bluuebunny/crossref_metadata_2025 huggingface dataset:
Demonstration of a molybdenum‐ and vanadium‐independent nitrogenase in a nifHDK‐deletion mutant of Rhodobacter capsulatus
Authors: SCHNEIDER Klaus , MÜLLER Achim , SCHRAMM Uwe , KLIPP Werner
Published: March 2005

In Rhodobacter capsulatus there exists, in addition to a conventional Mo‐containing nitrogenase, a second, Mo‐indendent nitrogenase which was demonstrated in wild‐type cells as well as in cells of a nifHDK− mutant. To construct this R. capsulatus mutant, a 4‐kb BglII–HindIII fragment encompassing nifK, nifD and most of the nifH coding region was substituted by an interposon coding for kanamycin resistance. The alternative nitrogenase is repressed by molybdenum. Mo concentration > 1 ppb in the growth medium prevented diazotrophic growth of nifHDK− cells and the expression of nitrogenase activity. The Mo‐independent nitrogenase was maximally derepressed in activated carbon‐treated media which contained < 0.05 ppb Mo, high concentrations of iron (1 mM ferric citrate) and serine as N source. Under N2‐fixing and optimal Mo‐deficient conditions, nifHDK− cells grew with a doubling time of 9 h. The highest activity achieved with whole cells was 1.2 nmol ethylene · min−1· mg protein−1. Vanadium neither stimulated nor inhibited growth and activity.

The alternative nitrogenase reduced acetylene to both ethylene and ethane. With whole cells (nifHDK−) the proportion of ethane varied over 2–5% depending on the amount of residual traces of Mo in the medium. The addition of Mo to a growing, nitrogenase‐active culture resulted in a slow decrease of total activity but also in a simultaneous increase of ethane production up to 40%. In contrast, cell‐free extracts and the purified enzyme did not show any or only very little ethane formation (0–0.4%).

Both enzyme components appeared to be very labile proteins. Component 2 lost almost all its activity during cell breakage. With component 1 in crude extracts, if complemented with the stable component 2 of the Mo‐nitrogenase from Xanthobacter autotrophicus, a recovery of 50% of the original whole cell activity could be achieved. During purification, component 1 (from the nifHDK− mutant) remained remarkably stable. The partially purified component 1 had a pH optimum (acetylene reduction) of 7.8–8.0, relatively high affinity to acetylene (Km= 0.055 mM) and was analyzed to contain 20 mol Fe atoms/mol protein, 0.2 mol Mo atoms and negligible amounts of V, W and Re. The dithionite‐reduced dinitrogenase appeared to be ESR‐silent. The results indicate that the alternative nitrogenase of R. capsulatus is not a vanadium enzyme but rather a heterometal‐free Fe‐nitrogenase or a nitrogenase with an as‐yet‐unidentified heterometal atom.
Efficient renaturation and fibrinolytic properties of prourokinase and a deletion mutant expressed in Escherichia coli as inclusion bodies
Authors: ORSINI Gaetano , BRANDAZZA Anna , SARMIENTOS Paolo , MOLINARI Antonio , LANSEN Jacqueline , CAUET Gilles
Published: March 2005

Prourokinase is a plasminogen activator of 411 amino acids which displays a clot‐lysis activity through a fibrin‐dependent mechanism, and which seems to be a promising agent for the treatment of acute myocardial infarction.

The preparation of recombinant prourokinase in bacteria has been hampered by its insolubility and by difficulty in refolding the polypeptide chain.

In this paper we describe the renaturation process of two recombinant proteins expressed in Escherichia coli as inclusion bodies: prourokinase and a deletion derivative (δ125‐prourokinase) in which 125 amino acids of the N‐terminal region have been removed. Deletion of this sequence brings to higher refolding yields and faster kinetics (first‐order rate constant of renaturation of 0.57 h−1 for δ125‐prourokinase and 0.25 h−1 for prourokinase).

Our process involves sequential steps of denaturation, reduction and controlled refolding of the polypeptide chain. When applied to pure, non‐glycosylated and active prourokinase, it gives a refolding yield of about 80%, demonstrating the efficiency of the renaturation procedure. Lower yields (15% and 30%, respectively, for prourokinase and δ125‐prourokinase) were obtained when the same refolding protocol was applied to inclusion bodies from bacteria.

After purification to homogeneity (as shown by HPLC and SDS/PAGE) specific activities were 160000 and 250000 IU/mg protein, respectively, for prourokinase and δ125‐prourokinase.

As with prourokinase, the deletion mutant δ125‐prourokinase displays a zymogenic nature, being activated by plasmin to the active two‐chain form; however, this mutant is approximately fourfold more resistant than prourokinase to plasmin activation, and consequently shows a different fibrinolytic profile.
Linking phospholipase A2 to phospholipid turnover and prostaglandin synthesis in mast cell granules
Authors: CHOCK Stephen P. , RHEE Sue Goo , TANG Lily C. , SCHMAUDER‐CHOCK Elsa A.
Published: March 2005

Rapid incorporation of exogenous arachidonic acid into phospholipid has been detected in conjunction with eicosanoid synthesis by purified mast cell granules [Chock, S. P. & Schmauder‐Chock, E. A. (1988) Biochem. Biophys. Res. Commun. 156, 1308 – 1315]. The species of phospholipid formed has now been identified primarily as phosphatidylinositol. A calcium‐dependent phospholipase A2 has also been detected in the secretory granule. This enzyme, like the cyclooxygenase [Schmauder‐Chock, E. A. & Chock, S. P. (1989) J. Histochem. Cytochem. 37, 1319 – 1328], appears to bind tightly to the granule matrix components. It is heat resistant and requires millimolar concentrations, of calcium for optimal activity. It prefers phosphatidylinositol over phosphatidylcholine as substrate. Since the granule contains a large amount of phospholipid, the action of this phospholipase A2 can provide the required substrate for the arachidonic acid cascade. These findings provide the basis for linking phospholipase A2 to the production of eicosanoids during granule exocytosis. Since the granule also contains both an active acylating system that can rapidly reacylate lysophosphatidylinositol to form phosphatidylinositol, and an active phospholipase A2 which hydrolyzes phosphatidylinositol, a rapid turnover involving the fatty acid at the sn‐2 position of phosphatidylinositol may occur. These findings are consistent with our postulation that the secretory granule is the source and/or the cause of many of the early biochemical events associated with the process of stimulus‐secretion coupling.
The substrate specificity of protein kinases which phosphorylate the α subunit of eukaryotic initiation factor 2
Authors: PROUD Christopher G. , COLTHURST David R. , FERRARI Stefano , PINNA Lorenzo A.
Published: March 2005

The α subunit of eukaryotic protein synthesis initiation factor (eIF‐2α) is phosphorylated at a single serine residue (Ser51) by two distinct and well‐characterised protein kinase, the haem‐controlled repressor (HCR) and the double‐stranded RNA‐activated inhibitor (dsI). The sequence adjacent to Ser51 is rich in basic residues (Ser51‐Arg‐Arg‐Arg‐Ile‐Arg) suggesting that they may be important in the substrate specificity of the two kinases, as is the case for several other protein kinases. A number of proteins and synthetic peptides containing clusters of basic residues were tested as substrates for HCR and dsI.

Both kinases were able to phosphorylate histones and protamines ar multiple sites as judged by two‐dimensional mapping of the tryptic phosphopeptides. These data also showed that the specificities of the two kinases were different from one another and from the specificities of two other protein kinases which recognise basic residues, cAMP‐dependent protein kinase and protein kinase C. In histones, HCR phosphorylated only serine residues while dsI phosphorylated serine and threonine. Based on phosphoamino acid analyses and gel filtration of tryptic fragments, dsI was capable of phosphorylating both ‘sites’ in clupeine Y1 and salmine A1, whereas HCR acted only on the N‐terminal cluster of serines in these protamines.

The specificities of HCR and dsI were further studied using synthetic peptides with differing configurations of basic residues. Both kinases phosphorylated peptides containing C‐terminal clusters of arginines on the ‘target’ serine residue, provided that they were present at positions + 3 and/or + 4 relative to Ser51. However, peptides containing only N‐terminal basic residues were poor and very poor substrates for dsI and HCR, respectively. These findings are consistent with the disposition of basic residues near the phosphorylation site in eIF‐2α and show that the specificities of HCR and dsI differ from other protein kinases whose specificities have been studied.
Characterization of the cellular binding domain and the effects of monoclonal antibodies and thrombin inhibitors on the binding and internalization of the antithrombin‐III — thrombin complex by cultured cells
Authors: KNOLLER Sarah , SAVION Naphtali
Published: March 2005

Antithrombin III (AT) binds to cultured cells mainly as a complex with thrombin or other serine proteases rather than in its free form. This implies that, upon complex formation, a new determinant appears on the AT molecule which is recognized by the cells. Fragmentation of AT by cyanogen bromide exposes this determinant and an 8‐kDa fragment is recognized by cultured cells. The binding of this fragment to cultured cells is inhibited by antithrombin‐III — thrombin (AT‐T) complex, but not by free AT. The putative cellular binding domain of AT‐T is located over amino acid residues 253–314 of AT, in the large loop close to the carboxy‐terminus of the molecule.

The cell‐associated AT‐T is internalized and degraded, forming the thrombin‐modified AT (ATM). This process is inhibited by lysosomal degradation inhibitors, such as chloroquine or benzamidine. Thus, the appearance of ATM in cells is not a result of its binding to the cell surface, but probably a result of lysosomal degradation of cell‐associated AT‐T. Another degradation product, with a molecular mass of 43 kDa, appears in cells along with the appearance of ATM. Hirudin, a specific inhibitor of thrombin, inhibits the cellular internalization of AT‐T complexes, indicating a possible role for thrombin activity in the internalization process of complexes.

Monoclonal antibodies (mAb) against AT, whose epitopes on the AT molecule have been located, affect the binding of AT‐T to cultured cells. Two mAb, A10 whose epitope is found outside of the cellular binding site, and B108 whose epitope may overlap this sequence, totally inhibit the binding of the complex to cells. Inhibition of binding results either from steric hindrance or induced changes in the binding site.
Three‐dimensional correlated NMR study of Megasphaera elsdenii flavodoxin in the oxidized state
Authors: WIJMENGA Sybren S. , van MIERLO Carlo P. M.
Published: March 2005

The value of a three‐dimensional (3D) non‐selective total correlation/nuclear Overhauser enhancement spectroscopy (TOCSY‐NOESY) spectrum for making sequential resonance assignments in proteins is demonstrated using the relatively large Megasphaera elsdenii flavodoxin (molecular mass 15 kDa) in the oxidized state. An easy and concise method for the analysis of 3D‐NMR spectra and a strategy for the resonance assignment of 3D‐NMR protein spectra is introduced. In this context, non‐selective TOCSY‐NOESY is compared with selective TOCSY‐NOESY and non‐selective NOESY‐TOCSY. Sequential assignments in various secondary structure elements of flavodoxin are made using the method of analysis introduced. NOEs not previously identified in 2D‐NMR spectra due to resonance overlap are found in the 3D Clean‐TOCSY‐NOESY spectrum. Also additional side‐chain assignments could be made.
A peptide‐sensitive channel of large conductance is localized on mitochondrial outer membrane
Authors: CHICH Jean‐François , GOLDSCHMIDT Denise , THIEFFRY Michel , HENRY Jean‐Pierre
Published: March 2005

Applying the technique of ‘tip‐dip’ to mitochondria, we have shown the existence in this organelle of a cationic channel of large conductance, which is blocked by a 13‐residue peptide possessing the sequence of the N‐terminal extremity of the cytochrome c oxidase subunit IV precursor. To study the submitochondrial localization of the channel, the effect of trypsin on isolated channels and on entire mitochondria were compared. One side of isolated channels is sensitive to trypsin, which eliminates the voltage dependence. Channels isolated from trypsinized mitochondria were devoid of voltage dependence and were blocked by the peptide. This suggests a localization of the channel on the outer membrane. Consistent with this hypothesis, the channel was observed with the highest frequency in outer membrane fractions purified by different procedures, either from bovine adrenal cortex or from rat liver mitochondria. Such a localization is also consistent with digitonin solubilization experiments. The channel was solubilized before the inner membrane marker, cytochrome c oxidase. The orientation of the channel was inferred from its trypsin sensitivity and its potential dependence: a transmembrane potential (inside negative) will close the channel.
NADPH—cytochrome c reductase from rabbit peritoneal neutrophils
Authors: LAPORTE François , DOUSSIERE Jacques , MECHIN Virginie , VIGNAIS Pierre V.
Published: March 2005

An NADPH‐dependent membrane‐bound flavoprotein dehydrogenase, assayed as a catalyst of electron transfer from NADPH to cytochrome c, was extracted from membranes of rabbit peritoneal neutrophils with Triton X‐100 and sodium deoxycholate in the presence of diisopropylfluorophosphate as antiprotease, and purified to electrophoretic homogeneity. The purified enzyme in detergent was able to enhance the rate of formation of the superoxide anion O−2 in a cell‐free system, consisting of membrane and cytosolic fractions from resting neutrophils complemented with arachidonic acid, guanosine 5′‐[γ‐thio]triphosphate and Mg2+. This suggested that the NADPH dehydrogenase was a component of the rabbit neutrophil oxidase complex. The purification factor of the enzyme with respect to the membrane fraction was close to 1000 and the recovery of activity was 33%. FMN and FAD were associated with the enzyme in a molar ratio close to 1. On SDS/PAGE, the enzyme migrated with a molecular mass of 77 kDa. A similar mass was determined by filtration on a molecular sieve. The isoelectric point of this enzyme was 4.7 ± 0.1. Its activity was maximal between pH 7.5 and pH 8.5, and depended on the ionic strength of the medium, with a maximum at an ionic strength of 0.5. Reduction of cytochrome c by NADPH obeyed Michaelis‐Menten kinetics with a KM value of 15 μM for cytochrome c. When NADPH was the variable substrate, a KM value of 1.9 μM for NADPH was found, but a significant deviation from Michaelis‐Menten kinetics was observed at high concentrations of NADPH. Mersalyl strongly inhibited the reductase activity when added to the enzyme prior to NADPH; preincubation of the enzyme with NADPH considerably reduced the inhibitory efficiency of mersalyl. A partially proteolyzed water‐soluble, active, form of the enzyme with a molecular mass of 67 kDa was prepared. The proteolyzed enzyme exhibited the same specificity, and kinetic behavior with respect to NADPH, and the same dependency on the ionic strength, as the native enzyme.
NMR and circular dichroic studies on the solution conformation of a synthetic peptide derived from the calmodulin‐binding domain of Bordetella pertussis adenylate cyclase
Authors: PRÊHEUR Bénédicte , SIFFERT Odile , BÂRZU Octavian , CRAESCU Constantin T.
Published: March 2005

The solution conformation of a synthetic peptide of 20 amino acids (P235–254) derived from the calmodulin‐binding domain of Bordetella pertussis adenylate cyclase was studied by proton two‐dimensional NMR spectroscopy and circular dichroism. Based on the standard techniques we have assigned all the resonances in the NMR spectrum to the corresponding protons of the peptide. Analysis of the secondary chemical shift distribution and of the nuclear Overhauser effect connectivities showed no evidence for a highly populated regular conformation but suggested the tendency to form an α‐helix arround the unique Trp residue. The propensity for a helical structure is in agreement with the results of circular dichroic spectroscopy showing a slight negative band at 222 nm which was cancelled by 6 M guanidine hydrochloride. Increasing amounts of 2,2,2‐trifluorethanol (up to 40%) increase considerably the helical population of the peptide as reflected in the circular dichroic spectra. Analysis of the present results shows that the free peptide P235–254 has the tendency to form a basic amphiphilic helix. The presence of two acid residues, Glu236 and Asp239, on the hydrophilic side of the α‐helix, which is mainly composed by basic residues, may explain the lower affinity of this peptide for calmodulin as compared with other peptides derived from calmodulin‐activated enzymes.
Amino acid 55 plays a central role in tetramerization and function of Escherichia coli single‐stranded DNA binding protein
Authors: CURTH Ute , BAYER Irene , GREIPEL Joachim , MAYER Frank , URBANKE Claus , MAASS Günter
Published: March 2005

The histidine at position 55 of the amino acid sequence of the Escherichia coli single‐stranded DNA binding protein was replaced by tyrosine, glutamic acid, lysine, phenylalanine, and isoleucine. The properties of the mutant proteins were determined using analytical ultracentrifugation, NMR spectroscopy, gel filtration, and fluorimetric detection of their single‐stranded DNA binding ability. While the phenylalanine and isoleucine substitutions did not change the properties of the protein measurably, tyrosine and lysine mutants dissociate into subunits and loose some of their binding affinity for poly(dT). For the lysine mutant we show by electron microscopy that the protein, although fully dissociated and possibly denatured in the free state, binds to poly(dT) as a tetramer indistinguishable from the wild‐type protein. The process of tetramerization as observed via single‐stranded DNA binding ability is composed of a variety of steps ranging in time from some milliseconds to several hours; it probably involves several forms of dissociated and non‐native protein.
Immunology, biosynthesis and in vivo assembly of the branched‐chain 2‐oxoacid dehydrogenase complex from bovine kidney
Authors: CLARKSON George H. D. , LINDSAY J. Gordon
Published: March 2005

Specific, polyclonal antisera have been raised to the native branched‐chain 2‐oxoacid dehydrogenase complex (BCOADC) from bovine kidney and each of its three constituent enzymes: E1, the substrate‐specific 2‐oxoacid dehydrogenase; E2, the multimeric dihydrolipoamide acyltransferase ‘core’ enzyme and E3, dihydrolipoamide dehydrogenase. Purified BCOADC, isolated by selective poly(ethyleneglycol) precipitation and hydroxyapatite chromatography, contains only traces of endogenous E3 as detected by a requirement for this enzyme in assaying overall complex activity and by immunoblotting criteria. A weak antibody response was elicited by the E1β subunit relative to the E2 and E1α polypeptides employing either purified E1 or BCOADC as antigens.

Anti‐BCOADC serum showed no cross‐reaction with high levels of pig heart E3 indicating the absence of antibody directed against this component. However, immunoprecipitates of mature BCOADC from detergent extracts of NBL‐1 (bovine kidney) or PK‐15 (porcine kidney) cell lines incubated for 3–4 h in the presence of [35S]methionine contained an additional 55000‐Mr species which was identified as E3 on the basis of immunocompetition studies.

Accumulation of newly synthesised [35S]methionine‐labelled precursors for E2, E1α and E3 was achieved by incubation of PK‐15 cells for 4 h in the presence of uncouplers of oxidative phosphorylation. Pre‐E2 exhibited an apparent Mr value of 56 500, pre‐E1α, 49 000 and pre‐E3, 57 000 compared to subunit Mr values of 50 000, 46 000 and 55 000, respectively, for the mature polypeptides. Thus, like the equivalent lipoate acyltransferases of the mammalian pyruvate dehydrogenase (PDC) and 2‐oxoglutarate dehydrogenase (OGDC) complexes, pre‐E2 of BCOADC characteristically contains an extended presequence.

In NBL‐1 cells, pre‐E2 was found to be unstable since no cytoplasmic pool of this precursor could be detected; moreover, processed E1α was not assembled into intact BCOADC as evidenced by the absence of E2 or E3 in immunoprecipitates with anti‐(BCOADC) serum after a 45‐min ‘chase’ period in the absence of uncoupler. Dihydrolipoamide dehydrogenase (E3), in its precursor state, was not present in immune complexes with anti‐(BCOADC) serum, indicating that its co‐precipitation with mature complex is by virtue of its high affinity for assembled complex in vivo whereas no equivalent interaction of pre‐E3 with its companion precursors occurs prior to mitochondrial import.
Exopolysaccharide structure from Bacillus circulans
Authors: FONTAINE Thierry , WIERUSZESKI Jean‐Michel , TALMONT Frank , SANIEZ Marie‐Héléne , DUFLOT Pierrick , LELEU Jean‐Bernard , FOURNET Bernard
Published: March 2005

The exopolysaccharide harvested from the liquid culture medium after Bacillus circulans fermentation consists of the following hexasaccharide repeating unit:
Two functional Na+/K+‐ATPase isoforms in the left ventricle of guinea pig heart
Authors: BERREBI‐BERTRAND Isabelle , MAIXENT Jean‐Michel , GUEDE Frederic G. , GERBI Alain , CHARLEMAGNE Danièle , LELIEVRE Lionel G.
Published: March 2005

Guinea pig left ventricular muscle contains two distinct molecular forms of the Na+/K+‐ATPase catalytic α subunit. Sarcolemmal vesicles highly enriched in Na+/K+‐ATPase were isolated by a new procedure that yielded specific activities of 60–100 μmol Pi· h−1· mg−1. SDS/PAGE of isolated sarcolemma after reduction and alkylation of the sulfhydryl groups and identification on immunoblots with specific anti‐(α subunit) antibodies indicated the presence of two major polypeptides of 100 kDa and 103 kDa, respectively. The two α subunits were functional: the dose/response curves of Na+/K+‐ATPase activity with ouabain, dihydrooubain and digitoxigenin were biphasic, revealing the presence of high‐affinity [concentration of drug causing 50% inhibition (IC50) = 10nM] and low‐affinity (IC50= 2 μM) forms with proportional contributions of 55% and 45%, respectively. The involvement of the high‐affinity form in the positive inotropic effect of digitalis and of the low‐affinity sites in both inotropy and toxicity are consistent with the literature data on rodents.
Effect of phenylephrine on pyruvate dehydrogenase in fasting rat livers
Authors: STERNICZUK Anna , HRENIUK Stan , SCADUTO Russell C. , LaNOUE Kathryn F.
Published: March 2005

Previous estimates of flux through the pyruvate‐dehydrogenase complex were made by measuring 14CO2 generated from oxidation of [1‐14C]pyruvate, assuming a 1:1 stoichiometry. However, this method fails to discriminate between 14CO2 produced from pyruvate dehydrogenase and 14CO2 generated from phospho‐enolpyruvate carboxykinase and citric‐acid‐cycle dehydrogenases. While some previous reports have attempted to correct for the additional 14CO2 production by comparing 14CO2 generated by [1‐14C]pyruvate with [2‐14C]pyruvate or [3‐14C]pyruvate, the estimates are flawed by failure to determine the radioactivity and distribution of the 14C label in the oxalacetate pool. The present method circumvents these problems by utilizing [1,4‐14C]succinate to radiolabel the oxalacetate pool and by directly measuring the specific radioactivity of malate. The results demonstrate that flux through the pyruvate‐dehydrogenase complex is negligible compared to the other reactions which generate 14CO2 from [1‐14C]lactate in the fasted state. Phenylephrine did not significantly alter this result in the fasted state. However, 14CO2 production via the pyruvate‐dehydrogenase complex is large (∼ 11.5 nmol · min−1· mg mitochondrial protein−1) compared to 14CO2 production via phosphoenolpyruvate carboxykinase and citric‐acid‐cycle dehydrogenases (∼ 6.4 nmol · min−1· mg−1) when the pyruvate‐dehydrogenase complex is activated, in the fed state with 1 mM dichloroacetate.
A second gene product of the inositol‐phospholipid‐specific phospholipase Cδ subclass
Authors: MELDRUM Eric , KRIZ Ronald W. , TOTTY Nicholas , PARKER Peter J.
Published: March 2005

Sequence analysis of a inositol‐phospholipid‐specific phospholipase C (PtdIns‐PLC) purified from bovine brain has led to the isolation of a novel cDNA that encodes this protein. While this cDNA contains two introns, these appear to be removed upon transfection of the cDNA into COS‐1 cells. The protein transiently expressed in COS‐1 cells shows phosphatidylinositol 4,5‐bisphosphate hydrolysing activity which distributes preferentially into the particulate fraction. Comparison of the predicted amino acid sequence of this PtdIns‐PLC with other Known PtdIns‐PLCs reveals a high degree of similarity, throughout all of its sequence, with PtdIns‐PLCδ. Thus, we believe that the identification of this cDNA represents evidence for multiple functional‐gene products within the δ subclass of PtdIns‐PLCs.
Molecular cloning and sequence analysis of bovine lactotransferrin
Authors: PIERCE Annick , COLAVIZZA Didier , BENAISSA Monique , MAES Pierrette , TARTAR André , MONTREUIL Jean , SPIK Geneviève
Published: March 2005

The screening of a bovine submaxillary gland cDNA library yielded 25 clones coding for bovine lactotransferrin. The nucleotide sequence of the longest insert contained a protein‐coding region of 2115 nucleotides and a 3′ non‐coding region of 194 nucleotides followed by a poly(A) tract of about 55 nucleotides. The predicted peptide sequence included a 16‐amino‐acid signal sequence upstream of the first amino acid of the native protein. The identity of the clone was confirmed by matching the amino acid sequence predicted from the cDNA with the N‐terminal and tryptic peptide sequences derived from purified bovine milk lactotransferrin, and also by similarity with human and murine lactotransferrins. The cDNA described corresponds to a 705‐amino‐acid‐long preprotein that lacks the start methionine. The sequence of the secreted protein is 689 amino acids long and contains five potential glycosylation sites. Bovine lactotransferrin is 69% and 64% identical to human and murine lactotransferrins, respectively.
Yeast fructose‐2,6‐bisphosphate 6‐phosphatase is encoded by PHO8, the gene for nonspecific repressible alkaline phosphatase
Authors: PLANKERT Uwe , PURWIN Claudio , HOLZER Helmut
Published: March 2005

Yeast fructose‐2,6‐bisphosphate 6‐phosphatase has been purified 7000‐fold by heat treatment, poly(ethylene glycol) precipitation, ion‐exchange chromatography with Q‐Sepharose Fast Flow and Mono Q followed by affinity chromatography with concanavalin‐A–Sepharose and gel filtration with Superose 12. The purified dimeric enzyme contains 1.5 mol zinc and 1.3 mol copper/mol subunit. It reacts with fructose 2,6‐bisphosphate [Fru(2,6)P2] as well as with p‐nitrophenyl phosphate (NpP) showing a pH optimum at pH 6–6.5 with Fru(2,6)P2 [Plankert, U., Purwin, C. & Holzer, H. (1988) FEBS Lett. 239, 69–72] and above pH 9.0 with NpP. The following observations suggest that activity with both substrates depends on the same protein. (a) During 7000‐fold purification, the ratio of activity with NpP to that with Fru(2,6)P2 remained constant. (b) The time course of inactivation of enzyme activity in dilute solution at 30 °C is similar for both substrates. (c) At increasing temperatures, inactivation of enzyme activity measured with both substrates proceeds at nearly identical rates. (d) Activity with both substrates is found preferentially in the vacuoles. (e) Mutants defective in the nonspecific alkaline phosphatase coded by the PHO8 gene are also defective in Fru(2,6)P2 6‐phosphatase activity. (f) A proteinase A mutant, defective in processing and activation of nonspecific alkaline phosphatase coded by the PHO8 gene, also fails to activate Fru(2,6)P2 6‐phosphatase.
Biochemical, cytotoxic and pharmacokinetic properties of an immunotoxin composed of a mouse monoclonal antibody Fib75 and the ribosome‐inactivating protein α‐sarcin from Aspergillus giganteus
Authors: WAWRZYNCZAK Edward J. , HENRY Raymond V. , CUMBER Alan J. , PARNELL Geoffrey D. , DERBYSHIRE Elaine J. , ULBRICH Norbert
Published: March 2005

An immunotoxin was synthesized by the attachment of α‐sarcin, the ribosome‐inactivating protein derived from the mould Aspergillus giganteus, to a monoclonal mouse IgG2a antibody Fib75. The α‐sarcin immunotoxin exerted toxic effects in tissue culture against the EJ human bladder carcinoma cell line, expressing the antigen recognised by the Fib75 antibody, inhibiting the incorporation of [3H]leucine by 50% at a concentration of 0.46 nM. The cytotoxic effects of the α‐sarcin immunotoxin were indistinguishable from those of a Fib75 immunotoxin made with ricin A chain. Fib75‐α‐sarcin was cleared from the circulation of the rat with biphasic kinetics following intravenous administration. The α‐ and β‐phase half‐lives were 0.8 h and 6 h, respectively, similar to the serum half‐lives of analogous Fib75 immunotoxins made with ribosome‐inactivating proteins derived from plants. α‐Sarcin was completely stable in physiological saline buffer at 37°C, whereas the ribosome‐inactivating activity of ricin A chain was gradually lost under identical conditions. α‐Sarcin may be a valuable alternative to ricin A chain for the construction of therapeutic immunotoxins because of its smaller size and greater thermostability.
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).
Deoxyribonucleotide metabolism in hydroxyurea‐resistant V79 hamster cells
Authors: HÖGLUND Lars , PONTIS Elisabet , REICHARD Peter
Published: March 2005

V79 hamster cells were made resistant against hydroxyurea by continuous culture at stepwise increasing drug concentrations. Two cell lines were cloned, resistant to 0.4 mM (V79/H0.4) and 4 mM (V79/H4) hydroxyurea, with a fivefold and a 20‐fold increase in soluble ribonucleotide reductase activity. We investigated how the increased amount of enzyme affected the in situ activity of ribonucleotide reductase and deoxyribonuleotide metabolism, in particular substrate cycles between pyrimidine deoxyribonucleosides and their 5′‐phosphates. The in situ activity of the reductase was only moderately elevated (1.3‐fold in V79/H4 cells). In the fully resistant line, the steady‐state level of dATP was increased fourfold, and that of dTTP twofold. These nucleotides are negative allosteric effectors of the reductase and we propose that the increased pools inhibit the enzyme and thereby maintain the in situ activity of the reductase at only a slightly increased level. The surplus deoxyribonucleotides was excreted from the cells as thymidine and deoxycytidine via substrate cycles. The data support and extend our previous model for the regulation of deoxyribonucleotide synthesis via the allosteric properties of ribonucleotide reductase and substrate cycles that link salvage and degradation of deoxyribonucleotides.
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.
Effect of deletion of the A1 domain of von Willebrand factor on its binding to heparin, collagen and platelets in the presence of ristocetin
Authors: SIXMA Jan J. , SCHIPHORST Marion E. , VERWEIJ Cor L. , PANNEKOEK Hans
Published: March 2005

In order to study the functional importance of the collagen, heparin and glycoprotein‐Ib‐binding domain, we deleted the A1 domain of von Willebrand factor (vWF), corresponding to residues 478–716, by oligonucleotide‐directed mutagenesis. The resulting ΔA1‐vWF cDNA was expressed in COS‐1 monkey kidney cells and compared to wild‐type vWF. The higher‐molecular‐mass multimers were decreased in ΔA1 recombinant von Willebrand factor (ΔA1‐rvWF) compared to plasma vWF and rvWF. The reactivity of ΔA1‐rvWF and rvWF with monoclonal antibodies directed against the collagen‐binding domain (residues 969–992), the vessel‐wall‐binding domain, and the binding site for glycoprotein IIb‐III a on platelets was identical. The interaction with vWF of the monoclonal antibody directed against the glycoprotein Ib binding domain was abolished for ΔA1‐rvWF, and similar to plasma vWF for rvWF. The binding of factor VIII to ΔA1‐rvWF and rvWF was similar. ΔA1‐rvWF and rvWF bound similarly to collagen, but the binding of ΔA1‐rvWF to heparin and to platelets in the presence of ristocetin were abolished. These data indicate that the heparin‐binding site in the A1 domain is essential. There is no second binding domain for glycoprotein Ib outside the A1 domain. The collagen‐binding domain in the A1 domain is either not active or its action can be compensated by the second collagen‐binding domain.
Chemical modification and 1H‐NMR studies on the receptor‐binding region of human interleukin 6
Authors: NISHIMURA Chiaki , EKIDA Teiji , MASUDA Susumu , FUTATSUGI Kensuke , ITOH Shun‐ichi , YASUKAWA Kiyoshi , KISHIMOTO Tadamitsu , ARATA Yoji
Published: March 2005

Oxidation of the Met residues of human interleukin 6 (IL‐6) molecule has been performed. Reactivity of Met for the oxidation reaction was found to decrease in the order of Met50, Met118, Met185, Met162, and Met68. Chemical modifications involving oxidation and carboxypeptidase A digestion of IL‐6 have led to the assignments of the methyl proton resonances of Met162 and Met185, respectively. The hydroxynitrobenzyl chromophore attached to Trp158 in the IL‐6 molecule showed a different absorption spectrum when the labeled IL‐6 was bound to the soluble IL‐6 receptor. This result indicates that Trp158 is near the receptor‐binding region in IL‐6. On the basis of the 1H‐NMR and chemical modification data, it has been concluded that Trp158 is in spatial proximity to Met162, His165 and Met185. The receptor‐binding activity decreased with an increase in the number of oxidized Met residues. Of these five Met residues, Met162 was the residue in which the receptor‐binding activity decreased in the most parallel degree with that of the oxidation reaction.
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.
Primary structure of toad sperm protamines and nucleotide sequence of their cDNAs
Authors: TAKAMUNE Kazufumi , NISHIDA Hiromi , TAKAI Masayuki , KATAGIRI Chiaki
Published: March 2005

Acid extract of mature sperm of the toad, Bufo japonicus, exclusively comprised sperm‐specific basic proteins which moved faster than somatic histones on acid/urea/Triton X‐100 polyacrylamide gel electrophoresis. When these proteins were purified by reversed‐phase high‐performance liquid chromatography they were found to consist of three components; one of these was a phosphorylated form of another, so that there were only two distinct components (P1 and P2). Amino acid sequence analyses indicated that the components both contained 39 amino acid residues, with 43.6% Arg, and differed only in the 28th amino acid residue (P1, Asp; P2, Glu). They had molecular masses of 5092 Da (P1) and 5106 Da (P2). The nucleotide sequences of cDNA clones encoding P1 (245 bases) and P2 (305 bases) showed that the difference in the amino acid residue between P1 and P2 was due to the difference of a nucleotide at position +87. Both cDNAs possessed a canonical signal (AATAAA) for polyadenylation and/or cleavage of transcript at the 3′ untranslational region. Statistical analyses of amino acid sequence similarities suggested that the Bufo protamines are homologous with the protamines of fishes rather than with those of avian/mammalians.
Induced plant responses to pathogen attack
Authors: WELLE Roland , SCHRÖDER Gudrun , SCHILTZ Emil , GRISEBACH Hans , SCHRÖDER Joachim
Published: March 2005

In soybean (Glycine max L.), pathogen attack induces the formation of glyceollin‐type phytoalexins. The biosynthetic key enzyme is a reductase which synthesizes 4,2′,4′‐trihydroxychalcone in co‐action with chalcone synthase. Screening of a soybean cDNA library from elicitor‐induced RNA in λ gt11 yielded two classes of reductase‐specific clones. The deduced proteins match to 100% and 95%, respectively, with 229 amino acids sequenced in the purified plant protein. Four clones of class A were expressed in Escherichia coli, and the proteins were tested for enzyme activity in extracts supplemented with chalcone synthase. All were active in 4,2′,4′‐trihydroxychalcone formation, and the quantification showed that shorter lengths of the cDNAs at the 5′ end correlated with progressively decreasing enzyme activities. Genomic blots with DNA from plants capable of 4,2′,4′‐trihydroxychalcone synthesis revealed related sequences in bean (Phaseolus vulgaris L.) and peanut (Arachis hypogaea L.), but not in pea (Pisum sativum L.). No hybridization was observed with parsley (Petroselinum crispum) and carrot (Daucus carota) which synthesize other phytoalexins. The reductase protein contains a leucine‐zipper motif and reveals a marked similarity with other oxidoreductases most of which are involved in carbohydrate metabolism.