we attempted to clarify how NH4+ is involved in the transport system. The bacterium acidifies the cytoplasm, which was suggested to result in NH4+ accumulation when NH4Cl was added to the medium. Increase of the NH4Cl concentration administered to the medium caused the acceleration of leucine and glucose transport, which was accompanied by an increase in the internal pH and the absolute internal concentration of NH4+, whereas a decrease in the concentration ratio of internal NH4+/external NH4+ was observed. The addition of 3 mM NH4Cl, which resulted in significant stimulation of leucine and glucose transport, raised the internal NH4+ concentration by 42 mM, but the internal pH only by 0.1 units. It seems more likely that leucine and glucose transport are accelerated depending on the increase in the internal NH4+ concentration rather than the increase in the internal pH.

By the imposition of an inwardly directed Na+ gradient, the K+‐loaded membrane vesicles accumulated leucine and glucose, indicating that a sodium chemical potential is available for active transport.

The membrane of the bacterium exhibited a Na+‐stimulated ATPase activity which was remarkably enhanced by the addition of NH4Cl, depending on its concentration, and was inhibited by vanadate. Leucine and glucose transport were inhibited by vanadate. Based on these results, we propose a mechanism in which NH4+ contributes internally to leucine and glucose transport, depending on its concentration, by the activation of a Na+‐translocating ATPase responsible for the generation of a sodium chemical potential.
Biotin biosynthesis in higher plant cells
Authors: BALDET Pierre , GERBLING Heidrun , AXIOTIS Stella , DOUCE Roland
Published: March 2005

Biotin biosynthesis was investigated in lavender cell cultures (Lavandula vera L.). Two different biological assays and two different HPLC procedures were used to identify all the intermediates involved in biotin biosynthesis. The pathway for biotin biosynthesis could be analyzed starting with [3H]pimelic acid as precursor, leading to labelled biotin and even to labelled biotinylated enzymes. Intermediates known from the bacterial pathway (7‐oxo‐8‐amino‐pelargonic acid, 7,8‐diamino‐pelargonic acid, dethiobiotin) were present in detectable amounts. Pimelic acid activation to pimeloyl‐CoA could be observed. In contrast to bacterial cells, an unknown stable labelled intermediate, named compound A, accumulated. This compound coeluted with an authentic sample of 9‐mercaptodethiobiotin from HPLC with an anion‐exchange column and was as effective as biotin in supporting the growth of the strain bioB105 of Escherichia coli. When 3H‐labelled compound A was added to the growth medium of the lavender cells it was incorporated in an acidomycin‐sensitive manner into biotin. [3H]Dethiobiotin was incorporated into both compound A and biotin. These results strongly suggest that, in higher plant cells, the reaction catalysed by biotin synthase may proceed in two distinct steps involving mercaptodethiobiotin (9‐mercaptodethiobiotin ?) as an intermediate.
Specific molecular interaction sites on factor VII involved in factor X activation
Authors: KUMAR Anuradha , FAIR Daryl S.
Published: March 2005

Factor VII, a serine‐protease zymogen, and tissue factor, the cellular receptor/coenzyme, are the protein components of the macromolecular complex which initiates the extrinsic pathway of the coagulation cascade. Previous studies were directed to the identification of functional sites on factor VII which mediate factor X activation, employing a series of potentially inhibitory synthetic peptides representing the primary structure of factor VII and antibodies to selected peptides. The involvement of at least four high‐affinity interactive regions [factor VII (44–50), (196–229), (285–305) and (376–396) peptides] on the surface of factor VII was clearly demonstrated. The minimal sequences for the expression of inhibitory activity of these four molecular recognition domains on factor VII were identified using short and overlapping peptides. The short factor VII‐(206–218)‐peptide (most inhibitory peptide in the sequence 196–229 on factor VII) inhibited the binding of factor VII to the tissue‐factor‐expressing J82 cell line. Furthermore, radiolabeled [Tyr201] factor VII‐(199–221)‐peptide, with a tyrosine substituted for the normal tryptophan residue, was specifically bound to J82 cells, and also the binding of the radiolabeled peptide to this cell line was specifically inhibited by a monoclonal antibody to tissue factor, confirming that the interaction site for tissue factor on factor VII is present within the peptide sequence 196–229. Kinetic analyses suggested that the regions represented by factor VII‐(285–305)‐ and factor VII‐(376–396)‐peptides are involved in factor X recognition and the chemical cross‐linking of the radiolabeled peptides resulted in specific binding to factor X, confirming that these two regions on factor VII represent the substrate‐recognition site. Furthermore, these radiolabeled peptides specifically interact with the heavy chain of factor X, suggesting that the complementary binding region for the substrate‐recognition site on factor VII are present on the heavy chain of factor X.
Limited proteolysis of yeast phosphofructokinase
Authors: KOPPERSCHLÄGER Gerhard , BÄR Jörg , STELLWAGEN Earle
Published: March 2005

Purified phosphofructokinase 1 from baker's yeast (Saccharomyces cerevisiae) was subjected to proteolysis by thermolysin, endoproteinase lys‐C, trypsin and chymotrypsin under defined solvent conditions. In the absence of substrates and allosteric effectors, the catalytic activity of phosphofructokinase rapidly disappeared in the presence of each proteolytic enzyme. The presence of a saturating concentration of ATP protected phosphofructokinase activity from proteolytic inactivation while the collective presence of fructose 6‐phosphate, AMP and fructose 2,6‐bisphosphate provided transient activation during proteolysis.

Changes in the quaternary structure of phosphofructokinase resulting from proteolysis were estimated by high performance size exclusion chromatography while changes in the primary sequence of the individual α and β polypeptide chains were estimated by polyacrylamide‐gel electrophoresis in sodium dodecylsulfate. The site(s) of proteolytic cleavage were identified by N‐terminal sequence analysis of resolved electrophoretic components.

The presence of ATP protects phosphofructokinase from thermolysin proteolysis, while the collective presence of fructose 6‐phosphate, AMP and fructose 2,6‐bisphosphate restricts proteolysis to one site in each polypeptide chain involving the peptide bonds preceding Leu199 in the α chain and Leu192 in the β chain. The truncated phosphofructokinase retains its octameric structure. The presence of ATP largely restricts endoproteinase lys‐C proteolysis to a single site in the α chain involving the peptide bond preceding Val914. This cleavage results in the dissociation of the octameric form of phosphofructokinase into two tetramers. The presence of ATP restricts both trypsin and chymotrypsin proteolysis to the N‐terminal and C‐terminal regions described above, resulting in the preferential stabilization of the tetrameric form of phosphofructokinase.

It would appear that the first 200 and last 80 residues which are unique to the sequence of the yeast phosphosphofructokinase are not directly involved in catalysis or its allosteric regulation. However, the last 80 residues of the α polypeptide chain do appear to stabilize an octameric structure which is unique to yeast phosphofructokinase.
Tyrosinase isoenzymes in mammalian melanocytes
Authors: JIMENEZ‐CERVANTES Celia , GARCIA‐BORRON Jose Carlos , VALVERDE Paloma , SOLANO Francisco , LOZANO Jose A.
Published: March 2005

B‐16 mouse melanoma melanosomes contain two forms of tyrosinase that can be resolved by SDS/PAGE. These forms interact to different extents with the ion‐exchanger DEAE‐Sephadex and with hydroxyapatite, and have different affinity for the melanosomal membrane and/or the intraorganular matrix. After partial purification and complete separation of the two tyrosinases, several kinetic parameters were analyzed. The form of lower electrophoretic mobility displayed a higher Km for 3,4‐dihydroxy‐L‐phenylalanine (L‐dopa) and L‐tyrosine, an absolute requirement for the cofactor L‐dopa in its tyrosine hydroxylase activity, and a lower ratio of tyrosine hydroxylation to Dopa oxidation. The form of higher electrophoretic mobility displayed lower values of Km for both substrates and was able to exhibit tyrosine hydroxylase activity after a lag period even in the absence of L‐dopa. Both forms were stereospecific for the L isomers and sensitive to the specific tyrosinase inhibitor 2‐phenylthiourea. These forms do not appear to result from different degrees of glycosylation, nor from limited proteolysis and are also present in the microsomal fraction of B16 mouse melanoma. They might correspond to different gene products, most likely derived from the b and c loci.
Structural flexibility of the calmodulin‐binding locus in Bordetella pertussis adenylate cyclase
Authors: MUNIER Hélène , BOUHSS Ahmed , GILLES Anne‐Marie , KRIN Evelyne , GLASER Philippe , DANCHIN Antoine , BÂRZU Octavian
Published: March 2005

The catalytic domain of Bordetella pertussis adenylate cyclase, a calmodulin‐activated enzyme with toxic properties, is a modular construct cleaved by trypsin into two subdomains of 224 (T25) and 175 (T18) amino acids. The calmodulin‐binding locus of the bacterial enzyme consists of approximately 70 amino acids and overlaps the C‐terminus of T25 and the N‐terminus of T18. This region, exposed to the solvent or proteases, also exhibits an unusual high flexibility and allows, as demonstrated in this study, reconstitution in the presence of calmodulin of active species of adenylate cyclase from overlapping inactive fragments of the enzyme. Moreover, several combinations of inactive variants of the bacterial enzyme obtained by site‐directed mutagenesis can yield active species. Heterodimers, resulting from a few selected combinations of inactive species of adenylate cyclase, exhibit specific activity similar to that of the native enzyme. Productive complementation from inactive fragments is a unique phenomenon among calmodulin‐activated enzymes and represents a new and helpful tool in the understanding of the molecular mechanism of activation of B. pertussis adenylate cyclase upon binding of calmodulin.
Properties of the two isoenzymes of methyl‐coenzyme M reductase in Methanobacterium thermoautotrophicum
Authors: BONACKER Lutz G. , BAUDNER Siegfried , MÖRSCHEL Erhard , BÖCHER Reinhard , THAUER Rudolf K.
Published: March 2005

Methyl‐coenzyme M reductase (MCR) catalyses the methane‐forming step in the energy metabolism of methanogenic Archaea. It brings about the reduction of methyl‐coenzyme M (CH3‐S‐CoM) by 7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP). Methanobacterium thermoautotrophicum contains two isoenzymes of MCR, designated MCR I and MCR II, which are expressed differentially under different conditions of growth. These two isoenzymes have been separated, purified and their catalytic and spectroscopic properties determined. Initial‐velocity measurements of the two‐substrate reaction showed that the kinetic mechanism for both isoenzymes involved ternary‐complex formation. Double reciprocal plots of initial rates versus the concentration of either one of the two substrates at different constant concentrations of the other substrate were linear and intersected on the abcissa to the left of the 1/v axis. The two purified isoenzymes differed in their Km values for H‐S‐HTP and for CH3‐S‐CoM and in Vmax. MCR I displayed a Km for H‐S‐HTP of 0.1–0.3 mM, a Km for CH3 S‐CoM of 0.6–0.8 mM and a Vmax of about 6 μmol · min−1· mg−1 (most active preparation). MCR II showed a Km for H‐S‐HTP of 0.4–0.6 mM, a Km for CH3‐S‐CoM of 1.3–1.5 mM and a Vmax of about 21 μmol · min−1· mg−1 (most active preparation). The pH optimum of MCR I was 7.0–7.5 and that of MCR II 7.5–8.0. Both isoenzymes exhibited very similar temperature activity optima and EPR properties.

The location of MCR I and of MCR II within the cell, determined via immunogold labeling, was found to be essentially identical.

The possible basis for the existance of MCR isoenzymes in M. thermoautotrophicum is discussed.
Core‐typing of O‐linked glycans from human gastric mucins
Authors: HANISCH Franz‐Georg , CHAI Wengang , ROSANKIEWICZ Jerzy R. , LAWSON Alexander M. , STOLL Mark S. , FEIZI Ten
Published: March 2005

Mucins from the pooled gastric juice of Lewis‐positive secretors were investigated to establish their glycosylation patterns with particular reference to the type and abundance of the glycan‐core structures. Following reductive β‐eliminination, the neutral glycan alditols from these mucins were fractionated by ion exchange and size‐exclusion chromatographies and subjected to structural analyses. It was possible to gain insights into the core sequences of the neutral O‐linked glycan alditols by matching (a) composition data from liquid secondary‐ion mass spectrometry of the native alditol fractions, (b) specific structural information on the core sequences by thin‐layer‐chromatography mass spectrometry of alditol‐derived neoglycolipids and (c) data from electron‐impact mass spectrometry of permethylated glycan alditols or their partially methylated alditol acetates. The predominant core structures detected among the neutral glycans representing about 77% (by mass) of the total carbohydrates released from gastric mucins were core 1, Galβ1‐3GalNAc (Ac, acetyl) and core 2, Galβ1–3(GlcNAcβ1–6)GalNAc in the approximate ratio 1:2. Core 3, GlcNAcβ1–3GalNAc, and core 4, GlcNAcβ1–3(GlcNAcβ1–6)GalNAc, were much less abundant (< 10%), while core 5, GalNAcα1–3GalNAc, core 6, GlcNAcβ1–6GalNAc, and a recently described sequence GalNAcα1–6GalNAc (core 7) were not detected. This investigation also addressed the question of the presence of the sequence Galβ1–6GalNAc which has been reported previously to occur as a core‐structure element in gastric mucins. This was greatly assisted by the availability of the authentic chemically synthetised disaccharide alditol which, when converted into a neoglycolipid after mild periodate oxidation, gives diagnostic ions in mass spectrometry and can be detected with high sensitivity. No evidence was found for the presence of this unusual sequence among the oligosaccharides in gastric mucins.
Aggregation of pyrene‐labeled microsomal glutathione S‐transferase
Authors: PIEMONTE Fiorella , CACCURI Anna M. , MORGENSTERN Ralf , ROSATO Nicola , FEDERICI Giorgio
Published: March 2005

Microsomal glutathione S‐transferase was labeled by the fluorescence probe N‐(1‐pyrenyl)maleimide which modified 1 mol thiol residue/mol protein. The enzyme activity increased about tenfold after the binding. The pyrene‐labeled microsomal glutathione S‐transferase exibited two fluorescence bands which are typical of pyrene; one at 393 nm attributable to unassociated pyrenes, the other at 480 nm attributable to pyrenes excimers (excited dimers). The excimeric fluorescence increased at high protein concentrations indicating a shift of the equilibrium of labeled polypeptide chains from trimeric complexes, the functional unit of microsomal glutathione S‐transferase, to larger aggregates. At 25°C and at a 1% Triton X‐100 concentration, the calculated equilibrium constant of this process is 65 μM. Along with the formation of large aggregates, a progressive increase of the enzymic activity was observed. Thus, N‐(1‐pyrenyl)maleimide appears to be a very useful probe to study the supramolecular structure of this enzyme.