bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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.
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.
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.
Comparison of X‐ray powder‐diffraction data of various bacterial lipopolysaccharide structures with theoretical model conformations
Authors: KASTOWSKY Manfred , GUTBERLET Thomas , BRADACZEK Hans 
Published: March 2005

X‐ray powder‐diffraction experiments have been performed on dry samples of lipid A and various rough‐mutant lipopolysaccharides (LPS) of Salmonella minnesota, Salmonella typhimurium and Escherichia coli. The diffraction patterns obtained indicated exclusively lamellar, bilayered arrangements in all samples. The periodicities were found to be in the range 4.5 nm for lipid A to 8.8 nm for Ra‐LPS. Upon treatment with water‐saturated air, swelling of the lamellar structures was achieved, as indicated by shifts of reflections. The increase in bilayer dimensions normally was about 0.3 nm.X‐ray intensities were used for the determination of the inner bilayer structure, i.e. for calculation of the one‐dimensional electron‐density distribution across the bilayer. For lipid A and several Re‐LPS, Rd2‐LPS, Rd1‐LPS and Rc‐LPS samples, a striking coincidence of the electron‐density distributions in the lipid‐A domain was found, suggesting that in all these structures the lipid‐A portion is similarly arranged. For Rb1 and Ra‐LPS, the lipid‐A domain could not be resolved due to the limited number of observed reflections. For other Re‐mutant lipopolysaccharide samples, quite different X‐ray patterns were obtained. Some samples yielded diffraction patterns indicating a very high state of order in the lipid‐A domain, whereas, in others, a significantly reduced order in the lipid‐A domain was infered.Comparison of the X‐ray data with features of a calculated three‐dimensional molecular model of lipopolysaccharide revealed reasonable agreement in molecular dimensions and bilayer structure.
Reconstruction of translation
Authors: GANOZA M. Clelia , AOKI Hiroyuki , KOZIERADZKI Ivona , SCHWARTZ Ira 
Published: March 2005

The in vitro reversal of conditionally lethal mutations has greatly aided the study of translation. N4316 is a mutant of Escherichia coli that has a temperature‐sensitive defect in a protein called the rescue protein. Without the rescue protein, translation in vivo and in vitro is drastically reduced and frameshift errors, as well as increased read‐through of nonsense codons, occurs. Using reversal of temperature‐sensitivity as an assay, the rescue protein was purified from a ribosomal eluate of the parental (D10) strain.Composite polyacrylamide/agarose gel electrophoresis and sedimentation on sucrose density gradients were employed to examine the distribution of 70S ribosomes and ribosomal subunits in the mutant (N4316) and the parental (D10) extracts at restrictive (43°C) and non‐restrictive (35°C) temperatures. Fewer polysomes and a larger proportion of 70S ribosomes relative to subunits were observed at 43°C with N4316, but not with D10 extracts. Addition of the rescue protein had no effect at 35°C with either strain, but restored the polysome pattern of N4316 at 43°C.The purified rescue protein labelied by methylation retained activity and bound preferentially to 30S subunits. Rescue bound to 30S particles prevented the action of IF‐3 fostering formation of 70S ribosomes. Thus the rescue protein enables formation of 70S ribosomes from 30S and 50S subunits. 70S ribosomes which contain the rescue protein are active in translation and resist dissociation induced by high centrifugal fields. We propose that the rescue protein alters the conformation of 70S ribosomes resulting in a tighter association of subunits which, in turn, fosters both higher rates and increased accuracy of translation.
Isolation, structural characterization and biological function of a lysine‐conopressin in the central nervous system of the Pharyngobdellid leech Erpobdella octoculata
Authors: SALZET Michel , BULET Philippe , VAN DORSSELAER Alain , MALECHA Jean 
Published: March 2005

Several neuropeptides are suspected to act on the control of hydric balance in leeches. One of these peptides, a peptide immunoreactive to an antibody against oxytocin, was previously characterized from the central nervous system of the leech Erpobdella octoculata [Salzet, M., Wattez, C., Verger‐Bocquet, M., Beauvillain, J.‐C. & Malecha, J. (1993) Brain Res. 601, 173–184].This paper reports the isolation from the central nervous system of E. octoculata of another peptide of the oxytocin/vasopressin family; a lysine‐vasopressin‐like molecule. Its purification was performed by reverse‐phase high‐performance liquid chromatography combined with both dot immunobinding assay and enzyme‐linked immunosorbent assay for lysine‐vasopressin. The amino acid sequence was established by Edman degradation and confirmed by electrospray‐mass‐spectrometry measurement. The nonapeptide obtained corresponded to the lysine‐conopressin previously isolated from the venom of the mollusc Conus geographus [Cruz, L. L., de Santos, V., Zafaralla, G. C., Ramilo, C. A., Zeikus, R., Gray, W. R. & Olivera, B. M. (1987) J. Biol. Chem. 262, 15821–15824]. In leeches, synthetic lysine‐conopressin exerts a diuretic effect which can be compared to that of the arginine‐vasopressin‐like peptide isolated in the Insect Locusta migratoria [Proux, J., Miller, C. A., Li, J. P., Carney, R. L., Girardie, A., Delaage, M. & Schooley, D. A. (1987) Biochem. Biophys. Res. Commun. 149, 180–186].
Site‐directed mutagenesis of β‐lactamase TEM‐1
Authors: LENFANT Françise , PETIT Agnès , LABIA Roger , MAVEYRAUD Laurent , SAMAMA Jean‐Pierre , MASSON Jean‐Michel 
Published: March 2005

From sequence alignments, two groups can be defined for the carbenicillin‐hydrolysing β‐lactamases (CARB enzymes). One group includes the Pseudomonas‐specific enzymes PSE‐1, PSE‐4, CARB‐3, CARB‐4 and also the Proteus mirabilis GN79, for which the well‐conserved residue Lys 234 in all class‐A β‐lactamases is changed to an arginine residue. The second group includes the enzymes PSE‐3 and AER‐1 which have an arginine or a lysine residue at position 165. All these enzymes also have leucine at position 68, threonine at position 104 and glycine at position 240. We engineered these mutations into the TEM‐1 β‐lactamase to study their potential role in defining the substrate profile of the CARB enzymes.The mutations K234R and E240G in TEM‐1 noticeably increased the hydrolysis of carboxypenicillins relative to other penicillins by approximately sixfold and twofold, respectively. The variant E240G also demonstrated an improved rate of second‐generation cephalosporin and cefotaxime hydrolysis. In contrast, the substitution of Trp165 by arginine does not extend the substrate profile to α‐carboxypenicillins nor does it noticeably modify the kinetic behavior of the enzyme. The mutations M68L and E104T do not have a large effect on the hydrolysis rate but the mutation E104T enhances the affinity of the enzyme for third‐generation cephalosporins. As the mutation K234R resulted in a severe decrease in the affinity for carboxypenicillins, the double mutant E240G/K234R was constructed in an attempt to enhance the CARB character of the enzyme. Contrary to what could be expected, the additional mutation E240G for the TEM‐1 K234R enzyme increases neither the catalytic constant for the carboxypenicillins nor the affinity towards these substrates. Consequently, this study strongly suggests that the three‐dimensional structures of the active site of the TEM‐1 enzyme and PSE‐3, PSE‐4 or other related enzymes are significantly different. This probably explains the discrepancy of the substrate profile between the CARB enzymes and the TEM‐1 protein variants.
The separate roles of plant cis and trans prenyl transferases in cis‐1,4‐polyisoprene biosynthesis
Authors: CORNISH Katrina 
Published: March 2005

In plants, the elongation of cis‐1,4‐polyisoprene (natural rubber, Mr > 106) requires a small trans‐allylic diphosphate (≦C20) initiator. The trans‐allylic diphosphates are hydrophilic cytosolic compounds, whereas cis‐1,4‐polyisoprene is hydrophobic and compartmentalised in subcellular rubber particles.In this paper, it is demonstrated that soluble trans‐prenyl transferase from latex of Hevea brasiliensis functions solely as farnesyl diphosphate synthase, and plays no direct role in cis‐1,4‐polyisoprene elongation. The cis‐1,4‐prenyl transferase is firmly associated with the H. brasiliensis rubber particle, as is also the case in other rubber‐producing species [Archer, B. L., Audley, B. G., Cockbain, E. G. & McSweeney, G. P. (1963) Biochem. J. 89, 565–574; Madhavan, S., Greenblatt, G. A., Foster, M. A. & Benedict, C. R. (1989) Plant Physiol. 89, 506–511; Siler, D. J. & Cornish, K. (1993) Phytochemistry 32, 1097–1102]. The experimental data explain and refute previous reports in which soluble trans‐prenyl transferase isolated from H. brasiliensis latex was attributed both trans‐prenyl transferase and cis‐prenyl transferase activities [Light, D. R. & Dennis, M. S. (1989) J. Biol. Chem. 264, 18589–18597; Light, D. R., Lazarus, R. A. & Dennis, M. S. (1989) J. Biol. Chem. 264, 18598–18607].Thus, it appears that plant prenyl transferases are comparable to animal enzyme systems in which trans‐prenyl transferases are soluble enzymes whilst cis‐prenyl transferases are membranebound [Ericcson, J., Runquist, M., Thelin, A., Andersson, M., Chojnacki, T. & Dallner, G. (1992) J. Biol Chem. 268, 832–838].
19F‐NMR study on the pH‐dependent regioselectivity and rate of the ortho‐hydroxylation of 3‐fluorophenol by phenol hydroxylase from Trichosporon cutaneum
Authors: PEELEN Sjaak , RIETJENS Ivonne M. C. M. , VAN BERKEL Willem J. H. , VAN WORKUM Wilbert A. T. , VERVOORT Jacques 
Published: March 2005

The regioselectivity and rate of the ortho‐hydroxylation of 3‐fluorophenol by phenol hydroxylase from Trichosporon cutaneum (EC 1.14.13.7) was studied using 19F‐NMR. The regioselective hydroxylation as well as the rate of ortho‐hydroxylation are pH dependent with a pKa of 6.5. At pH values below 6.5, 3‐fluorophenol preferentially becomes hydroxylated at the C6 ortho position, resulting in a maximum C6/C2 hydroxylation ratio of 6.7. Upon increasing the pH, the total rate of conversion increases. Also, the C2 ortho‐hydroxylation increases relatively to the C6 ortho‐hydroxylation and yields a minimum C6/C2 hydroxylation ratio of 2.2 at pH values above 7.5.Based on data from 19F‐NMR binding studies and molecular orbital calculations, a hypothesis is put forward which explains the pH‐dependent effects observed. A mechanism is proposed involving an active‐site amino acid residue acting as a base in the reduced form of the protein. Deprotonation of this residue results in hydrogen bond formation with the hydroxyl moiety of the phenolic substrate, leading to (partial) deprotonation of the substrate. Molecular orbital calculations demonstrate that such a (partial) deprotonation increases (a) the overall reactivity of 3‐fluorophenol for an electrophilic attack and (b) the reactivity of C2 relative to the C6 position. The hypothesis may explain the decrease in the C6/C2 hydroxylation ratio. Furthermore the increased amount of ortho‐hydroxylated products formed with increasing pH can also be explained by this hypothesis.
NAD turnover and utilisation of metabolites for RNA synthesis in a reaction sensing the redox state of the cytochrome b6f complex in isolated chloroplasts
Authors: PEARSON Colin K. , WILSON Stanley B. , SCHAFFER Robert , ROSS Alexander W. 
Published: March 2005

NAD is normally regarded as a redox molecule or as the substrate for ADP‐ribosylation reactions. In this study, we describe the rapid metabolism of NAD by Percoll‐gradient‐purified lettuce chloroplasts and show that the adenine moiety can be incorporated into RNA in a dark‐activated reaction that senses the redox state of the cytochrome b6f complex. Isolated chloroplasts rapidly metabolised radiolabelled NAD+ to 5′‐AMP (within seconds) and adenosine during a 60‐min incubation in vitro; the products were analysed by high‐performance liquid chromatography. No radiolabelled ADP‐ribose was detected. Radioactivity was incorporated into trichloroacetic‐acid‐insoluble material during this period, with approximately 2–4‐fold more incorporation occurring in the dark. Most of this radiolabel was rendered acid‐soluble by dilute alkaline digestion at 37°C, yielding an approximately equal mixture of 2′‐AMP and 3′‐AMP, and by RNase digestion, identifying the acid‐insoluble radioactive material as RNA. Protein‐bound ADP‐ribose would have yielded 5′‐AMP and/ or oligomeric/polymeric ADP‐ribose after alkali digestion. The utilisation of NAD metabolites for RNA synthesis was restricted to the thylakoid compartment of the chloroplast.The use of a variety of electron‐transport inhibitors such as 2,5‐dibromo‐3‐methyl‐6‐isopropyl‐p‐benzoquinone, bromanil (tetrabromo‐1,4‐benzoquinone), electron donors (dithiothreitol), electron acceptors (ferricyanide) and an uncoupler showed that the incorporation of radiolabel from NAD into acid‐insoluble material was favoured when the cytochrome b6f complex was in the oxidised state (as pertaining to incubations in the dark).
General occurrence of binding synergism in zinc proteases and its possible significance
Authors: CHAN William W.‐C. , PFUETZNER Richard A. 
Published: March 2005

The observation of binding synergism has been successfully extended to include carboxypeptidases A and B. The behaviour of these two enzymes follows the same pattern previously found for three other Zn‐proteases. Thus in all cases examined, the affinity of a suitable Zn‐ligand is increased in the presence of a compound (specificity probe) which contains the key structural features of specific substrates. A bifunctional ligand such as phosphonoacetate is particularly useful for generating synergism in both carboxypeptidases. Presumably the carboxylate moiety binds to the C‐terminal recognition site while the other functional group interacts with the metal ion. Several basic compounds (e.g. methyl guanidine) act as effective specificity probes for carboxypeptidase B while phenol and other hydrophobic substances serve this purpose in carboxypeptidase A.The above phenomenon appears to be a mechanism designed to enhance catalytic efficiency through a substrate‐induced conformational change. We postulate that there is a requirement for at least one ionizable group at the active site. The proposed mechanism keeps this group in the correct ionization state in the presence of water and increases its reactivity after exclusion of water by substrate binding. We suggest the term xerophilic shift for this process. Since proton transfer is a common process in enzyme reactions, the xerophilic‐shift mechanism may play a similar role in many instances. It should therefore be possibe to detect binding synergism in a wide variety of enzymes.