bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of exposed polypeptide loops in trimeric stability and membrane insertion of Escherichia coli OmpF porin
Authors: FOUREL Didier , BERNADAC Alain , PAGÈS Jean‐Marie
Published: March 2005

Different ompF–ompC gene fusions were used to analyse the regions involved in the stable trimerization and membrane insertion of the Escherichia coli OmpF porin. The stability of the trimers formed from the various hybrids was analysed. Three classes of trimer instability are observed related to the presence of different exposed polypeptide loops of OmpF. In all cases, amino acids located between residue 115 and residue 144 of OmpF are necessary to promote a correct and stable trimeric conformation. However, immunogold labelling studies indicate the correct insertion of the protein in the outer membrane despite a marked instability of some hybrid porins. The location of the residues involved in trimer stability is discussed with regards to both the three‐dimensional structure and the folding of OmpF.
Reversible and non‐denaturing replacement of iron by cadmium in Clostridium pasteurianum ferredoxin
Authors: BONOMI Francesco , GANADU Maria Luisa , LUBINU Giuseppe , PAGANI Silvia
Published: March 2005

Incubation of native, reduced Clostridium pasteurianum ferredoxin with different metals gave a range of modifications in the electronic and EPR spectrum of the protein, or made the signals disappear. The reduced protein, isolated after incubation with different metals under identical conditions (50 μM protein, 1 mM metal, 1 h incubation) was found to contain amounts of foreign metals increasing with their thiophylicity, i.e. Cd2+≫ Zn2+ > Co2+. Little, if any, incorporation was observed for Ni2+, Cu2+, Mn2+ or in the absence of reductant. The activity of substituted ferredoxins in a hydrogenase‐coupled assay was proportional to the amount of residual iron, suggesting that the residual iron is present in a population of intact active molecules rather than in partially substituted clusters distributed among individual molecules. The cadmium‐substituted ferredoxin did not contain iron, but contained eight cadmium atoms and six labile sulfide atoms/mol. Folding of the isolated, substituted proteins was investigated by CD and 1H‐NMR. Both techniques showed retention of the main structural features of the protein upon metal substitution. The rate and extent of the substitution of iron by cadmium were essentially independent of pH, but were found to decrease with increasing ionic strength and to increase with the cadmium concentration. In the cadmium‐substituted protein, cadmium was replaced by iron upon incubation with iron and mercaptoethanol in the absence of dithionite. In the presence of dithionite, cadmium was not replaced by iron upon incubation of the cadmium‐substituted protein with excess iron and mercaptoethanol. In competition experiments, incubation of iron‐containing ferredoxin with stoichiometric amounts of cadmium in the presence of dithionite and excess iron and mercaptoethanol resulted in quantifiable replacement of iron by cadmium. Therefore, substitution of iron by cadmium was only achieved under reducing conditions, and was only reversible in the absence of strong reductants.
C‐type natriuretic peptide stimulates secretion of growth hormone from rat‐pituitary‐derived GH3 cells via a cyclic‐GMP‐mediated pathway
Authors: SHIMEKAKE Yoshiyuki , OHTA Shigeki , NAGATA Kiyoshi
Published: March 2005

Although C‐type natriuretic peptide (CNP) has been shown to exist at the highest concentration in the anterior pituitary in rat tissues, its physiological role(s) there is (are) not clear. In this study, we report a novel function of CNP examined with anterior pituitary‐derived cell lines, GH3 and AtT20/D16v‐F2. Both CNP and atrial natriuretic peptide (ANP) increased cellular cGMP levels in both cell lines in dose‐dependent manners. CNP, but not ANP, stimulated growth hormone (GH) release from GH3 cells. In contrast, neither ANP nor CNP had any significant effect on the corticotropin release from AtT20/D16v‐F2 cells. An activator for cGMP‐dependent protein kinase (cGK), dibutyryl cGMP, mimicked the stimulation of GH release from GH3 cells by CNP. Constitutive GH release from GH3 cells was greatly diminished in the presence of inhibitors for cAMP‐dependent protein kinase, while stimulative GH release by CNP was not affected. However, inhibitors which can block cGK almost completely diminished the stimulative effect of CNP. An inhibitor for protein kinase C did not show any effect on either constitutive or CNP‐stimulative GH release. Our observations indicate that the stimulation of GH release from GH3 cells by CNP is mediated mainly by the cGK signal‐transduction pathway, not by cAMP‐dependent protein kinase or protein kinase C, through a CNP‐specific receptor (possibly ANP‐B receptor). Thus, CNP may act as a local modulator in the anterior pituitary.
Conformational analysis of elcatonin in solution
Authors: OGAWA Kiyoshi , NISHIMURA Shigenori , DOI Masamitsu , KYOGOKU Yoshimasa , HAYASHI Mitsuo , KOBAYASHI Yuji
Published: March 2005

The conformational characteristics of elcatonin, an analogue of eel calcitonin having a disulfide bond Cys1‐Cys7 replaced by an ethylene linkage between residues 1 and 7, have been analyzed in aqueous trifluoroethanol solutions. Circular dichroic spectra of elcatonin and eel calcitonin itself reveal the presence of α‐helices at trifluoroethanol concentrations above 15%. The spectral changes caused by the trifluoroethanol content of the solutions are interesting. An isosbetic point is detected for eel calcitonin indicating that a conformational transition occurs between two states, namely α‐helical and random coil states. On the other hand, the CD curves of elcatonin at less than 15% trifluoroethanol deviate from the isosbetic point while those at higher concentration are similar to those of eCT. This can probably be attributed to the third element of the ordered structure of elcatonin which is formed in 15% trifluoroethanol.

The solution conformation of elcatonin in a mixture of 60% water and 40% trifluoroethanol has been determined by the combined use of 1H‐NMR spectroscopy and distance geometry calculations. The conformation is characterized by an amphiphilic α‐helix between Thr6 and Thr21, which extends into the constrained cyclic portion of the molecule to Thr6. The third structural element of elcatonin found in the CD analysis is detected by some turn structures in the region between residue 1 and Ser5 in the calculated structure.
Ca2+ efflux from platelets
Authors: CAVALLINI Lucia , ALEXANDRE Adolfo
Published: March 2005

Large amounts of Ca2+ (almost 20 nmol/108 cells) are released from platelets by exocytosis. This secretory‐granule‐associated Ca2+ does not contribute to the cytosolic free Ca2+ ([Ca2+]i), which is controlled by the much smaller agonist‐sensitive Ca2+ pool, unless high (1 μM), but not low (0.04 μM) concentrations of ionomycin are present. Low concentrations of ionomycin release Ca2+ almost exclusively from the agonist‐sensitive stores. In aspirinated platelets incubated in the presence of 0.5 mM EGTA the extensive depletion of the agonist‐sensitive stores is obtained by the combined action of low ionomycin and the endomembrane Ca2+‐ATPase inhibitor thapsigargin (which individually promote only a partial depletion). The subsequent decay of [Ca2+]i is increased by phorbol‐myristate acetate, confirming that Ca2+ efflux from platelets is potentiated by the activation of protein kinase C [Pollock, W. K, Sage, S. O. & Rink, T. J. (1987) FEBS Lett. 210, 132–140]. A novel type of control of Ca2+ efflux appears to be exerted by the filling state of the stores. Treatment with low ionomycin or thapsigargin determines the release of a fraction of the stores‐associated Ca2+; the subsequent decay of [Ca2+]i is slow. The decay rate of [Ca2+]i accelerates after extensive depletion of the stores following the addition of thapsigargin or ionomycin. If the depletion of the stores is induced by thrombin, added alone or in combination with thapsigargin, the increases of [Ca2+]i are the same and the subsequent decay rates are largely superimposable; however a large fraction of [Ca2+]i is reaccumulated into the stores in the absence, but not in the presence of thapsigargin, indicating that Ca2+ efflux is activated when the stores are empty. Ca2+ efflux can proceed against a concentration gradient. In 45Ca‐loaded platelets, the thrombin‐promoted 45Ca efflux is potentiated by thapsigargin. The protein‐kinase‐C‐dependent and store‐depletion‐dependent stimulations of 45Ca efflux are additive. These observations indicate that, in addition to being activated by protein kinase C, Ca2+ efflux from platelets is activated by the depletion of the stores. The two activations appear to be additive.
Induction, localization and metal content of hydrogenase in the green alga Chlamydomonas reinhardtii
Authors: HAPPE Thomas , MOSLER Britta , NABER J. Dirk
Published: March 2005

The hydrogenase enzyme occurring in Chlamydomonas reinhardtii is induced by anaerobic adaptation of the cells. In aerobically growing cells, antibodies against the hydrogenase failed to detect either active or inactive enzyme. However, already 10 min after the onset of anaerobic adaptation, the protein could be detected. The maximal amount of enzyme was reached after 2–3 hours anaerobiosis. Addition of nickel or iron to the growth medium did not influence activity. In atomic absorption experiments, a Ni/Fe ratio of about 1:250 was measured. We, therefore, propose the hydrogenase from C. reinhardtii to be of the Fe‐only type. Adaptation in the presence of uncouplers of phosphorylation showed this process to be energy‐dependent. From protein synthesis inhibition experiments, it is concluded that the protein is synthesized on cytoplasmic ribosomes and, therefore, must be nuclear encoded. After isolation of intact chloroplasts from adapted cells, the active enzyme was shown, by Western‐blotting analysis, to be located in the chloroplasts.
Apparent cooperativity for carbamoylphosphate in Escherichia coli aspartate transcarbamoylase only reflects cooperativity for aspartate
Authors: ENGLAND Patrick , LECONTE Claire , TAUC Patrick , HERVÉ Guy
Published: March 2005

The reaction catalyzed by Escherichia coli aspartate transcarbamoylase (ATCase) proceeds through an ordered mechanism, in which carbamoylphosphate binds first, followed by aspartate; upon binding of this second substrate, the enzyme undergoes a concerted transition from a low‐affinity T state to a high‐affinity R state. In various studies, conflicting results were obtained concerning the existence of positive cooperativity for the first substrate, carbamoylphosphate. It is shown here that cooperativity for this substrate is only apparent. Indeed, saturation curves for carbamoylphosphate display sigmoidicity only if the aspartate concentration used is high enough to shift ATCase into the R state. Furthermore, it is shown that succinate, an unreactive aspartate analogue which is able to promote the T→R conformational transition, also induces the appearance of cooperativity for carbamoylphosphate. Similar results were obtained in the course of continuous‐flow‐dialysis experiments, which show that the binding of carbamoylphosphate is apparently cooperative only in the presence of a concentration of succinate high enough to shift the enzyme into the R state. Taken together, these data show that the apparent cooperativity for carbamoylphosphate is not an intrinsic property of ATCase, as it only reflects the cooperativity for the second substrate, aspartate, as a consequence of the process of ordered substrate binding.
Probing the interaction of the multidrug‐resistance phenotype with the polypeptide ionophore gramicidin D via functional channel formation
Authors: ASSARAF Yehuda G. , BORGNIA Mario J.
Published: March 2005

It has been proposed that the multidrug resistance (MDR) transporter, P‐glycoprotein (P‐170), may be physiologically involved in the transport of polypeptides. As a step towards understanding the interaction of P‐170 with polypeptides, we isolated various gramicidin‐d‐resistant mammalian cell lines. Gramicidin D is a hydrophobic pentadecapeptide ionophore that forms proton and alkali metal cation‐permeable channels in lipid bilayers. Gramicidin‐d‐resistant cells displayed a prominent MDR gene amplification, P‐170 overexpression, reduced drug accumulation, and consequent resistance to MDR‐type cytotoxic agents. Modulators of the MDR phenotype, including verapamil, reserpine and quinidine, rendered these cells sensitive to gramicidin D. Using these cell lines, we established an assay that probes for the intra‐membranal interaction between P‐170 and gramicidin D. Gramicidin‐D channel formation was followed by cellular accumulation of 86Rb+. Ionophore‐resistant cells, and other MDR cells, did not show an appreciable increase in 86Rb+ influx rates, in the presence of increasing gramicidin‐D concentrations. In contrast, parental cells displayed a dose‐dependent increase in the 86Rb+ influx rates. Interestingly, in the absence of serum, gramicidin‐d‐resistant cells resumed the wild‐type, ionophore‐dose‐dependent increase in 86Rb+ influx rates. MDR modulators caused a resumption of channel formation in ionophore‐resistant cells. We conclude that acquisition of the MDR phenotype is an efficient means of cellular protection against gramicidin D. Hence, a new approach is offered in which P‐170 interaction with gramicidin D is quantitatively followed by a rapid assessment of the biological activity (i.e. channel formation) of the substrate itself. Possible mechanisms of P‐170 interaction with free ionophore monomers, and membrane‐associated gramicidin D are discussed.
Cloning and expression of eel natriuretic‐peptide receptor B and comparison with its mammalian counterparts
Authors: KATAFUCHI Takeshi , TAKASHIMA Akira , KASHIWAGI Masahide , HAGIWARA Hiromi , TAKEI Yoshio , HIROSE Shigehisa
Published: March 2005

A comparative study of the natriuretic‐peptide receptor NPR‐B was performed by cloning and expressing, in COS‐1 cells, the NPR‐B receptor subtype from the eel gill which exhibited a strong C‐type‐natriuretic‐peptide(CNP)‐induced guanylate cyclase activity. Like other mammalian NPR‐B receptors, the eel NPR‐B receptor consisted of a ligand‐binding extracellular domain, a hydrophobic transmembrane domain, a kinase‐like domain and a guanylate cyclase domain. Sequence comparison among the eel and mammalian receptors revealed a relatively low similarity (∼44%) in the extracellular domain compared to a very high similarity (∼ 84 %) in the cytoplasmic regulatory and catalytic domains. This low similarity allowed identification of the amino acid residues or candidate regions important for the ligand‐binding activity. RNase protection analysis of the eel NPR‐B mRNA demonstrated that the message was predominantly expressed in the liver and atrium as well as in the gill with moderate‐to‐small amounts in the brain, ventricle, esophageal sphincter, stomach, posterior intestine and kidney.

The high NPR‐B mRNA levels in the liver, atrium and gill were found to decrease markedly when eels were transferred from fresh water to seawater and kept there for 2 weeks. Since similar changes are known to occur in the ligand CNP levels when eels are facing osmotic challenges, the CNP/NPR‐B system appears to play an important role in their successful adaptation to salinity changes.
The adsorption protein of bacteriophage fd and its neighbour minor coat protein build a structural entity
Authors: GAILUS Valérie , RASCHED Ihab
Published: March 2005

The adsorption protein g3p and another minor coat protein, g6p, are located at one end of the filamentous bacteriophage fd [Grant, R. A., Lin, T. C., Konigsberg, W. E. & Webster, R. E. (1981) J. Biol. Chem. 256, 539–546]. Both proteins, representing the proximal tip, were detached as an entity by a technique that allowed for gentle solubilization. Disrupting the phage particle with the detergent sodium deoxycholate and chloroform dissociates the major coat protein g8p, frees the phage DNA, but leaves g3p and g6p associated with each other. The g3p‐g6p complex, which we termed the adsorption complex, and an oligomeric form of g3p with lower molecular mass were isolated and purified by gel‐filtration chromatography in the presence of deoxycholate. These different oligomeric structures of g3p showed a different mobility in non‐denaturing polyacrylamidegel electrophoresis. Both forms were also found in non‐denaturing polyacrylamide‐gel electrophoresis from deoxycholate‐ and Triton‐X‐100‐solubilized phage without prior chromatographic separation. The two oligomeric forms of g3p are composed of two g3p polypeptide chains in the case of the low‐molecular‐mass species, and four g3p and four g6p polypeptide chains for the adsorption complex.
Purification of a sixth ferredoxin from Rhodobacter capsulatus
Authors: NAUD Isabelle , VINÇON Mathilde , GARIN Jérôme , GAILLARD Jacques , FOREST Eric , JOUANNEAU Yves
Published: March 2005

A new ferredoxin has been purified from the photosynthetic bacterium Rhodobacter capsulatus. It is the sixth ferredoxin to be isolated from this bacterium and it was called FdVI.

Its primary structure was established based on amino acid sequence analysis of the protein and of peptides derived from it. It is composed of 106 residues including five cysteines. The calculated mass of the polypeptide is 11402.6 Da which matches the experimental value determined by electrospray mass spectrometry. Amino acid sequence comparison revealed that ferredoxin VI (FdVI) is strikingly similar to a ferredoxin from Caulobacter crescentus and to the putidaredoxin from Pseudomonas putida.

FdVI exhibited an ultraviolet‐visible absorption spectrum typical for a [2Fe‐2S] ferredoxin. EPR spectroscopy of the reduced protein showed a nearly axial signal similar to that of mitochondrial and P. putida ferredoxins.

FdVI is biosynthesized in cells growing anaerobically under either nitrogen‐sufficient or nitrogen‐deficient conditions. Although the function of FdVI is unknown, its structural resemblance to [2Fe‐2S] ferredoxins known to transfer electrons to oxygenases such as P‐450 cytochromes, suggests that FdVI may have a similar role in R. capsulatus.
Primary structure of maize chloroplast adenylate kinase
Authors: SCHILTZ Emile , BURGER Sabine , GRAFMÜLLER Rita , DEPPERT Wolfgang R. , HAEHNEL Wolfgang , WAGNER Edgar
Published: March 2005

This paper describes the sequence of adenylate kinase (Mg‐ATP + AMP ⇄ Mg‐ADP + ADP) from maize chloroplasts. This light‐inducible enzyme is important for efficient CO2fixation in the C4cycle, by removing and recycling AMP produced in the reversible pyruvate phosphate dikinase reaction.

The complete sequence was determined by analyzing peptides from cleavages with trypsin, Asp‐N protease and CNBr and subcleavage of a major CNBr peptide with chymotrypsin. N‐terminal Edman degradation and carboxypeptidase digestion established the terminal residues. Electrospray mass spectrometry confirmed the final sequence of 222 residues (Mr= 24867) including one cysteine and one tryptophan.

The sequence shows this enzyme to be a long‐variant‐type adenylate kinase, the nearest relatives being adenylate kinases from Enterobacteriaceae. Alignment of the sequence with the adenylate kinase fromEscherichia colireveals 44% identical residues. Since theE. colistructure has been published recently at 0.19‐nm resolution with the inhibitor adenosine(5′)pentaphospho(5′)adenosine (Ap5A) [Müller, C. W. & Schulz, G. E. (1992)J. Mol. Biol. 224, 159–177], catalytically essential residues could be compared and were found to be mostly conserved. Surprisingly, in the nucleotide‐binding Gly‐rich loop Gly‐Xaa‐Pro‐Gly‐Xaa‐Gly‐Lys the middle Gly is replaced by Ala. This is, however, compensated by an Ile→Val exchange in the nearest spatial neighborhood. A Thr→Ala exchange explains the unusual tolerance of the enzyme for pyrimidine nucleotides in the acceptor site.
Two‐dimensional 1H‐NMR studies of maize lipid‐transfer protein
Authors: PETIT Marie‐Christine , SODANO Patrick , MARION Didier , PTAK Marius
Published: March 2005

Correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY) and NOE spectroscopy (NOESY) experiments have been used to assign sequentially the 1H 500‐MHz NMR spectra of a non‐specific (ns) lipid‐transfer protein extracted from maize seeds. The spin‐system identification and sequential assignment were combined with secondary‐structure determination to identify most of the proton resonances of this 93‐residue protein. From the sequential connectivities it was established that the secondary structure mainly involved four helical fragments: H1, H2, H3 and H4. This secondary structure was compared with that of wheat ns‐lipid‐transfer protein recently determined. The four helices are located in nearly the same regions, but helix H4 is appreciably longer in the maize protein than in the wheat protein. Comparison of the transfer activities reveals that the maize protein is more efficient than the wheat ns‐lipid‐transfer protein and that this difference is probably due to the affinity of the lipid for the binding site and not to the interfacial activation, i.e. adsorption of the ns‐lipid‐transfer protein to the membrane. From these results, it is suggested that helix H4 is a part of the lipid‐binding site or contributes to the folding of this site. The present data define the basis for a further modelling of the three‐dimensional structure of the maize ns‐lipid‐transfer protein which will be compared with that of the wheat ns‐lipid‐transfer protein in order to establish structure/activity relationships for this class of carriers by using natural ns‐lipid‐transfer protein mutants.
Carotenoid biosynthesis in microorganisms and plants
Authors: SANDMANN Gerhard
Published: March 2005

CONTENTS. Carotenogenic in vitro systems – Strategies for the cloning of carotenogenic genes – Enzymology and molecular genetics of the reactions leading to cyclic carotenoids– Synthesis of GGPP – Phytoene synthesis – Desaturation sequence of phytoene to lycopene –Mechanism – Isomerization – Genes and phylogenetic relationship – Enzyme purification – Localization – Regulation of the pathway and modulation of activity – Developmental and physiological regulation– Lycopene cyclization – Formation of xanthophylls derived from cyclic carotenoids –Hydroxy carotenoids – Keto carotenoids – Epoxy carotenoids–Formation of acyclic xanthophylls in Rhodobacter– Conclusion and outlook
Generation of reactive‐oxygen species induced by electropermeabilization of Chinese hamster ovary cells and their consequence on cell viability
Authors: GABRIEL Bruno , TEISSIÉ Justin
Published: March 2005

Cells can be permeabilized transiently when pulsed by a brief intense external electric field. The molecular and cellular bases of cell electropermeabilization are still unclear. This process can be described by a local transient membrane organisation in which high permeability exists. In this study, using the chemiluminescent probe lucigenin, we showed that electropulsation of Chinese hamster ovary cells induced generation of reactive oxygen species at the electropermeabilized cell level. This generation was directly associated with the part of the membrane surface which is electrically restructured, as shown by its dependence on electric parameters. The electroinduced cell process was activated by Ca2+ and Mg2+ ions, and by exogenous adenosine 5′‐triphosphate. A metal‐ion‐catalyzed Haber‐Weiss reaction was thought to occur in the process, as shown by the action of effector molecules of Haber‐Weiss reaction such as the chelating agent EDTA, and the hydroxyl radical scavenger dimethylsulfoxide. The modulation of the oxygen species electroinduction and the use of antioxidant products (dimethylsulfoxide, sodium l‐ascorbate) showed that cell survival after electric treatment was directly correlated to the oxidative jump intensity. This observation had to be associated with the cell‐damaging action of oxygen‐reactive species.
Inhibition of cell adhesion to glycoproteins of the extracellular matrix by peptides corresponding to serum amyloid A
Authors: PRECIADO‐PATT Liana , LEVARTOWSKY David , PRASS Mordechai , HERSHKOVIZ Rami , LIDER Ofer , FRIDKIN Mati
Published: March 2005

Synthetic peptides related to amino acid residues 29–42 of human serum amyloid A (SAA), Tyr‐Ile‐Gly‐Ser‐Asp‐Lys‐Tyr‐Phe‐His‐Ala‐Arg‐Gly‐Asn‐Tyr, were found to inhibit the adhesion of human T‐lymphocytes and of mouse M4 melanoma cells to surfaces coated with the major cell adhesive glycoproteins of the extracellular matrix, laminin or fibronectin. Correspondingly inhibitory activity was manifested by the entire 14‐residue peptide, by its YIGSD laminin‐related domain, and by RGN, the fibronectin‐related domain. Intact recombinant SAA (rSAA) and its 1–76 fragment, an amyloid A (AA) protein, also inhibited cell adhesion. The peptides did not inhibit collagen and ADP‐induced aggregation of human platelets. Proteolysis of SAA by lysosomal enzymes originating from human neutrophils led to generation of specific peptide segments some of which pertain to the 29–42 domain. It is suggested that the acute‐phase protein SAA might be involved, either directly or via its peptide fragments, in inhibition of inflammatory reactions or metastatic processes which depend on integrin and possibly other extracellular‐matrix‐specific receptors mediated specific recognition and interactions with immobilized components of blood‐vessel walls.
Soluble forms of α‐d‐mannosidases from rat liver
Authors: GRARD Thierry , SAINT‐POL Agnès , HAEUW Jean‐François , ALONSO Catherine , WIERUSZESKI Jean‐Michel , STRECKER Gérard , MICHALSKI Jean‐Claude
Published: March 2005

We have previously reported the substrate specificity of the rat liver cytosolic α‐d‐mannosidase [Haeuw, J. F., Strecker, G., Wieruszeski, J. M., Montreuil, J. & Michalski, J.‐C. (1991) Eur. J. Biochem. 202, 1257–1268]. Here, we report the characterization and the purification of this α‐d‐mannosidase and the presence of two soluble forms of α‐d‐mannosidases from rat liver. The cytosolic α‐d‐mannosidase was purified nearly 660‐fold with 2.66% recovery to a state approaching homogeneity using: (a) (NH4)2SO4 precipitation; (b) concanavalin‐A–Sepharose chromatography; (c) affinity chromatography on a cobalt‐chelating Sepharose column; (d) ion‐exchange (DEAE‐trisacryl M) column chromatography; (e) molecular‐size chromatography (Sephacryl S 200). The enzyme was eluted from the final column at an apparent molecular mass of 113 kDa. SDS/PAGE analysis yielded a major protein band at 108 kDa. Moreover, the purification allowed to distinguish two mannosidase activities with different kinetic properties. The first cytosolic activity retained on the cobalt‐chelating column was optimally active at neutral pH, was activated by Co2+, was strongly inhibited by swainsonine (Ki= 3.7 μM) but not by deoxymannojirimycin and was active with p‐nitrophenyl α‐d‐mannoside (Km= 0.072 mM). Man9GlcNAc was hydrolysed by the purified enzyme down to a Man5GlcNAc structure, i.e. Man(α1‐2)Man(α1‐2)Man(α1‐3)[Man(α1‐6)]Man(β1‐4)GlcNA c, which represents the Man5 oligosaccharide chain of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N‐glycosylprotein glycans. The second activity not retained on the cobalt‐chelating column was optimally active at neutral pH, was inhibited by swainsonine (Ki= 28.4 μM) but not by deoxymannojirimycin and was active with p‐nitrophenyl α‐d‐mannoside (Km= 0.633 mM). Man9GlcNAc was broken by this enzymic activity down to Man8GlcNAc and Man7GlcNAc structures. Similitaries with endoplasmic reticulum α‐d‐mannosidase exist and this enzyme could be the cytosolic form of the endoplasmic reticulum α‐d‐mannosidase.
Mutations of iso‐1‐cytochrome c at positions 13 and 90 Separate effects on physical and functional properties
Authors: HUANG Yue , BEESER Scott , GUILLEMETTE J. Guy , STORMS Reginald K. , KORNBLATT Jack A.
Published: March 2005

Residues at positions 13 (lysine or arginine) and 90 (glutamate or aspartate) of eukaryotic cytochromes c have been conserved during evolution; Cys102, however, is found only in yeast cytochrome c. The positively charged residue at position 13 and the negatively charged residue at position 90 are close together in those cytochromes c for which three‐dimensional structures are available. We have replaced the amino acids at these two positions by cysteine in Saccharomyces cerevisiae iso‐1‐cytochrome c; in an earlier study, Cys102 was replaced by threonine without negatively influencing the physical or enzymic properties of the protein. The mutated proteins [R13C, C102T]cytochrome c (iso‐1‐cytochrome c containing Arg13→Cys and Cys102→Thr mutations), [D90C, C102T]cytochrome c (iso‐1‐cytochrome c containing Asp90→Cys and Cys102→Thr mutations) and [R13C, D90C, C102T]cytochrome c (iso‐1‐cytochrome c containing Arg13→Cys, Asp90→Cys, and Cys102→Thr mutations) are functional in vivo. Free sulfhydryl titration shows that the doubly mutated forms each contain one sulfhydryl group while the triple mutant contains two sulfhydryl groups. The stability of mutant [R13C, C102T]cytochrome c resembles that of [C102T] cytochrome c, whereas the stability of [D90C, C102T]cytochrome c resembles the stability of [R13C, D90C, C102T]cytochrome c. The activity of cytochrome‐c oxidase using cytochrome c was monitored polarographically. Compared to the wild‐type or [C102T]cytochrome c, which shows two kinetic phases with cytochrome‐c oxidase, [D90C, C102T]cytochrome c has much the same profile; [R13C, C102T]cytochrome c and [R13C, D90C, C102T]cytochrome c exhibit one kinetic phase with decreased activity. Electron‐transfer activity of the mutant cytochromes c is inhibited by Hg2+. The inhibition is highest for the triple mutant, less for [R13C, C102T]cytochrome c, even less for [D90C, C102T]cytochrome c and insignificant for the wild type. It would appear as though the stability of the triple mutant follows the changes that result from the Asp90→Cys mutation while the activity changes follow those of the Arg13→Cys mutation.
P2 protamines are phosphorylated in vitro by protein kinase C, whereas P1 protamines prefer cAMP‐dependent protein kinase
Authors: PIRHONEN Arja , LINNALA‐KANKKUNEN Annikka , MÄENPÄÄ Pekka H.
Published: March 2005

P1 protamines isolated from ejaculated human, stallion, bull, boar and ram spermatozoa and P2 protamines from human and stallion spermatozoa were subjected, after alkaline phosphatase treatment, to in vitro phosphorylation reactions using cAMP‐dependent protein kinase (PKA) and protein kinase C (PKC). All P1 protamines were phosphorylated by PKA, whereas P2 protamines were phosphorylated only by PKC. In addition, human, stallion and boar, but not bull and ram, P1 protamines were phosphorylated by PKC. After phosphoamino acid analysis, the protamines showing positive signals for phosphoserine (P‐Ser) were subjected to P‐Ser conversion reaction and protein sequencing. Only stallion (St1) and human (HP1) P1 protamines contained P‐Ser after PKA phosphorylation, located in the middle region of the molecule, i.e., at Ser29 in St1 and Ser28 in HP1. All other phosphorylated P1 protamines contained only P‐Thr, which could not be further localized in the sequence with the present methods. After PKC phosphorylation, the internally located Ser residues in human (Ser21) and stallion (Ser29) P1 protamines were phosphorylated and, in boar P1 protamine, only Thr43 was slightly phosphorylated. The N‐terminally located Ser residues in P1 protamines, which are known to be phosphorylated in vivo, were not phosphorylated by either kinase, indicating that there must still be other types of protamine kinases in sperm cells responsible for their phosphorylation. Within P2 protamines, HP2 was equally well phosphorylated at all Ser residues in addition to some Thr phosphorylation, whereas, in St2, Ser32 was the main target for PKC phosphorylation in vitro. Collectively, PKC is a good candidate for in vivo phosphorylation of P2 protamines and PKA for phosphorylation of some hydroxyamino acid residues in P1 protamines.
Analysis of heparan‐sulphate chains and oligosaccharides from proliferating and quiescent fibroblasts
Authors: SCHMIDTCHEN Artur , FRANSSON Lars‐Åke
Published: March 2005

Human skin fibroblasts in different growth states were incubated with [3H]glucosamine and/or Na235SO4 and extracted with Triton X‐100 for various periods of time. Free heparan‐sulphate oligosaccharides and protein‐bound heparan‐sulphate chains were separated by chromatography on octyl‐Sepharose and analyzed. A pool of endogenously produced oligosaccharides, present in the cultured cells and isolated after brief extraction, contained fragments of uniform size (approximately 7–10 kDa corresponding to approximately 14–20 disaccharides). Analysis by heparinase I and heparinase III degradations followed by electrophoretic separation (oligosaccharide mapping) showed that the oligosaccharides were rich in glucuronic acid but had a few sulphated iduronic acid residues at the periphery of each molecule. These results indicated that endoheparanase cleavage points were located close to linkages between N‐sulphated glucosamine and sulphated iduronic acid, generating fragments that comprise a major portion of the unmodified segments and a minor portion of the highly modified segments.

Prolonged extraction (24–48 h) of cells with Triton X‐100 at 4°C in the presence of proteinase inhibitors resulted in further degradation. There was an increase in the amount of heparan‐sulphate oligosaccharides and a concomitant decrease in the amount of protein‐bound heparan‐sulphate chains present in the same extract. The heparan‐sulphate oligosaccharides obtained after prolonged extraction were more heterogeneous in size comprising, in addition to the major species of approximately 7–10 kDa, intermediate and larger fragments of approximately 17 kDa and 30–40 kDa. This observation suggests that endoheparanase acted at periodically appearing, specific regions in the intact heparan‐sulphate chain. Furthermore, the enzyme and substrate should remain closely associated during cold Triton X‐100 extraction.

To determine if the endogenously produced heparan‐sulphate oligosaccharides were derived from a particular heparan‐sulphate species degraded during the growth phase, proteoglycan‐derived heparan‐sulphate chains obtained from proliferating or quiescent fibroblasts were also examined. These chains showed similar oligosaccharide maps, except for a small increase in the amount of glucuronic acid as cell growth was arrested. Hence, an endoheparanase with restricted specificity may generate slightly different oligosaccharides in the various growth states.
Purification of ATP synthase from Acetobacterium woodii and identification as a Na+‐translocating F1FO‐type enzyme
Authors: REIDLINGER Jutta , MÜLLER Volker
Published: March 2005

The ATPase of Acetobacterium woodii was purified after solubilization of membranes with Triton X‐100 by poly(ethylene glycol) precipitation and gel filtration. The enzyme consists of at least six subunits of apparent molecular masses of 57, 52, 35, 19, 15 and 4.8 kDa, as determined by SDS/PAGE. The 52‐kDa band is immunologically related to the F1FO‐ATPase β subunit of Escherichia coli. The enzyme is not inhibited by vanadate but is inhibited by nitrate, azide and N,N′‐dicyclohexylcarbodiimide; the 4.8‐kDa subunit specifically reacts with N,N′‐dicyclohexyl[14C]carbodiimide, indicating that the enzyme is of the F1FO type. The enzyme activity is dependent on MgATP (Km= 0.4), has a pH optimum of pH 7–9 and is stimulated by sulfite. ATP hydrolysis is strictly dependent on sodium ions with a Km for Na+ of 0.4 mM. The purified enzyme was reconstituted into liposomes. Upon addition of ATP, primary and electrogenic 22Na+ transport into the lumen of the proteoliposomes was determined. These experiments demonstrate that the ATPase of Acetobacterium woodii is a Na+‐translocating F1FO‐type ATPase.
Hemoglobin function under extreme life conditions
Authors: CLEMENTI Maria E. , CONDÒ Saverio G. , CASTAGNOLA Massimo , GIARDINA Bruno
Published: March 2005

Considering the variety of species that depend on hemoglobin for oxygen transport, these molecules must execute their primary function under extreme environmental conditions. Hence, a thermodynamic analysis of oxygen binding with hemoglobins from different species reveals a series of adaptive mechanisms which are based on the thermodynamic connection between the binding of heterotropic effectors and the reaction with oxygen.

The examples reported, from fishes to human fetus, illustrate how evolution can alter the structural basis of the heterotropic interactions to optimize the oxygenation‐deoxygenation cycle in dependence of the physiological needs of the particular organisms. Moreover they show that a thermodynamic analysis of the reaction with oxygen overcomes the meaning of a detailed structural and functional characterization going deeper into the physiology of the specific organism.
Transcriptional activation of human leukosialin (CD43) gene by Sp1 through binding to a GGGTGG motif
Authors: KUDO Shinichi , FUKUDA Minoru
Published: March 2005

Human leukosialin (CD43) is expressed on the surface of hematopoietic cells in cell‐type specific and differentiation‐stage‐specific manners. Previously we found that the sequence from –53 to –40 was critically involved in the promoter function [Kudo, S. & Fukuda, M. (1991) J. Biol. Chem. 266, 8483–8489]. A transient‐expression assay using a chloramphenicol acetyltransferase reporter gene revealed that the promoter could confer a high basal transcriptional activity in both leukosialin‐producing and non‐producing cells. The transcription factor interacting with the promoter sequence was determined by DNase I footprinting and gel‐mobility‐shift assays. The nuclear extracts from both leukosialin‐producing Jurkat cells and non‐producing Hela cells showed a footprint on the 5′ flanking region from −58 to −34. Gel‐mobility‐shift assays revealed that DNA‐protein complexes were formed with both nuclear extracts, and these complex formations were inhibited by an oligonucleotide containing the Sp1‐binding consensus sequence. Prior incubation of anti‐Sp1 antibody with nuclear extracts in this assay resulted in the supershift of the band for the DNA‐protein complex. In addition, the footprint produced by the purified Sp1 transcription factor was identical to those produced by nuclear extracts of Jurkat and Hela cells. The mutational analyses revealed that the binding affinities of Sp1 to mutated promoter sequences were parallel to the transcriptional activity of these promoter sequences. Transient expression analyses in Drosophila Schneider cells demonstrated that cotransfection with Sp1 expression plasmid increased the transcriptional activity. These results establish that Sp1 can bind to the promoter and positively regulates the expression of the leukosialin gene. Even the stable expression of CAT constructs in non‐producing Hela cells showed high transcriptional activity. The leukosialin expression thus appears to be regulated by the unique mechanism, that is the repression of high basal transcriptional activity rather than the activation of the basal transcriptional level. Tissue‐specific expression is probably achieved by suppression of the basal transcriptional activity in non‐producing cells.
F420H2: quinone oxidoreductase from Archaeoglobus fulgidus
Authors: KUNOW Jasper , LINDER Dietmar , STETTER Karl O. , THAUER Rudolf K.
Published: March 2005

Archaeoglobus fulgidus, a hyperthermophilic sulfate‐reducing archaeon, was found to contain a membrane‐bound F420H2:quinone oxidoreductase complex presumed to be involved in energy conservation during growth on lactate plus sulfate. After solubilization with dodecyl‐β‐d‐maltoside the complex was purified 32‐fold with a yield of 24%. Using both gel filtration and native PAGE, an apparent molecular mass of approximately 270 kDa was determined. SDS/PAGE revealed the presence of at least seven polypeptides with apparent molecular masses 56, 45, 41, 39, 37, 33, and 32 kDa. The purified complex contained 1.6 mol FAD, 9 mol non‐heme iron and 7 mol acid‐labile sulfur/mol complex. It did not contain cytochromes, which were, however, present in the membrane fraction of A. fulgidus (3 nmol/mg membrane protein). The purified F420H2:quinone oxidoreductase complex catalyzed the reduction of 2,3‐dimethyl‐1,4‐naphthoquinone (apparent Km 190 μM) with reduced coenzyme F420 (apparent Km 50 μM) exhibiting a specific activity of 500 U/mg (apparent Vmax) at pH 8.0 (pH optimum) and 65°C (temperature optimum). 2‐Methyl‐1,4‐naphthoquinone (menadione), 2‐hydroxy‐1,4‐naphthoquinone, 1,4‐naphthoquinone, 2,3‐dimethoxy‐5‐methyl‐1,4‐benzoquinone, and 2,3‐dimethoxy‐5‐methyl‐6‐decyl‐1,4‐benzoquinone (decyl‐ubiquinone) were also reduced with F420H2, albeit with lower rates. The physiological electron acceptor of the F420H2:quinone oxidoreductase complex is most likely the menaquinone found in the membrane fraction of A. fulgidus.
The mechanism of iron release from transferrin
Authors: EL HAGE CHAHINE Jean‐Michel , FAIN Dominique
Published: March 2005

The role of protonation of amino acid ligands involved in iron release from human serum transferrin, previously saturated with nitrilotriacetatoiron(III) complex, has been elucidated in acidic media. Iron loss occurs first from the N‐terminal site at pH < 6 and is followed at pH < 4 by iron release from the C‐terminal iron‐binding site. Nitrilotriacetatoiron(III) release from the N‐terminal site is controlled by the slow protonation of the mixed protein/nitrilotriacetatoiron(III) complex; the second‐order rate constant was k3a= 9.95 ± 0.35 × 104M−1· s−1. Protonation of an amino acid ligand in the C‐terminal site leads to a new protein‐site‐C‐loaded mixed complex with dissociation constant K4= 0.300 ± 0.025 mM. Nitrilotriacetatoiron(III) release is the result of mixed complex dissociation and the slow rate‐limiting protonation of the iron‐free protein with a proton dissociation constant K5a= 0.100 ± 0.010 mM and a second‐order rate constant k5a= 4.20 ± 0.40 × 103M−1· s−1. The mechanism of iron uptake and release seems to imply that slow proton transfers can induce complex formation between iron and the amino acid ligands of each of the protein iron‐binding sites. These slow proton transfers may be controlled by the change of conformation of the binding sites upon iron loss.
β‐Ureidopropionase with N‐carbamoyl‐α‐l‐amino acid amidohydrolase activity from an aerobic bacterium, Pseudomonas putida IFO 12996
Authors: OGAWA Jun , SHIMIZU Sakayu
Published: March 2005

β‐Ureidopropionase of aerobic bacterial origin was purified to homogeneity from Pseudomonas putida IFO 12996. The enzyme shows a broad substrate specificity. In addition to β‐ureidopropionate (Km 3.74 mM, Vmax 4.12 U/mg), γ‐ureido‐n‐butyrate (Km 11.6 mM, Vmax 19.4 U/mg), and several N‐carbamoyl‐α‐amino acids, such as N‐carbamoylglycine (Km 0.68 mM, Vmax 9.14 × 10−2U/mg), N‐carbamoyl‐l‐alanine (Km 1.56 mM, Vmax 1.00 U/mg), N‐carbamoyl‐l‐serine (Km 75.1 mM, Vmax 3.78 U/mg), and N‐carbamoyl‐dl‐α‐amino‐n‐butyrate (Km 2.81 mM, Vmax 1.08 U/mg), are also hydrolyzed. The hydrolysis of N‐carbamoyl‐α‐amino acids is strictly l enantiomer specific. N‐Formyl‐l‐alanine and N‐acetyl‐l‐alanine are also hydrolyzed by the enzyme, but the rate of hydrolysis is lower than the rate for N‐carbamoyl‐l‐alanine. The enzyme requires a divalent metal ion, such as Co2+, Ni2+ or Mn2+, for activity, and is significantly affected by sulfhydryl reagents. The enzyme consists of two polypeptide chains with identical relative molecular mass Mr 45000. The broad substrate specificity and metal ion dependence of the enzyme show that the β‐ureidopropionase of this aerobic bacterium is quite different from the β‐ureidopropionases of mammals and anaerobic bacteria.
The stabilizing effects of hydrophobic cores on peptide folding of bovine‐pancreatic‐trypsin‐inhibitor folding‐intermediate model
Authors: KWON Dae Young , KIM Peter S.
Published: March 2005

A synthetic peptide model composed of α‐helical and β‐sheet portions (PαPβ) is a crucial folding intermediate in the folding of bovine pancreatic trypsin inhibitor (BPTI), which contains 30 amino acid residues, and provides a good model for studying the folding structure. Using this model the roles of hydrophobic cores, such as non‐polar amino acids and a short β strand, in the folding structure stability were investigated by deleting the hydrophobic amino acids or substituting with Ala. As a first step, a mutant peptide, Pα6Pβ1, was made by removing the four residues (NNFK46) from the N‐terminus of Pα which make a short β strand in native BPTI, and each of the hydrophobic cores, Phe45 of Pα and Tyr21 of Pβ, were substituted by Ala. Without the short β strand the peptide still folds in spite of its low solubility, suggesting that a simple α helix and central antiparallel β sheet contain sufficient information to direct the folding of this region of molecule. The peptide without Tyr21 was much more unstable than Pα6Pβ1, such that most of the folding structure collapsed even at 0°C, whereas without Phe45 the structure was more stable than Pα6Pβ1. This indicates that the hydrophobic interactions of Tyr21 in Pβ are more important in BPTI folding.
Identification of gastrin component I as gastrin‐71
Authors: REHFELD Jens F. , JOHNSEN Anders H.
Published: March 2005

Gastrin component I is the largest hormonally active form of gastrin. In order to determine its structure, we isolated progastrin‐derived peptides from normal human antral tissue. A radioimmunoassay specific for sequence 20–25 of human progastrin was developed to monitor the purifications. After four or five steps of reverse‐phase chromatography, the peptides were pure and could be identified by combination of microsequence, amino acid and mass spectral analysis as well as by a library of sequence‐specific immunoassays. In addition to intact progastrin 1–80, fragments 1–71, 1–35, 6–35, 20–35, and 20–36 of progastrin were identified. Only the 71‐amino‐acid peptide contained at its C‐terminus the α‐amidated bioactive site (Trp‐Met‐Asp‐Phe‐NH2). This unoheptacontapeptide amide (gastrin‐71) corresponds to component I and is the largest possible bioactive product of progastrin. Its structure shows that progastrin is used in its entirety for biosynthesis of active peptides. The occurrence of fragments 6–35, 20–35, and 20–36 demonstrate that antral progastrin is partially cleaved at two monobasic sites (Arg5 and Arg19) in addition to processing at the three C‐terminal dibasic sites. The results show that both the N‐ and C‐terminal parts of antral progastrin undergo extensive processing. The results also suggest that progastrin may follow two different processing pathways of which the less trafficked releases gastrin‐71.
Determination of common structural features in Escherichia coli promoters by computer analysis
Authors: LISSER Shlomit , MARGALIT Hanah
Published: March 2005

Escherichia coli promoters show a large degree of sequence variation. However, they are all recognized specifically by RNA polymerase as the sites for transcription initiation, suggesting that they share common basic structural features distinguishing them from the rest of the sequence. Our hypothesis is that the promoter is determined not only by the two consensus sequences at –10 and –35, but also by the surrounding nucleotides, and that it is not only the identity of the nucleotides that is important for promoter function but the presence of specific physical‐chemical and structural characteristics that are sequence dependent. This approach is supported by accumulating evidence indicating the role that the DNA conformation may play in modulating protein‐DNA interaction. In this study, four intrinsic sequence‐dependent characteristics are examined in E. coli promoter regions: helix stability, helix flexibility, and two conformational parameters represented by the DNA tendencies for B→Z and B→A transition. The promoter is defined by the consensus sequences and their vicinity and the examined properties are compared between promoter and random sequences. It is demonstrated that both the consensus and flanking regions are less stable, more flexible and show a higher tendency for the B conformation in comparison to random sequences. Discriminant analysis is used to evaluate the relative contributions of the various characteristics.