bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrostatic interactions in the cellular dynamics of the interferon‐receptor complex
Authors: UZE Gilles , BANDU Marie‐Thérèse , EID Pierre , GRÜTTER Markus , MOGENSEN Knud E.
Published: March 2005

Using membrane preparations of the interferon receptor, prepared from cells of the Burkitt line, Daudi, we have examined the binding of three human recombinant α‐interferons.

We discovered a binding titration of the interferons IFN‐αA and IFN‐αD in the pH range 6–9. Receptor binding, negligible at pH 6, rises to a maximum close to pH 9. We have shown that binding of IFN‐αA at basic pH is to the same receptors as at neutrality and that IFN‐receptor complexes extracted with digitonin are more stable at basic pH than they are at neutrality.

The recombinant interferon, IFN‐αB, shows little change of binding in the pH range 6–9. At its basic optimum the binding of IFN‐αA approaches that of IFN‐αB, while at neutral pH the binding of IFN‐αA is 3–4 times less. This difference at neutral pH is seen on intact cells as well as on membrane preparations. The specific activity of IFN‐αB is close to that of IFN‐αA, both of which are 10–20 times more active than IFN‐αD; and the binding titration is, therefore, independent of the initial binding affinities.

Using hybrid IFNs constructed from the DNA sequences of αD and αB, we have isolated the sequence responsible for the binding titration to the segment comprising amino acids 61–92. Examination of these sequences reveals that Lys‐84 is present in all the IFN‐α except IFN‐αB where it is replaced by Glu; and Tyr‐90, present in most of the common IFN‐α including αA and αD, is replaced by Asp in IFN‐αB. Lys and Tyr would normally titrate in the pH range 6–9. We conclude that the binding titration is due to an electrostatic interaction and we propose that the interaction is between IFN‐receptor complexes. The role of the interaction in the binding losses that accompany the antiproliferative effects of IFN is discussed.
Role of tyrosine‐80 in the stability of kanamycin nucleotidyltransferase analyzed by site‐directed mutagenesis
Authors: MATSUMURA Masazumi , YAHANDA Saburo , YASUMURA Shigeyoshi , YUTANI Katsuhide , AIBA Shuichi
Published: March 2005

A thermostable mutant of kanamycin nucleotidyltransferase (KNTase) with a single amino acid replacement of Asp at position 80 by Tyr has been isolated by a novel screening method in a previous study [Matsumura, M. & Aiba, S. (1985) J. Biol. Chem. 260, 15298–15303]. To elucidate the role of Tyr80 in stabilizing the enzyme, the KNTase gene was modified by site‐directed mutagenesis so that the codon for Asp80 of the wild type was replaced by that for Ser, Thr, Ala, Val, Leu, Phe and Trp, respectively. The eight mutant KNTases including Tyr80 were all purified, as well as the wild‐type enzyme. The heat‐inactivation rate constants were determined at 58°C and the half‐life values were found to be correlated with the hydrophobicity of the amino acid residues replaced at the unique position. The Gibbs energy change of unfolding in water of KNTase assessed from urea denaturation (25 °C, pH 7.0) was also found to be correlated with hydrophobicity. The results suggest that different amino acids at position 80 of KNTase contribute to the stability of the protein by hydrophobic interactions. In the case of tyrosine at position 80 the unusually high stability of the enzyme compared to the Phe80 enzyme suggests that the hydroxyl group also contributes to the conformational stability.
Highly sensitive peptide‐4‐methylcoumaryl‐7‐amide substrates for blood‐clotting proteases and trypsin
Authors: KAWABATA Shun‐Ichiro , MIURA Takako , MORITA Takashi , KATO Hisao , FUJIKAWA Kazuo , IWANAGA Sadaaki , TAKADA Katsumi , KIMURA Terutoshi , SAKAKIBARA Shumpei
Published: March 2005

Seventy‐four peptide amides of 7‐amino‐4‐methylcoumarine (Mec) of the type Boc‐Xaa‐Yaa‐Arg‐NH‐Mec were newly synthesized and tested to find specific substrates for blood‐clotting proteases and trypsin. The Xaa and Yaa residues of these substrates have been replaced by 12 and 15 different amino acids, respectively. Among these peptides, the followings were found to be most sensitive substrates for individual enzymes: Boc‐Asp(OBzl)‐Pro‐Arg‐NH‐Mec (kcat= 160 s−1, Km= 11 μM, kcat/Km=15000000 M−1 s−1) for human α‐thrombin, Z‐< Glu‐Gly‐Arg‐NH‐Mec(kcat= 19 s−1, Km= 59 μM, kcat/Km= 320000 M−1 s−1) for bovine factor Xa, Boc‐Gln‐Gly‐Arg‐NH‐Mec (kcat= 5.8 s−1, Km= 140 μM, kcat/Km= 42000) for bovine factor XIIa, Boc‐Asp(OBzl)‐Ala‐Arg‐NH‐Mec (kcat= 9.2 s−1, Km= 120 μM, kcat/Km= 77000 M−1 s−1) for bovine activated protein C, and Boc‐Gly‐Phe‐Arg‐NH‐Mec (kcat= 29 s−1, Km= 230 μM, kcat/Km= 130000 M−1 s−1) for bovine plasma kallikrein. Moreover, Boc‐Glu(OBzl)‐Ala‐Arg‐NH‐Mec (kcat= 46 s−1, Km= 370 μM, kcat/Km= 120000 M−1 s−1) was newly found as a good substrate for human factor XIa. Bovine trypsin effectively hydrolyzed peptide‐NH‐Mec substrates containing Ala and Pro at the P2 site. The most reactive substrate was Boc‐Gln‐Ala‐Arg‐NH‐Mec (kcat= 120 s−1, Km= 6.0 μM, kcat/Km= 20000000 M−1 s−1).
Specific inhibition of simian virus 40 protein synthesis by heat and arsenite treatment
Authors: ANGELIDIS Charalampos E. , LAZARIDIS Ioannis , PAGOULATOS Gerassimos N.
Published: March 2005

The effects of heat treatment of CV1 cells infected with simian virus 40 (SV40) on viral and cellular protein synthesis were investigated by one‐dimensional and two‐dimensional polyacrylamide gel electrophoresis.

A 12‐h heat treatment during the late phase of the viral life‐cycle inhibits VP1 synthesis. No inhibition of normal cellular proteins is apparent, but heat‐shock proteins are strongly induced and accumulate in the cells. Inhibition of VP1 synthesis in infected cells is demonstrated to occur also after arsenite treatment, another agent known to induce heat‐shock proteins. Northern blot analysis of cytoplasmic RNA demonstrated a decrease in the abundance of late SV40 mRNAs thus showing that the inhibition occurs at the transcriptional or immediately post‐transcriptional level. Cumulative labeling with [3H]thymidine of viral DNA showed that the decrease in the abundance of late mRNAs is not due to a blocking of viral DNA synthesis. Immunofluorescence microscopy and immunoprecipitation analysis show that heat and arsenite treatments also affect the synthesis of T antigen.

These results suggest that heat‐shock proteins may play a role in the inhibition of SV40 virus gene functions.
Human basement membrane collagen (type IV)
Authors: BRAZEL Dieter , POLLNER Reinhold , OBERBÄUMER Ilse , KÖHN Klaus
Published: March 2005

The cDNA and protein sequences of the N‐terminal 60% of the α2(IV) chain of human basement membrane collagen have been determined. By repeated primer extension with synthetic oligodeoxynucleotides and mRNA from either HT1080 cells or human placenta overlapping clones were obtained which cover 3414 bp. The derived protein sequence allows for the first time a comparison and alignment of both α chains of type IV collagen from the N terminus. This alignment reveals an additional 43 amino acid residues in the α2(IV) chain as compared to the α1(IV) chain. 21 of these additional residues form a disulfide‐bridged loop within the triple helix which is unique among all known collagens.
Purification of the LSTRA tyrosine protein kinase (p56lck)
Authors: VAN TAN Huynh , FISCHER Siegmund , FAGARD Remi
Published: March 2005

p56lck, a p60src related tyrosine protein kinase was recently described in a murine lymphoma cell line (LSTRA), in several human lymphomas and in normal peripheral lymphocytes. We have purifed p56lck to homogeneity by electrofocusing followed by two‐step SDS/polyacrylamide gel electrophoresis. The pure protein was identical to the p56 characterized in LSTRA cells and used to raise antibodies in rabbits. This simple procedure is applicable for the rapid purification of minor proteins.
Rat liver alcohol dehydrogenase of class III
Authors: JULIÀ Pere , PARÉS Xavier , JÖRNVALL Hans
Published: March 2005

The amino acid sequence of alcohol dehydrogenase of class III from rat liver (the enzyme ADH‐2) has been determined. This type of structure is quite different from those of both the class I and the class II alcohol dehydrogenases. The rat class III structure differs from the rat and human class I structures by 133–138 residues (exact value depending on species and isozyme type); and from that of human class II by 132 residues. In contrast, the rat/human species difference within the class III enzymes is only 21 residues. The protein was carboxymethylated with iodo[214C]acetate, and cleaved with CNBr and proteolytic enzymes. Peptides purified by exclusion chromatography and reverse‐phase high‐performance liquid chromatography were analyzed by degradation with a gas‐phase sequencer and with the manual 4‐N,N‐dimethylaminoazobenzene‐4′‐isothiocyanate double‐coupling method.

The protein chain has 373 residues with a blocked N terminus. No evidence was obtained for heterogeneity. The rat ADH‐2 enzyme of class III contains an insertion of Cys at position 60 in relation to the class I enzymes, while the latter alcohol dehydrogenase in rat (ADH‐3) has another Cys insertion (at position 111) relative to ADH‐2. The structure deduced explains the characteristic differences of the class III alcohol dehydrogenase in relation to the other classes of alcohol dehydrogenase, including a high absorbance, an anodic electrophoretic mobility and special kinetic properties. The main amino acid substitutions are found in the catalytic domain and in the subunit interacting segments of the coenzyme‐binding domain, the latter explaining the lack of hybrid dimers between subunits of different classes. Several substitutions provide an enlarged and more hydrophilic substrate‐binding pocket, which appears compatible with a higher water content in the pocket and hence could possibly explain the higher Km for all substrates as compared with the corresponding values for the class I enzymes. Finally the class III structure supports evolutionary relationships suggesting that the three classes constitute clearly separate enzymes within the group of mammalian zinc‐containing alcohol dehydrogenases.
Isocitrate lyase from Pinus pinea
Authors: GIACHETTI Eugenio , PINZAUTI Giancarlo , BONACCORSI Riccardo , VANNI Paolo
Published: March 2005

We found that the Mg‐isocitrate complex is the true substrate for pine isocitrate lyase and that magnesium acts as a non‐essential activator. Both the non‐activated and the activated enzyme forms are catalytically active. Our model is consistent with the presence of two Mg‐binding sites with different affinities: an activator site with high affinity in addition to the catalytic site with lower affinity. This may result in a complex, fine regulation of isocitrate lyase activity by magnesium. The affinity of the free enzyme for isocitrate is very low. Moreover, free isocitrate does not bind to the activated enzyme, nor it can yield a catalytically active form by binding to an enzyme species whose catalytic site has already been bound by magnesium.
Purification of bovine liver cytosolic 5′‐nucleotidase
Authors: ZEKRI Mustapha , HARB Jean , BERNARD Serge , MEFLAH Khaled
Published: March 2005

Cytosolic 5′‐nucleotidase from bovine liver has been purified to homogeneity. Two affinity chromatographies on concanavalin A and 5'AMP‐Sepharose columns result in a 12000‐fold purification. The sequential elution of glycoproteins from the concanavalin‐A – Sepharose column with methyl α‐D‐glucoside and methyl α‐D‐mannoside greatly increases the degree of purification of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows two subunits having apparent molecular masses of 65 kDa and 57 kDa respectively, while only one band at 70 kDa is observed in the case of the membrane‐bound 5′‐nucleotidase. Both the Stokes radii, measured by gel exclusion HPLC, and the sedimentation coefficient, determined by density gradient ultracentrifugation, indicate that the cytosolic enzyme is a heterodimer of about 130 kDa. This contrasts with the membrane‐bound 5′‐nucleotidase which is a homodimer of 140 kDa. Moreover, the antibodies raised against the membrane 5′‐nucleotidase inhibited the cytosolic form indicating that a common antigenic determinant(s) exists between the two isoenzymes. However, structural differences are revealed by immunoblotting. In the same way, the effect of lectins suggests that differences in the structure of the carbohydrate chains exist between the two isoenzymes.

The purified cytosolic enzyme has lower affinity for the nucleotides than does the membrane enzyme. In addition, while ADP, [α,β‐CH2]ADP and ATP were strong competitive inhibitors of the membrane enzyme, ADP and ATP activate the cytosolic form and [α,β‐CH2]ADP has no effect. Moreover, two pH optima at 7.5 and 9.5 are observed in the cytosolic enzyme while only one at 7.5 occurred in the membrane form. Finally the exogenous cations, MgCl2 and MnCl2, are necessary for the maximal activity of the cytosolic but not of the membrane 5′‐nucleotidase. All these observations indicate that the two isoenzymes are different.
1H nuclear‐magnetic‐resonance studies of the three‐dimensional structure of the cardiotoxin CTXIIb from Naja mossambica mossambica in aqueous solution and comparison with the crystal structures of homologous toxins
Authors: STEINMETZ Wayne E. , BOUGIS Pierre E. , ROCHAT Hervé , REDWINE O. David , BRAUN Werner , WÜTHRICH Kurt
Published: March 2005

Using the previously reported sequence‐specific 1H‐NMR assignments, structural constraints for the cardiotoxin CTXIIb from Naja mossambica mossambica were collected. These include distance constraints from nuclear Overhauser enhancement measurements both in the laboratory and in the rotating frame, dihedral angle constraints derived from spin‐spin coupling constants, and constraints from hydrogen bonds and disulfide bridges. Structure calculations with the distance geometry program DISMAN confirmed the presence of the previously identified antiparallel β‐sheets formed by residues 1–5 and 10–14, and by 20–27, 35–39 and 49–55, and established the nature of the connections between the individual β‐strands. These include a right‐handed crossover between the two peripheral strands in the triple‐stranded β‐sheet, and a type I tight turn immediately preceding the β‐strand 49–55. The spatial arrangement of the polypeptide backbone in the solution structure of CTXIIb is closely similar to that in the crystal structure of the homologous cardiotoxin VII4 from the same species. In an Appendix the origin of the large pH dependence of two amide proton chemical shifts in CTXIIb is explained.
The heat‐shock response in Trypanosoma cruzi
Authors: ALCINA Antonio , URZAINQUI Ana , CARRASCO Luis
Published: March 2005

When Trypanosoma cruzi epimastigotes are exposed to temperatures of 37–41°C there is a drastic decline in total protein synthesis. Analysis of the proteins synthesized at 41°C by one‐dimensional gel electrophoresis showed three major bands of Mr 83000, 70000 and 60000. A similar pattern of heat‐shock proteins was found in two different strains of T. cruzi (Tulahuen and GM strains) and in exponentially growing or in stationary epimastigotes. Actinomycin D prevented the appearance of these polypeptide bands, suggesting that the heat‐shock proteins in T. cruzi epimastigotes are induced at the level of transcription. Analysis of the proteins synthesized by metacyclic forms at different temperatures suggests that heat‐shock proteins in these cells are already synthesized at 27°C. Elevation of temperature above 37°C blocks the synthesis of most proteins in metacyclic forms except for major bands of Mr 83000, 70000, 60000 and 55000. More detailed analyses by high‐resolution two‐dimensional gel electrophoresis of the proteins synthesized at 27°C or 37°C by epimastigotes indicates that the heat‐shock protein pattern is more complex than that demonstrated by one dimension, and at least ten new polypeptides are identified in two‐dimensional gels. A similar analysis of metacyclic forms shows that most if not all the proteins present at 39°C are also present at 27°C. This result led us to the suggestion that the differentiation of T. cruzi to metacyclic forms involves the induction of heat‐shock proteins, which prepares the parasite to infect the mammalian host.
Characterization of cDNA clones for human glycophorin A
Authors: RAHUEL Cécile , LONDON Jacqueline , d'AURIOL Luc , MATTEI Marie‐Geneviève , TOURNAMILLE Christophe , SKRZYNIA Cécile , LEBOUC Yves , GALIBERT Francis , CARTRON Jean‐Pierre
Published: March 2005

Glycophorin A is the major membrane sialoglycoprotein of human erythrocytes and represents a typical example of a transmembrane glycoprotein. The functional role of this cell‐surface component is not known but it represents a receptor for viruses, bacteria and parasites like Plasmodium falciparum.

Two cDNA clones encoding glycophorin A have been characterized from human fetal cDNA libraries. The longer cDNA extended from the coding region of glycophorin A (residues 4–131) to the 3′ untranslated region which included two polyadenylation signals and a poly(A) tail.

The structural gene for glycophorin A is located on chromosome 4, q28–q31 as shown by in situ hybridization, thus confirming the previous localization by genetic linkage analysis.

Three distinct mRNA species (1.0 kb, 1.7 kb and 2.2 kb) have been identified in erythroid spleen. Northern blot analyses with a probe directed against the 3′ untranslated region of the mRNAs indicated that all these species share a homologous 3′ non‐coding region and that the first polyadenylation signal downstream the stop codon is not used.

Preliminary studies by Southern blot analysis of the genomic DNA from normal En(a+) and rare En(a‐) donors suggest that the glycophorin A gene has a complex organization and is largely deleted in donors of the En(a‐) phenotype (Finnish type) who lack glycophorin A on their red cells.
Excision of apurinic and/or apyrimidinic sites from DNA by nucleolytical enzymes from rat brain
Authors: IVANOV Vladimir A. , TRETYAK Tatyana M. , AFONIN Yury N.
Published: March 2005

Apurinic and/or apyrimidinic (AP) sites were excised from PM2 phage DNA by two enzymes: an AP endodeoxyribonuclease isolated from rat neocortex chromatin and a rat brain exodeoxyribonuclease, DNase B III. The resulting gap was filled with DNA polymerase β prepared from rat liver and finally ligated by Escherichia coli DNA ligase.
Spectroscopic studies on cadmium(II)‐ and cobalt(II)‐substituted metallothionein from the crab Cancer pagurus
Authors: OVERNELL Julian , GOOD Meinrad , VAŠÀK Milan
Published: March 2005

The binding of diamagnetic Cd(II) and paramagnetic Co(II) ions to the metal‐free form of crab, Cancer pagurus, metallothionein (MT) was studied by various spectroscopic techniques. Both reconstituted and native Cd(II)‐MT containing 6 mol Cd(II)/mol protein display electronic absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra which were indistinguishable. The stoichiometric replacement of Cd(II) ions in native Cd(II)6‐MT by paramagnetic Co(II) ions enabled the geometry of the metal‐binding sites to be probed. The electronic absorption and MCD spectra of Co(II)6‐MT revealed features characteristic of distorted tetrahedral tetrathiolate Co(II) coordination for all six metal‐binding sites. The stepwise incorporation of Cd(II) and Co(II) ions into this protein was monitored by electronic absorption and CD, and by electronic absorption and EPR spectroscopy, respectively. The results indicate that the metal‐thiolate cluster structure is generated when more than four metal ions are bound. Below this titration point separate tetrahedral tetrathiolate complexes exist. This suggests that the cluster formation occurs in a two‐step process. Furthermore, the spectroscopic features in both Cd(II)‐ and Co(II)‐metal derivatives above the full metal occupancy of six suggest the existence of one additional metal‐binding site. The subsequent loss of one Cd(II) ion from crab Cancer Cd(II)7‐MT in the gel filtration studies demonstrate the low metal‐binding affinity of the latter site. While the spectroscopic properties indicate an exclusively tetrahedral type of metal‐thiolate sulfur coordination for the binding of the first six metal ions, they suggest that the seventh metal ion is coordinated in a different fashion.
Inactivation of the gene encoding the 11‐kDa subunit VIII of the ubiquinol–cytochrome‐c oxidoreductase in Saccharomyces cerevisiae
Authors: MAARSE Ammy C. , DE HAAN Muus , SCHOPPINK Peter J. , BERDEN Jan A. , GRIVELL Leslie A.
Published: March 2005

The single nuclear gene encoding the 11‐kDa subunit VIII of the ubiquinol– cytochrome‐c oxidoreductase (complex III) in Saccharomyces cerevisiae has been inactivated by a one‐step gene disruption procedure. Inactivation results in a loss of ubiquinol–cytochrome‐c oxidoreductase activity (< 1 % wild type) and respiratory deficiency. Cells lacking the 11‐kDa protein also display lowered steady‐state levels of other complex‐III subunits encoded by nuclear genes including the 14‐kDa subunit VII and the Rieske Fe‐S protein and of the mitochondrially encoded cytochrome b. The steady‐state levels of the transcripts from the genes encoding these proteins are however not reduced. The results strongly imply that the 11‐kDa protein plays an important role in regulating the synthesis of complex III at the post‐transcriptional level, most likely assembly. Separation of chromosomes by pulsed‐field gel electrophoresis of DNA of wild‐type and of the mutant lacking the 11‐kDa‐protein gene followed by Southern blot analysis reveals that the latter gene is located on chromosome X rather than on XII as reported by Van Loon et al. [Mol. Gen. Genet. 197 (1984) 219–241].
Interaction of a spin‐labelled cholesterol derivative with the cytochrome P‐450SCC active site
Authors: LANGE Reinhard , MAURIN Luc , LARROQUE Christian , BIENVENÜE Alain
Published: March 2005

The cholesterol analogue 25‐doxyl‐27‐nor‐cholesterol (CNO), was found to be a substrate for cytochrome P‐450scc. Upon incubation with the cytochrome P‐450scc electron transfer system, CNO is transformed to pregnenolone (Km= 33 μM, Vmax= 0.32 min−1). The pregnenolone formation from endogenous cholesterol is strongly inhibited by CNO (50% at 5 μM). It binds tightly to cytochrome P‐450scc as evidenced by a reversed type I spectral absorbance change (Kd= 5.9 μM) which is paralleled by a greater hyperfine splitting of the room‐temperature CNO ESR spectrum due to an enhanced probe immobilization (Kd= 1.9 μM). This finding is in accord with a rotational correlation time of about 10−7 s, which is close to the tumbling rate of the protein. At 110 K the CNO‐bound cytochrome P‐450ssc displays the ESR g‐values gx= 2.404/2.456, g,y= 2.245 and gz= 1.916; these are different from those of cholesterol‐liganded cytochrome P‐450scc and may thus serve as a marker for cytochrome P‐450ssc.

Our data indicate that the stereospecificity of the cytochrome P‐450scc, side‐chain‐cleaving activity is not dependent on the nature of the cholesterol side‐chain termination (C25 to C27). The substrate binding site is however rather sensitive to a modification of the side chain. The doxyl ring confers a stronger affinity of the substrate to the enzyme. Upon binding it becomes embedded in the protein matrix, and we estimate that its final position is 0.6 – 1.0 nm from the heme moiety.
Purified subunit δ of chloroplast coupling factor CF1 reconstitutes photophosphorylation in partially CF1‐depleted membranes
Authors: ENGELBRECHT Siegfried , JUNGE Wolfgang
Published: March 2005

The ATP synthase of chloroplasts consists of the proton channel, CF0, and the catalytic part, CF1, which carries nucleotide‐binding sites on subunits α and β. The still poorly understood interaction between CF0 and the catalytic sites on CF1 is mediated by the smaller subunits γ, δ and ɛ of CF1. We investigated the ability of purified δ to block proton leakage through CF0 channels after their exposure by removal of the CF1 counterpart.

Thylakoids were partially depleted of CF1 by EDTA treatment. This increased their proton permeability and thereby reduced the rate of photophosphorylation. Subunit δ was isolated and purified by FPLC [Engelbrecht, S. and Junge, W. (1987) FEBS Lett. 219, 321–325]. Addition of δ to EDTA‐treated thylakoids reconstituted high rates of phenazine‐methosulfate‐mediated photophosphorylation. Since δ does not interact with nucleotides by itself, the reconstitution was due to a reduction of the proton leakage through open CF0, channels. The molar ratio of purified δ over exposed CF0, which started to elicit this effect, was 3:1. However, if δ was added together with purified CF1 lacking δ, in a 1:1 molar ratio, the relative amount over exposed CF0 was as low as 0. 06. This corroborated our previous conclusion [Lill, H., Engelbrecht, S., Schönknecht, G. and Junge, W. (1986) Eur. J. Biochem. 160, 627–634] that only a very small fraction of exposed CF0 was actually proton‐conducting but with a very high unit conductance. CF1 including δ was apparently rebound preferentially to open CF0 channels.

Although the ability of δ to control proton conduction through CF0 was evident, it remains to be established whether δ acts as a gated proton valve or as a conformational transducer in the integral CF0CF1 ATPase.
Analysis of degradation of the basement membrane protein nidogen, using a specific monoclonal antibody
Authors: DZIADEK Marie , CLEMENTS Richard , MITRANGAS Katherine , REITER Hildegard , FOWLER Kerry
Published: March 2005

A monoclonal antibody was produced against purified nidogen extracted from a mouse basement‐membrane‐producing tumor. This antibody reacted with a determinant on Nd‐40, a rod which separates the globular domains of nidogen. Antigenicity depends on intrachain disulfide bonds within this rod. The monoclonal antibody was used to detect nidogen fragments after proteolytic cleavage of isolated nidogen, and nidogen complexed to laminin. The data indicate that thrombin and thermolysin generated very different patterns of degradation, but in both cases no differences were found between isolated and complexed nidogen. In contrast, nidogen in the laminin‐nidogen complex was much less degraded by trypsin than isolated nidogen, indicating that an interaction between these basement membrane components reduces the susceptibility of nidogen to trypsin digestion. Immunofluorescent studies, using the monoclonal antibody on sections of the EHS tumor after proteolytic digestion, showed that the retention or disappearance of the Nd‐40 determinant correlated with the in vitro digestion pattern of the laminin‐nidogen complex.
Thrombin‐like inhibitory action of trypsin and trypsin‐like proteases on human platelet adenylate cyclase
Authors: JAKOBS Karl H. , GRANDT Rüdiger
Published: March 2005

The effects of trypsin, acrosin and a recently described trypsin‐like protease from bovine sperm were studied on adenylate cyclase activity in membranes of human platelets. These proteases caused an immediate decrease in adenylate cyclase activity, which was independent of the platelet membrane concentration used and which was constant for up to 20 min of incubation at 25°C. When the incubation was prolonged, the proteases eliminated their own inhibitory action as well as that of the inhibitory hormone epinephrine. The adenylate cyclase inhibition caused by the proteases was strictly dependent on the presence of GTP (EC50∼ 0.1 μM), whereas in the absence of GTP only minor changes in enzyme activity were observed at the conditions and protease concentrations used. Maximal inhibition caused by the proteases was between 40% and 60%. Half‐maximal inhibition by the purified proteases trypsin and acrosin was observed at about 30 ng/ml and 2 μg/ml respectively. Inhibition of platelet adenylate cyclase by the proteases was partially additive with that caused by epinephrine, while with thrombin no additivity was observed. The serine protease inhibitor leupeptin blocked the actions of the proteases when added simultaneously with the enzymes, but was ineffective when added later on. Treatment of platelet membranes with the alkylating N‐ethylmaleimide at low concentrations and Mn2+ ions (≥ 1 mM), both agents known to abolish inhibition of adenylate cyclase via the inhibitory guanine‐nucleotide‐binding protein Gi, eliminated the inhibitory action of the proteases. The data indicate that trypsin and trypsin‐like proteases have two opposite effects on the platelet adenylate cyclase system, the well‐documented elimination of Gi action and, as shown here, an immediate activation of Gi with subsequent adenylate cyclase inhibition. The data are consistent with the hypothesis that the activation of Gi caused by the proteases is due to an interaction of the proteases with specific cell‐surface receptor sites in a manner similar to thrombin.
Differential inhibition of various deoxyribonucleic and ribonucleic acid polymerases by suramin
Authors: ONO Katsuhiko , NAKANE Hideo , FUKUSHIMA Masanori
Published: March 2005

The inhibitory effects of hexasodium sym‐bis(m‐aminobenzoyl‐m‐amino‐p‐methylbenzoyl‐1‐naphthylamino‐4,6,8‐trisulfonate)carbamide (trivial name: suramin) on the activities of various deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) polymerases from mammalian cells, bacteria and retrovirus were examined and compared with each other. Among the various DNA and RNA polymerases tested, the activities of DNA primase, DNA polymerase α, reverse transcriptase and Escherichia coli RNA polymerase were strongly inhibited by suramin, while the activities of other enzymes including DNA polymerases β and γ, terminal deoxynucleotidyl‐transferase and DNA polymerase I were relatively resistant to inhibition by this drug. The inhibition by suramin of DNA polymerase α from KB cells and Rauscher murine leukemia virus (RLV) reverse transcriptase was due to competition with the respective template · primer (activated DNA for α polymerase and (rA)n· (dT)12–18 for reverse transcriptase) for the template · primer‐binding site of the enzyme, while the inhibition of DNA primase and E. coli RNA polymerase was due to competition with the ribonucleoside triphosphate substrate. The inhibition constants (Ki) of suramin were determined to be 2.6 μM, 0.35 μM, 0.54 μM and 0.70 μM for DNA primase, DNA polymerase α, RLV reverse transcriptase and E. coli RNA polymerase respectively. The observed inhibitions of these polynucleotide‐synthesizing enzymes by suramin seem to explain, at least in part, an as yet unknown mechanism of trypanocidal action of this drug.
The structure of the gene for cecropin B, an antibacterial immune protein from Hyalophora cecropia
Authors: XANTHOPOULOS Kleanthis G. , LEE Jong‐Youn , GAN Renbao , KOCKUM Kerstin , FAYE Ingrid , BOMAN Hans G.
Published: March 2005

Pupae of the moth Hyalophora cecropia respond to an injection of live bacteria by the production of a potent antibacterial activity. The broad‐spectrum property of this activity is due chiefly to two small proteins, cecropins A and B. Sequences of the proteins showed them to be homologous and to contain 31 and 35 amino acid residues respectively. The subsequent isolation of two cDNA clones for cecropin B showed that this protein is made as a prepro molecule composed of 62 amino acid residues. We have now prepared a genomic bank and studied four genomic clones for cecropin B. The coding regions were found in two neighbouring BglII fragments, one 0.79 kb and another varying in size from 3.1 kb to 4.9 kb for different clones. One transcriptional unit for preprocecropin B was sequenced and found to be 1035 bp long with a single intron, 514 bp in size. A conserved, insect specific cap site, ATCATTC, was identified by S1 mapping and primer extension experiments. Indications were found for the presence of multigene families and multicopy genes.
Sulfite oxidase from chicken liver
Authors: RITZMANN Martin , BOSSHARD Hans Rudolf
Published: March 2005

The mitochondrial enzyme sulfite oxidase catalyzes the oxidation of cytochrome c by sulfite. The reaction is inhibited when the enzyme is treated with N‐cyclohexyl‐N′‐[2‐(N‐methylmorpholino)‐ethyl]carbodiimide p‐toluenesulfonate (CMC). Inhibition follows the conversion of two carboxyl groups to N‐acylurea derivatives. The two groups are about equally reactive toward this inhibitor and blocking of either group abolishes electron transfer to cytochrome c.

The rate of inactivation is almost the same in the presence of cytochrome c and under conditions where, on average, 89% of the enzyme is bound to cytochrome c. Therefore, the functional groups are not likely to be at the cytochrome c binding site. There are two equal and non‐interacting cytochrome c binding sites per sulfite oxidase monomer. The Kd is 7.5 μM at pH 6.0 and low ionic strength.

The data are difficult to reconcile with binding of cytochrome c to a cluster of acidic residues in the area of the heme b prosthetic group, as was envisaged for the cytochrome‐bs–cytochrome c complex [Salemme, F. R. (1976) J. Mol. Biol. 102, 563–568].

An improved method for the purification of sulfite oxidase from chicken liver, using affinity chromatography on cytochrome c–Sepharose, is described.
The interaction of troponin‐I with the N‐terminal region of actin
Authors: LEVINE Barry A. , MOIR Arthur J. G. , PERRY S. Victor
Published: March 2005

The interaction between troponin‐I and actin that underlies thin‐filament regulation in striated muscle has been studied using proton magnetic resonance spectroscopy. A restricted portion of skeletal muscle troponin‐I (residues 96–116) has previously been shown to be capable of inhibiting the MgATPase activity of actomyosin in a manner enhanced by tropomyosin [Syska et al. (1976) Biochem. J. 153, 375–387]. On the basis of homologous spectral effects for signals of specific groups observed in different complexes formed using the native proteins and a variety of defined peptides, it is concluded that the segment of troponin‐I which has inhibitory activity interacts with the N‐terminal region of actin. The surface of contact of the inhibitory segment of troponin‐I with actin involves two regions of the N‐terminal of actin. These are located between residues 1–7 and 19–44. The data are discussed in the context of a structural mechanism for the inhibition of myosin ATPase activation.
Purification, properties and complete amino acid sequence of the ferredoxin from a green alga, Chlamydomonas reinhardtii
Authors: SCHMITTER Jean‐Mane , JACQUOT Jean‐Pierre , de LAMOTTE‐GUÉRY Frédéric , BEAUVALLET Christian , DUTKA Sophie , GADAL Pierre , DECOTTIGNIES Paulette
Published: March 2005

The ferredoxin was purified from the green alga, Chlamydomonas reinhardtii. The protein showed typical absorption and circular dichroism spectra of a [2Fe‐2S] ferredoxin. When compared with spinach ferredoxin, the C. reinhardtii protein was less effective in the catalysis of NADP+ photoreduction, but its activity was higher in the light activation of C. reinhardtii malate dehydrogenase (NADP). The complete amino acid sequence was determined by automated Edman degradation of the whole protein and of peptides obtained by trypsin and chymotrypsin digestions and by CNBr cleavage. The protein consists of 94 residues, with Tyr at both NH2 and COOH termini. The positions of the four cysteines binding the two iron atoms are similar to those found in other [2Fe‐2S] ferredoxins. The primary structure of C. reinhardtii ferredoxin showed a great homology (about 80%) with ferredoxins from two other green algae.
Amino acid sequence determination and three‐dimensional modelling of thioredoxin from the photosynthetic bacterium Rhodobacter sphaeroides Y
Authors: CLEMENT‐METRAL Jenny D. , HOLMGREN Arne , CAMBILLAU Christian , JÖRNVALL Hans , EKLUND Hans , THOMAS Daniel , LEDERER Florence
Published: March 2005

The complete primary structure of thioredoxin from Rhodobacter sphaeroides Y has been determined by analysis of peptides after cleavage with cyanogen bromide, chymotrypsin and trypsin. Peptides were separated by HPLC and analyzed by liquid‐phase and gas‐phase sequencer degradations. The protein consists of 105 residues (Mr= 11180); its amino acid sequence shows a clear homology to the five known thioredoxins from plant or bacterial sources, with 40–56% residue identity when the proteins are aligned at the active‐site disulfide. Not only the active‐site regions are conserved, but also residues which belong to the hydrophobic surface suggested to be important for binding of procaryote thioredoxins in redox interactions with other proteins (residues 75–76; 91–93 in Escherichia coli). A three‐dimensional model of Rb. sphaeroides thioredoxin has been derived from the E. coli crystallographic structure with computer graphics. This model indicates that the overall structures as well as the active sites are closely similar; however, the residue substitutions allow both proteins to adopt different local folding as shown in the hydrophobic core.
Characterization of the protein which binds insulin‐like growth factor in human serum
Authors: RAPP Reinhard , DEGER Arno , BLUM Werner , KOCH Rüdiger , WEBER Ulrich
Published: March 2005

The binding of the 125I‐labelled insulin‐like growth factors I and II (l25I‐IGF I and 125I‐IGF II) to the high‐molecular‐mass binding protein of human serum was characterized. With diluted human serum both growth factors showed optimal specific binding at 4°C and pH 5–6. When 0.1% Triton X‐100 was present in the incubation buffer an increase in the affinity of the IGF‐binding protein was induced, which produced an enhanced binding of IGF I and IGF II. Competition experiments with various peptide hormones revealed that the native IGF‐binding protein complex binds both the IGF I and IGF II with high specificity. Analysis of binding data according to the method of Scatchard resulted in linear plots for IGF I and IGF II respectively, indicating that in human serum only a single class of non‐interacting binding sites is present. At optimal binding conditions the dissociation constants were determined to be 0.28 × 10−9 M for IGF I binding and 0.66 × 10−9 M for IGF II.

Human serum was gel‐filtered on Sepharose CL‐6B at neutral pH and the eluate was assayed for binding activity with both IGF I and IGF II. One peak with an apparent molecular mass of 175 kDa and a Stokes radius of 4.8 nm was determined for both growth factors. Thus, our data suggest that human serum contains one class of high‐molecular‐mass binding protein with comparable binding characteristics for IGF I and IGF II.
Mechanism of glyceraldehyde‐3‐phosphate transfer from aldolase to glyceraldehyde‐3‐phosphate dehydrogenase
Authors: KVASSMAN Jan , PETTERSSON Gösta , RYDE‐PETTERSSON Ulf
Published: March 2005

The catalytic interaction of glyceraldehyde‐3‐phosphate dehydrogenase with glyceraldehydes‐3‐phosphate has been examined by transient‐state kinetic methods. The results confirm previous reports that the apparent Km for oxidative phosphorylation of glyceraldehydes‐3‐phosphate decreases at least 50‐fold when the substrate is generated in a coupled reaction system through the action of aldolase on fructose 1,6‐bisphosphate, but lend no support to the proposal that glyceraldehydes 3‐phosphate is directly transferred between the two enzymes without prior release to the reaction medium. A theoretical analysis is presented which shows that the kinetic behaviour of the coupled two‐enzyme system is compatible in all respects tested with a free‐diffusion mechanism for the transfer of glyceraldehydes 3‐phosphate from the producing enzyme to the consuming one.
Characterization of four tyrosine protein kinases from the particulate fraction of rat spleen
Authors: BRUNATI Anna Maria , PINNA Lorenzo A.
Published: March 2005

Four distinct tyrosine protein kinases active on poly(Glu4,Tyr1) and angiotensin II, and operationally termed TPK‐I, TPK‐IIA, TPK‐IIB and TPK‐III have been resolved and partially purified from rat spleen particulate fraction by combining DEAE‐Sepharose, heparin‐Sepharose, phosphocellulose and polylysine‐agarose chromatographies. Once partially purified all of them are free of Ser/Thr‐specific protein kinase activity as judged using casein, histones, protamine and the peptide Arg‐Arg‐Ala‐Ser‐Val‐Ala as substrates.

TPK‐I (apparent molecular mass 64 kDa, by gel filtration) and TPK‐IIA (54 kDa) share several properties, including substrate specificity and stimulation by heparin; the latter however is much more responsive to polylysine then the former (10‐ and 3‐fold maximum stimulation, respectively). Conversely TPK‐IIB (51 kDa) is markedly inhibited by heparin and it is also characterized by its unique substrate specificity: unlike the other three tyrosine protein kinases it by far prefers the tetrapeptide Glu‐Tyr‐Ala‐Ala over the decapeptide Asp‐Ala‐Glu‐Tyr‐Ala‐Ala‐Arg‐Arg‐Arg‐Gly and readily phosphorylates band‐3 protein of red cell membrane. The unusual preference for Mg2+ over Mn2+ as activator and the capability to phosphorylate calmodulin distinguish TPK‐III (61 kDa) from the other isoenzymes. Moreover TPK‐III is insensitive to heparin and polylysine and is inhibited by quercetin much more efficiently than the other enzymes (I50=10 μM).

Upon incubation with [γ‐32P]ATP, TPK‐I, TPK‐IIA and TPK‐III give rise to alkali‐stable radiolabeled components of 61, 55 and 52 kDa respectively, as evaluated by PAGE/SDS. In every case such a radiolabeling takes place also in the presence of a large excess of phosphorylatable substrate (angiotensin II) while it is readily reversed by isotopic dilution with 10‐fold excess unlabeled ATP, supporting the view that it represents an autophosphorylation process. No (auto)phosphorylation product(s) could be detected in TPK‐IIB even if its amount, in terms of catalytic activity, was 10‐fold higher than that of the others.
Hydrodynamic properties of colicin A
Authors: CAVARD Danièle , SAUVE Paul , HEITZ Frédéric , PATTUS Franc , MARTINEZ Carmen , DIJKMAN Ruud , LAZDUNSKI Claude
Published: March 2005

The hydrodynamic properties of colicin A have been studied. The molecular mass of colicin A was determined from sedimentation equilibrium centrifugation to be 63 ± 1.2 kDa, in agreement with that determined from the primary amino acid sequence [Morlon et al. (1983) J. Mol. Biol. 110, 271–289]. The sedimentation coefficient has been analyzed over a wide range of ionic strength (NaCl 0.06 – 0.56 M) and pH (8 – 4) and was found to remain almost constant. However, below pH 5 an oligomerization of colicin A to tetramers occurred. The frictional coefficient value indicated that the shape of the colicin A monomer was very asymmetric. Analysis of the pH dependence of circular dichroism of colicin A and of its COOH‐terminal domain indicated that a sharp transition occurred between pH 4 and 3. This transition was very much reduced for the COOH‐terminal domain in the presence of a non‐ionic detergent. The presence of a lipid‐binding site in colicin A at neutral pH was demonstrated both by hydrodynamic studies with micelles of n‐hexadecanoyl and n‐octadecanoylphosphocholine and by differential sensitivity to a proteolytic enzyme in the presence or absence of detergent micelles. About 75 molecules of lipid were bound under these conditions suggesting that colicin A was bound to lipid micelles. In contrast, at acid pH, in the presence of an excess of lipid the tetramer was dissociated into monomers complexed to 20 – 30 lipid molecules, indicating the exposure of a high‐affinity lipid‐binding site.
Low‐molecular‐mass surfactant protein type 1
Authors: CURSTEDT Tore , JOHANSSON Jan , BARROS‐SÖDERLING Jane , ROBERTSON Bengt , NILSSON Gunhild , WESTBERG Marie , JÖRNVALL Hans
Published: March 2005

The low‐molecular‐mass surfactant protein fraction, soluble in chloroform/methanol, contains at least two separate polypeptide chains. The 8‐kDa form (type I) was isolated, [14C]carboxymethylated after reduction, and submitted to structural analysis. Its highly hydrophobic nature complicated purification, proteolytic cleavages, and sequence analysis. Acid hydrolysis in 6 M HCl for 7 days was necessary for release of branched‐chain residues in full yield. Pepsin was the only enzyme found to cleave the surfactant protein and was used to complement peptide generation by chemical cleavage with CNBr. The primary structure deduced consists of 79 residues with 8 half‐cystine residues, and a total of 39% branched‐chain hydrophobic residues. However, 11 residues are charged at physiological pH, and all properties of the primary structure are not entirely outstanding in relation to those of other proteins. Hydrophobic segments, coupled with a presumably tight folding from the presence of disulfide bridges, probably explain the unusual properties and the solubility in organic solvents.
Mitochondrial aldehyde dehydrogenase from horse liver
Authors: JOHANSSON Jan , von BAHR‐LINDSTRÖM Hedvig , JECK Reinhard , WOENCKHAUS Christoph , JÖRNVALL Hans
Published: March 2005

The primary structure of the mitochondrial form of horse liver aldehyde dehydrogenase has been determined, utilizing peptide analyses and homology with other enzyme forms. The subunit exhibits N‐terminal heterogeneity in size similar to that for the corresponding human mitochondrial protein, the longest form having 500 residues. Catalase was identified as a contaminant of the preparations. All four pairs within a set of aldehyde dehydrogenases can now be compared, including the same two species variants (horse and human) for both the cytosolic and mitochondrial enzyme, revealing characteristic differences although Cys‐302 and other segments of presumed functional importance are unchanged. The cytosolic and mitochondrial enzymes are clearly different (172 exchanges in the horse pair; 160 exchanges in the human pair) and the mitochondrial forms are more conserved (28 exchanges of 500 residues) than the cytosolic ones (43 exchanges). Distributions of the residue substitutions also differ between the two enzyme types. These results suggest a comparatively distant separation of the cytosolic and mitochondrial enzymes into forms with separate functional constraints that are more strict on the mitochondrial than the cytosolic enzyme. Unexpectedly, positions with residues unique to one of the four enzymes are about twice as common in both of the horse proteins than in either of the human proteins. This difference may reflect a general pattern for human/non‐human proteins, showing that not only functional properties of the protein, but also other factors, such as generation time (longer in man than in horse), are important for enzyme divergence.
Dual mechanism of phosphatidylinositol hydrolysis by substance P in brain
Authors: CATALÁN R. Edgardo , MARTÍNEZ Ana M. , ARAGONÉS M. Dolores , MIGUEL Begoña G. , ROBLES Antonio , HERNANDEZ Félix
Published: March 2005

The treatment of cerebral cortex slices with substance P caused alterations in the phospholipid levels. A significant loss of phosphatidylinositol in a dose‐dependent manner was observed. In contrast, the levels of the major phospholipids, phosphatidylcholine and phosphatidylethanolamine, were enhanced by the peptide.

The effect of substance P on the fatty acid composition of phospholipids was also studied. The most relevant event was the decrease in the content of both stearic and arachidonic acids of phosphatidylinositol. This decrease was more evident at the lowest substance P concentration tested (65 pM).

These results are consistent with the phosphatidylinositol breakdown caused by substance P in some tissues. Furthermore, our data indicate that this breakdown is selective depending on the peptide dose. Thus, in the presence of very low doses of substance P (65 pM) a preferential degradation of 1‐acyl(predominantly stearoyl)‐2‐arachidonoylglycerophosphoinositol molecular species occurs, whereas high doses of the peptide (0.65 μM) induce a generalized hydrolysis of phosphatidylinositol without showing any preference towards molecular species rich in arachidonic acid.

Hence we describe for the first time a dual, dose‐dependent mechanism for phosphatidylinositol hydrolysis by substance P, suggesting the possibility that either phospholipase A2 or phospholipase C activation is involved.