bluuebunny/crossref_metadata_2025_split huggingface dataset:
Actin—caldesmon—myosin‐subfragment‐1 ternary complex viewed by electron microscopy
Authors: HARRICANE Marie‐Cécile , BONET‐KERRACHE Armelle , CAVADORE Claude , MORNET Dominique 
Published: March 2005

An earlier electron microscopic study using different caldesmon forms complexed with actin revealed that the aggregates produced display regular periodic striation after antibody labeling of the 35‐kDa caldesmon fragment. This approach provides further evidence that a caldesmon fragment, even as small as 15 kDa, can induce actin filaments to assemble into bundles. The observed difference in the compactness of these structures, depending on the use of the 15‐kDa fragment instead of the 35‐kDa fragment, suggests the existence of more than one actin‐binding site in the caldesmon molecule.In this study, the caldesmon‐induced process of F‐actin association was investigated in the presence of skeletal myosin subfragment‐1, using light‐scattering methods, cosedimentation experiments and electron microscopic techniques.We show that the actin‐caldesmon association is partially destabilized in the presence of subfragment‐1 and this leads to a ternary complex formation. Immunogold labelling of the actin filaments still reveals the presence of caldesmon within this structure. This latter result strengthens the hypothesis that actin has a site(s) able to bind both caldesmon and myosin subfragment‐1, as detected by recent NMR observations. This evidence is discussed with respect to the regulatory function of caldesmon during smooth muscle contraction.
Activation and binding volumes of the sarcoplasmic reticulum transport enzyme activated by calcium or strontium
Authors: STEPHAN Sibylle , HASSELBACH Wilhelm 
Published: March 2005

The effect of pressure on the hydrolysis of dinitrophenyl phosphate (DnpP) and p‐nitrophenyl phosphate (NpP) by the sarcoplasmatic reticulum transport enzyme in permeabilized and native closed vesicles activated by calcium or strontium, respectively, in aqueous and Me2SO‐containing media has been studied. At atmospheric pressure, the enzyme in permeabilized vesicles, saturated with respect to substrates and activating ions, hydrolyzes DnpP ten times faster than NpP; for both substrates, calcium activation exceeds that by strontium only a little (20%). In aqueous media the enzyme displays, under all activating conditions, an almost identical curvilinear relationship between the logarithm of enzyme activity and pressure. The data were analysed on the basis of a simplified reaction scheme, in which two unidirectionally proceeding substrate‐driven pressure‐dependent reactions (k2, k4) cyclically transfer high‐affinity into low‐affinity binding sites which are assumed to be in equilibrium with either calcium or strontium. The fitting procedure yielded two sets of positive activation volumes ΔV*2= 90–110 ml/mol and ΔV*4= 15–25 ml/mol. Substrate specificity, as well as the effect of temperature, are exclusively localized in the pressure‐independent rate constants k'2 and k'4. Considerably different pressure/activity relations characterized by a single activation volume of 20 ml/mol were obtained for the strongly suppressed substrate hydrolysis of native closed vesicles.At atmospheric pressure DnpP hydrolysis of open vesicles is inhibited by Me2SO, while NpP hydrolysis is considerably activated, irrespective of its activation by calcium or strontium. In the presence of 22.5% Me2SO, the activation volumes are reduced by 50–70 ml/mol. The rate constants of DnpP and NpP hydrolysis are either augmented or reduced by rising Me2SO concentrations, depending on the corresponding supporting substrate. Me2SO has only a slight effect on the pressure dependence of substrate hydrolysis by native vesicles.The small activation volume observed for the activity of native vesicles could be assigned on account of the simplified reaction scheme of the slow reaction step k4, by which the enzyme is transferred from its low‐affinity into its high‐affinity binding state. Volume changes connected with the binding of calcium or strontium to the luminal binding site of the enzyme were deduced from the observed activation volume and the computed volume change of the slow reaction step (ΔV*4).
Multiple apomucin translation products from human respiratory mucosa mRNA
Authors: PERINI Jean‐Marc , VANDAMME‐CUBADDA Nathalie , AUBERT Jean‐Pierre , PORCHET Nicole , MAZZUCA Marc , LAMBLIN Geneviève , HERSCOVICS Annette , ROUSSEL Philippe 
Published: March 2005

Poly(A)‐rich RNA was purified from a pool of five human tracheobronchial mucosa. After in vitro translation in a reticulocyte lysate and immunoprecipitation of the translated products, using either a polyclonal antiserum or a monoclonal antibody to deglycosylated respiratory mucin peptides, the products were characterized by SDS/PAGE. The respiratory mucin precursors migrated as a very large smear from almost the top of the resolving polyacrylamide gel to an area corresponding to a molecular mass of about 100 kDa.After hybridization with mucin cDNA probe TH 29 described by Crepin et al. [Crepin, M., Porchet, N., Aubert, J. P. & Degand, P. (1990) Biorheology 27, 471–484] respiratory mucin mRNAs also appeared polydisperse.Although degradation or incomplete translation of high‐molecular‐mass mRNA cannot be entirely ruled out, these results suggest that human respiratory apomucins consist of a family of peptides which share some common epitopes.This possibility is in agreement with (a) the diversity of mucin precursors observed previously with pulse/ chase experiments performed with explants of human respiratory mucosa and (b) the polydispersity of secreted respiratory mucins observed by electron microscopy.
Characterization of a murine β1‐4galactosyltransferase expressed in COS‐1 cells
Authors: NAKAZAWA Kazutoshi , FURUKAWA Kiyoshi , KOBATA Akira , NARIMATSU Hisashi 
Published: March 2005

We inserted a full‐length murine cDNA, which had been isolated from F9 embryonal carcinoma cells by using a bovine lactose synthetase A protein cDNA as a probe, in a mammalian expression vector (pCMGT1) and expressed it in COS‐1 cells to characterize the pCMGT1‐directed enzyme. The galactosyltransferase activity toward asialo‐agalacto‐transferrin (AsAg‐Tf) in the pCMGT1‐transfected cells was approximately eightfold higher than that in mock‐ or non‐transfected cells. In contrast, no difference was observed in the specific activity of galactose transfer between pCMGT1‐transfected cells and mock‐ or non‐transfected cells when asialo‐ovine submaxillary mucin were used as an acceptor. Since almost all [3H]galactose incorporated into the AsAg‐Tf was released by digestion with streptococcal β‐galactosidase, most of the linkage created by this enzyme was in the Galβ1‐4GlcNAc group.The acceptor specificity of the pCMGT1‐directed enzyme was changed from N‐acetylglucosamine to glucose by adding α‐lactalbumin in the reaction mixture. α‐Lactalbumin also partially inhibited the galactose transfer to AsAg‐Tf. The kinetic study revealed that the apparent Km values of the pCMGT1‐directed enzyme for N‐acetylglucosamine, AsAg‐Tf and UDP‐Gal are 2 mM, 60 μM and 24 μM, respectively.These results indicated that the murine cDNA isolated from F9 cells encodes an active enzyme which catalyzes not only the lactose synthesis but also the transfer of galactose to N‐acetylglucosamine residues of Asn‐linked sugar chains of glycoproteins in a β1‐4 linkage.
Functional residues at the active site of aminopeptidase N
Authors: HELENE Armelle , BEAUMONT Ann , ROQUES Bernard P. 
Published: March 2005

Sequence analysis of aminopeptidase N has shown that this zinc exopeptidase contains a consensus sequence (Val‐Xaa‐Xaa‐His‐Glu‐Xaa‐Xaa‐His), generally found at the active site of zinc endopeptidases [Jongeneel, C. V., Bouvier, J. and Bairoch, A. (1989) FEBS Lett. 242, 211–214]. This suggests that the active site of aminopeptidase N may be closer to that of a classical zinc endopeptidase, such as thermolysin, than to that of an exopeptidase, such as carboxypeptidase A, which does not contain the above sequence. However, the nature of the other amino acids involved in the enzymatic activity of the eukaryotic aminopeptidase N remains unknown.Chemical modifying agents have now been used to characterize the active site of aminopeptidase N further. The location of the modified residues was also determined by comparing the protection given by three competitive inhibitors which interact with different subsites of the active site. Aminopeptidase N was rapidly inactivated by 2,3‐butanedione and diethylpyrocarbonate and partially inactivated by N‐acetylimidazole, diazoacetamide and a soluble carbodiimide, suggesting the presence of functional arginyl, histidyl, tyrosyl and aspartyl/glutamyl residues. In each case the reaction kinetics showed that the inactivation could be correlated with modification of a single residue. The protection experiments indicated that the residues are at the active site of the enzyme and that the arginine and tyrosine are probably located in the S′1‐S′2 subsites, histidine in the S1 subsite and the acidic residue near the zinc binding site and the S′1 subsite. Steady‐state kinetics showed that the arginine, histidine and acidic residues are involved in substrate binding, while the tyrosine may play a role in the catalytic process. All these data support an endopeptidase‐like structure for the active site of aminopeptidase N.
Differences in primary structure among five phospholipases A2 from Heloderma suspectum
Authors: VANDERMEERS André , VANDERMEERS‐PIRET Marie‐Claire , VIGNERON Laurence , RATHE Jean , STIEVENART Michel , CHRISTOPHE Jean 
Published: March 2005

Five increasingly anionic phospholipases A2 (Pa1– Pa5) exist in the venom of the lizard Heloderma suspectum. We recently elucidated the sequence of Pa5, the most abundant and most active variant, towards emulsfied phosphatidylcholines. Here we present the primary structures of Pa2, Pa3 (subvariants a and b) and Pa4, based on Edman degradation of tryptic, endoproteinase Arg‐C and chymotryptic fragments of the reduced and S‐carboxymethylated proteins. Pa1– Pa5, considered collectively, belong to an original class of secretory phospholipases A2 with 141 – 143 residues, a short hydrophobic N‐terminus, 10 half‐cystine residues and an extended C‐terminus. The only known phospholipase A2 with characteristics close enough to be a member of the same class is that present in the venom from the insect Apis mellifera.More specifically, the sequences of Pa3 and Pa5 are almost identical, and those of Pa2 and Pa4 are also quite similar. Both groups diverge enough to indicate the translation of two mRNA species in the venom gland. The primary structure of Pa3 reveals the existence of subvariants a and b, the sequence of which is identical to that previously defined for Pa5, except that the C‐terminal tripeptide GEG in Pa5 is replaced by the dipeptide GE in Pa3a and the tetrapeptide GEGR in Pa3b. Pa4, when compared to Pa5, shows 21 substitutions with a cluster of five modified amino acids in positions 40 – 44, immediately after the catalytic segment amino acids 30 – 39, and added changes scattered before the C‐terminus. Pa2 differs from Pa4 only by the absence of the Gly142 C‐terminal residue. The 15% difference in primary structure observed between the Pa3– Pa5 and Pa2– Pa4 subgroups might be largely responsible for their distinct biological properties.
15N‐ and 13C‐NMR investigations of glucose oxidase from Aspergillus niger
Authors: SANNER Christoph , MACHEROUX Peter , RÜTERJANS Heinz , MÜLLER Franz , BACHER Adalbert 
Published: March 2005

The apoprotein of glucose oxidase from Aspergillus niger was reconstituted with specifically 15N‐ and 13C‐enriched FAD derivatives and investigated by 15N‐ and 13C‐NMR spectroscopy. On the basis of the 15N‐NMR results it is suggested that, in the oxidized state of glucose oxidase, hydrogen bonds are formed to the N(3) and N(5) positions of the isoalloxazine system. The hydrogen bond to N(3) is more pronounced than that to N(5) as compared with the respective hydrogen bonds formed between FMN and water. The resonance position of N(10) indicates a small decrease in sp2 hybridization compared to free flavin in water. Apparently the isoalloxazine ring is not planar at this position in glucose oxidase.Additional hydrogen bonds at the carbonyl groups of the oxidized enzyme‐bound FAD were derived from the 13C_NMR results. A strong downfield shift observed for the C(4a) resonance may be ascribed in part to the decrease in sp2 hybridization at the N(10) position and to the polarization of the carbonyl groups at C(2) and C(4). The polarization of the isoalloxazine ring in glucose oxidase is more similar to FMN in water than to that of tetraacetyl‐riboflavin in apolar solvents.In the reduced enzyme the N(1) position is anionic at pH 5.6. The pKa is shifted to lower pH values by at least 1 owing to the interaction of the FAD with the apoprotein.As in the oxidized state of the enzyme, a hydrogen bond is also formed at the N(3) position of the reduced flavin. The N(5) and N(10) resonances of the enzymes‐bound reduced FAD indicate a decrease in the sp2 character of these atoms as compared with that of reduced FMN in aqueous solution.Some of the 15N‐ and 13C‐resonance positions of the enzyme‐bound reduced cofactor are markedly pH‐dependent. The pH dependence of the N(5) and C(10a) resonances indicates a decrease in sp2 hybridization of the N(5) atom with increasing pH of the enzyme solution.
Monoclonal antibodies specific for the core protein of the β‐subunit of the gastric proton pump (H+/K+ ATPase)
Authors: JONES Claerwen M. , TOH Ban‐Hock , PETTITT John M. , MARTINELLI Teresa M. , HUMPHRIS Danielle C. , CALLAGHAN Judy M. , GOLDKORN Isabela , MU Fi‐Tjen , GLEESON Paul A. 
Published: March 2005

The gastric H+/K+ ‐transporting adenosine triphosphatase (H+/K+ ATPase) (proton pump) consists of a catalytic α‐subunit and a recently proposed 60–90‐kDa glycoprotein β‐subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60–90‐kDa glycoprotein.The monoclonal antibodies (1) specifically stained the cytoplasm of unfixed and formalin‐fixed dog gastric parietal cells; (2) specifically reacted by ELISA with gastric tubulovesicular membranes; (3) recognised epitopes located on the luminal face of parietal cell tubulovesicular membranes, the site of the proton pump, by immunogold electron microscopy; (4) immunoblotted a 60–90‐kDa molecule from tubulovesicular membranes and a 35‐kDa component from peptide N‐glycosidase‐F‐treated membrane extracts; (5) immunoblotted the 60–90‐kDa parietal cell autoantibody‐ or tomato‐lectin–Sepharose 4B affinity columns, and the 35‐kDa protein core of this autoantigen; this autoantigen has amino acid sequence similarity to the β‐subunit of the related NH+/K+ ‐transporting adenosine triphosphatase (Na+/K+ ATPase) [Toh et al. (1990) Proc. Natl Acad. Sci. 87, 6418–6422]; (6) co‐precipitated a molecule of 95kDa with the 60–90‐kDa molecule from 125I‐labelled detergent extracts of dog tubulovesicular membranes; and (7) co‐purified the catalytic α‐subunit of the H+/K+ ATPase with the 60–90‐kDa molecule by immunoaffinity chromatography of tubulovesicular membrane extracts on a monoclonal antibody 3A6‐Sepharose 4B column, indicating a physical association between the two molecules.These results provide further evidence that the 60–90‐kDa glycoprotein is the β‐subunit of the gastric H+/K+ ATPase. We conclude that the monoclonal antibodies specifically recognise luminal epitopes on the 35‐kDa core protein of the 60–90‐kDa β‐subunit of the gastric proton pump, a major target molecule in autoimmune gastritis and pernicious anaemia. These monoclonal antibodies will be valuable probes to study the structure and function of this associated β‐subunit, as well as the ontogeny of the gastric proton pump.
Release of proteinases from stimulated polymorphonuclear leukocytes
Authors: SCHETTLER Almut , THORN Hans , JOCKUSCH Brigitte M. , TSCHESCHE Harald 
Published: March 2005

The secretion of matrix‐degrading proteinases and protein components involved in the production of cytotoxic metabolites is an important step in the sequence of defense reactions executed by polymorphonuclear leukocytes (PMNL) in response to stimulation. In the present report, we have analyzed degranulation of PMNL stimulated either with soluble synthetic peptides fLeu‐Phe (fMet, formylmethionyl), or fAhx‐Leu‐Phe‐Ahx‐Tyr‐Phe (Ahx, aminohexyl) which trigger chemotaxis and degranulation, or with opsonized zymosan which induces phagocytosis.The release of elastase, myeloperoxidase and lactoferrin‐containing granules was not at all or only slightly (< 6%) induced either by fAhx‐Leu‐Phe‐Ahx‐Tyr‐Leu or by zymosan particles. In contrast, type‐I collagenase and gelatinase were secreted in significant amounts after treatment with these agents. The disruption of microfilaments by cytochalasin B and subsequent stimulation of PMNL with the formyl‐peptide led to the secretion of elastase, myeloperoxidase and lactoferrin, and enhanced the release of gelatinase. Disruption of microtubules by incubation with colcemid resulted in inhibition of fAhx‐Leu‐Phe‐Ahx‐Thyr‐Leu and fAhx‐Leu‐Phe‐Ahx‐Tyr‐Leu/cytochalasin‐B‐induced granule release. Furthermore, we found different patterns of enzyme distribution after fractionation by centrifugation: most (> 90%) type‐I collagenase and gelastinase was measured in the supernatant whereas 60–90% of elastase, myeloperoxidase and lactoferrin had partitioned into the cytoskeleton‐containing pellet. Our results suggest that the two main types of secretory vesicles identified in PMNL (specific and azurophilic granules) consist of subpopulations. The differential association of the various types of granules with cytoskeletal elements may serve to control their sequential discharge.
Heparinase II from Flavobacterium heparinum
Authors: MOFFAT Colin F. , McLEAN Maitland W. , LONG William F. , WILLIAMSON Frank B. 
Published: March 2005

Five chemically modified heparins were derived from native pig mucosal heparin (pig heparin Is). These were de‐N‐sulphated heparin (heparin Ih), N‐acetylheparin (heparin Ia), de‐N/O‐sulphated heparin (heparin IVh), de‐O‐sulphated heparin (heparin IVs) and de‐O‐sulphated N‐acetyl‐heparin (heparin IVa). Their structures were studied by 13C‐NMR spectroscopy at 90.56 MHz. Native heparin and the derivatives were incubated with Flavobacterium heparinase II at 25°C. The progress of degradation was followed by the δA235 and the final composition examined by gel filtration with Bio‐Gel P‐4.Native heparin (Is) was readily degraded by heparinase II and, with the exception of heparin IVh for which degradation was negligible, the chemically modified derivatives were also degraded. Approximately 90% of the saccharides from heparins Is, Ia, IVs and IVa were disaccharides and tetrasaccharides. For heparin Ih, which was degraded more slowly, the proportion was 65%. Heparins Is, IVs and IVa underwent initial rapid degradation. The digestion of heparin Ia proceeded rapidly after an initial lag phase.The undegraded polymers produced similar elution profiles from Bio‐Gel P‐4. Following the action of heparinase II on heparins Is, Ia, IVs and IVa, the elution profiles revealed a major peak of disaccharides and minor peaks of higher oligomers. The profile of heparin Ih revealed a greater proportion of intermediate‐molecular‐mass saccharides.Our results demonstrate a broad specificity for heparinase II. It is capable of lysing both N‐acetylated and N‐sulphated heparins independent of O‐sulphation. Heparinase II will also degrade heparin derivatives that are non‐N‐substituted provided that they are O‐sulphated.
Isolation and characterization of a membrane glycoprotein from human brain with sequence similarities to cell adhesion proteins from chicken and mouse
Authors: BERGLUND Erik , STIGBRAND Torgny , CARLSSON Sven R. 
Published: March 2005

We previously described the production of monoclonal antibodies against a preparation of membrane glyco‐proteins from human brain [Berglund et al. (1987) J. Neurochem. 48, 809–815]. One of the glycoproteins, recognized by monoclonal antibody CF3, was specifically expressed in the brain. We now report the isolation and characterization of this glycoprotein, called glycoprotein 135 (Gp135).Gp135 was purified by means of lentil lectin affinity chromatography and immunoaffinity chromatography, using monoclonal antibody CF3, from a crude membrane extract of human brain cortex. Gp135 was shown to consist of a glycosylated single polypeptide chain with an apparent molecular mass of 135 kDa. The size of the polypeptide moiety was estimated to 115 kDa following N‐glycanase digestion. The glycoprotein is anchored in the membrane by a glycosylphosphatidylinositol tail, as shown by phospholipase C digestion and liposome incorporation experiments.Amino acid sequence analysis of the amino terminal, and of an internal peptide obtained by V8 protease digestion of the glycoprotein, revealed a strong similarity to three previously described glycoproteins from chicken (contactin and F11) and mouse (F3) brains. These glycoproteins belong to the immunoglobulin superfamily and are implicated in cell adhesion phenomena in the developing brain. Gp135 may be the human counterpart to one or several of these glycoproteins.
Interaction of Zn2+ with the bovine‐heart mitochondrial bc1 complex
Authors: LORUSSO Michele , COCCO Tiziana , SARDANELLI Anna Maria , MINUTO Michele , BONOMI Francesco , PAPA Sergio 
Published: March 2005

A study is presented of the effect of Zn2+ on the enzymatic properties of the bovine‐heart cytochrome‐bc1 complex. Micromolar concentrations of Zn2+ reversibly inhibit the cytochrome‐c reductase activity of either the cholate‐solubilized or liposome‐reconstituted complex. Kinetic analysis of the redox reactions of the cytochromes indicate that Zn2+ affects the activity of the complex at the quinol oxidation site. The following have been determined: (a) Zn2+ inhibits the pre‐steady‐state reduction of cytochrome c1 by duroquinol either in the absence or in the presence of antimycin, (b) it does not inhibit the reduction of b cytochromes in the absence of antimycin or in the presence of myxothiazol, (c) it inhibits cytochrome‐b reduction in the presence of antimycin. Furthermore Zn2+ inhibits the antimycin‐promoted oxidant‐induced extrareduction of b cytochromes.Addition of Zn2+ to reduced bc1 complex causes a red shift in the absorption spectrum of cytochrome b566 and a substantial decrease in the signal intensity of the EPR spectrum of the Fe‐S protein. This is interpreted as an interaction of Zn2+ with the 2Fe–2S‐cluster region of the Fe‐S protein, thus giving rise to inhibition of the reductase activity and of the antimycin‐insensitive reduction route of b cytochromes.A Scatchard‐plot of 65Zn2+ binding to the native isolated complex gave a straight line from which a value of three binding sites and a single dissociation constant of 3 × 10−6 M can be calculated, which is practically equal to the concentration causing 50% inhibition of electron flow.