bluuebunny/crossref_metadata_2025 huggingface dataset:
15N‐ and 1H‐NMR investigations of the active‐site amino acids in semisynthetic RNase S′ and RNase A
Authors: KNOBLAUCH Herbert , RÜTERJANS Heinz , BLOEMHOFF Wim , KERLING Karel E. T.
Published: March 2005

Extensive 15N‐NMR investigations of active‐site amino acids were made possible by the solid‐phase synthesis of the N‐terminal pentadecapeptide of RNase A with selectively 15N‐enriched amino acids. On complexation with S‐protein a fully active RNase S′ complex was obtained. The 15N resonances of the side chains of lysine‐7 (Nɛ), glutamine‐11 (Nγ), and histidine‐12 (Nπ,τ) were studied in the free synthetic peptide, in the RNase S′ complex and in the nucleotide complexes RNase S′ with 2′CMP, 3′CMP, and 5′AMP.

The analysis of the 15N‐1H couplings, the 15N line broadenings due to proton exchange, and the chemical shift values showed that, while the imidazole ring is directly involved in the peptide‐protein interaction, the side chains of Lys‐7 and Gln‐11 do not contribute to this interaction. In the nucleotide complexes the resonances of His‐12 and Gln‐11 are shifted downfield. In the 2′CMP complex a doublet for the Nτ signal of His‐12 indicates a stable H bond between this nitrogen and the phosphate group of the nucleotide. The other nucleotide influence the resonances of the imidazole group much less, possibly due to a slightly different orientation of the phosphate group. The downfield shift of the Gln‐11 resonance indicates an interaction between the carbonyl oxygen of the amide group and the phosphate moiety of the nucleotide. The only observable effect of nucleotide complexation on the Lys‐7 signal is line broadening due to reduced proton exchange.

For comparison with the 15N‐NMR titration curves of His‐12 in RNase S′ the 1H‐NMR titration curves of RNase A were also recorded. Both shape and pK values were very similar for the 15N and the 1H titration curves. An extensive analysis of the protonation equilibria with several fitting models showed that a mutual interaction of the imidazole groups of the active‐site histidines results in flat titration curves. The Hill plots of all resonances of the imidazole rings, including the 15N resonances, show a small inflection in the pH range 5.8–6.4. Since the existence of a diimidazole system is most likely in this pH range, the inflection could be interpreted as a disturbance of the mutual electrostatic interaction of the active‐site histidines by a partial H‐bond formation between the imidazole groups.
Knowledge‐based protein modelling and design
Authors: BLUNDELL Tom , CARNEY Devon , GARDNER Stephen , HAYES Fiona , HOWLIN Brendan , HUBBARD Tim , OVERINGTON John , SINGH Diljeet Athwal , SIBANDA Bancinyane Lynn , SUTCLIFFE Michael
Published: March 2005

A systematic technique for protein modelling that is applicable to the design of drugs, peptide vaccines and novel proteins is described. Our approach is knowledge‐based, depending on the structures of homologous or analogous proteins and more generally on a relational data base of protein three‐dimensional structures. The procedure simultaneously aligns the known tertiary structures, selects fragments from the structurally conserved regions on the basis of sequence homology, aligns these with the ‘average structure’ or ‘framework’, builds on the loops selected from homologous proteins or a wider database, substitutes sidechains and energy minimises the resultant model. Applications to modelling an homologous structure, tissue plasminogen activator on the basis of another serine proteinase, and to modelling an analogous protein, HIV viral proteinase on the basis of aspartic proteinases, are described. The converse problem of ab initio design is also addressed: this involves the selection of an amino acid sequence to give a particular tertiary structure, in this case a symmetrical domain of two Greek‐key motifs.
Characterisation of two differently processed forms of human recombinant factor IX synthesised in CHO cells transformed with a polycistronic vector
Authors: BALLAND Alain , FAURE Thérèse , CARVALLO Dorothée , CORDIER Pascale , ULRICH Pascale , FOURNET Bernard , DE LA SALLE Henri , LECOCQ Jean‐Pierre
Published: March 2005

A stable transformed cell line constitutively expressing human factor IX has been established. Wild‐type Chinese hamster ovary cells (CHO cells) were transformed using a polycistronic expression vector carrying a previously isolated factor IX cDNA and a selection gene encoding the Escherichia coli xanthine‐guanine phosphoribosyl transferase. One clone, CHO 622.4, contains a high number of genomically integrated plasmids and secretes 1–3 mg factor IX l−1 day−1 into the culture medium with a biological activity ranging from 25% to 40%. The recombinant molecule was purified either by conventional chromatography or by immunoaffinity chromatography using antibodies specific to a calcium‐induced factor IX conformer. The purified recombinant protein migrates as a single band with the same mobility as that of natural factor IX on SDS/polyacrylamide gels. N‐terminal sequencing shows two differently processed forms of recombinant factor IX: whereas the majority of the zymogen is correctly processed, approximately 20% of the purified recombinant molecule contains an 18‐amino‐acid NH2‐extension corresponding to the precursor form of factor IX. Analysis of the 4‐carboxyglutamic acid content indicates a high but incomplete carboxylation (70%) of the recombinant molecule as compared to natural factor IX.

The carbohydrate composition of both the natural and recombinant molecules has been determined. Both molecules have a N‐glycan structure of similar complexity, indicating that factor IX contains all the information to direct the same glycosylation pattern in human liver cells and in an unrelated cell line such as CHO‐K1.
On the importance of the support material for bioorganic synthesis
Authors: RESLOW Mats , ADLERCREUTZ Patrick , MATTIASSON Bo
Published: March 2005

α‐Chymotrypsin was adsorbed on solid support materials and the catalytic activity of the preparations in organic solvents was studied. The activity was highly dependent on the nature of the support material and on the amount of water present in the reaction mixture. There appears to be competition for the water in the system between the enzyme, the support material and the solvent. The support materials were characterized by measuring their ability to absorb water from water‐saturated diisopropyl ether. For the quotient: (amount of water on the support)/(amount of water in the solvent) in the model system the term aquaphilicity was proposed. The activity of adsorbed chymotrypsin in diisopropyl ether decreased with increasing aquaphilicity of the support material. The same trend was observed when the activity of horse liver alcohol dehydrogenase adsorbed on different supports was measured in diisopropyl ether.
Binding characteristics of Ah receptors from rats and mice before and after separation from hepatic cytosols
Authors: FERNANDEZ Nadia , ROY Marianne , LESCA Pierre
Published: March 2005

The Ah receptor, a soluble protein implicated in the mechanism of action of the toxic halogenated aryl hydrocarbons has been examined in rodent livers. Due to the difficulty of making reliable quantitative determinations on binding parameters for hydrophobic compounds in cytosols that contain several components, Ah receptors from livers of Sprague‐Dawley rats and C57BL/6 mice have been separated, in a preparative manner, using sucrose density gradient centrifugation in a vertical rotor. The binding characteristics of Ah receptors, before and after separation, were assessed by competition of various chemicals as 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin, 2,3,7,8‐tetrachlorodibenzofuran, 3‐methylcholanthrene, benzo[a]pyrene, β‐naphthoflavone and ellipticines with [3H]3‐methylcholanthrene and 2,3,7,8‐tetrachloro[3H]dibenzo‐p‐dioxin as radioligands.

The rationale of this approach is supported by the results obtained and the major conclusions are as follows.

1. The intrinsic binding characteristics of Ah receptors were dependent on the presence or absence of other cytosolic binding components (light‐density component and 4‐S carcinogen‐binding protein).

2. In contrast with many previous unsuccessful attempts, the separation of the C57BL/6 Ah receptor allowed the unambiguous detection of a 9‐S binding peak with [3H]benzo[a]pyene as a radioligand.

3. The intrinsic binding characteristics of the separated Ah receptors of Sprague‐Dawley rats and C57BL/6 mice were similar if not identical.

4. A good correlation exists between the competitive potency (IC150) of chemicals and their ability to induce aryl hydrocarbon hydroxylase activity, except for 7‐hydroxyellipticine which binds to the Ah receptors of rat and mouse liver (IC50∼ 5–10 μM) without inducing aryl hydrocarbon hydroxylase.

5. When coadministered with various inducers, 7‐hydroxyellipticine antagonizes (from about 20% to 65%) the inducing ability of chemicals displaying similar (ellipticines) or weaker (chlorpromazine, phenothiazine) binding affinities for the Ah receptor.
Binding characteristics of 4‐S proteins from rat and mouse liver High affinity of ellipticines
Authors: ROY Marianne , FERNANDEZ Nadia , LESCA Pierre
Published: March 2005

The binding characteristics of 4‐S components (carcinogen‐binding protein) from livers of Sprague‐Dawley rats, C57BL/6 and DBA/2 mice have been examined before and after separation from other binding components presents in the cytosol. Competitive potency of 3‐methylcholanthrene, benzo[a]pyrene, β‐naphthoflavone and 20 ellipticines, a series of compounds differently substituted on the dimethyl‐pyrido‐carbazole nucleus and deprived of carcinogenic activity, has been determined with [3H]3‐methylcholanthrene and/or [3H]benzo[a]pyrene as radioligands. The inducing ability of the same compounds for aryl hydrocarbon hydroxylase and for ethoxyresorufin‐O‐deethylase has been compared to their affinity for the 4‐S protein and the Ah receptor respectively. The main results of this study are as follows.

1. The intrinsic binding characteristics of 4‐S proteins were dependent on both the nature of the radioligand used and the presence or absence of other cytosolic binding components.

2. The heterocyclic ellipticines were revealed as strong ligands for the carcinogen‐binding protein (stronger than benzo[a]pyrene for five derivatives substitiuted in the A ring of ellipticine), with IC50 values ranging from 0.047 μM (8‐hydroxyellipticine) to 5.8 μM (N2‐ethyl‐9‐hydroxyellipticinium).

3. When the affinity of ellipticines was plotted versus their inducing ability of aryl hydrocarbon hydroxylase and ethoxyresorufin‐O‐deethylase, it appears that a good correlation exists for the Ah receptor but not for the 4‐S protein.

It is concluded that these data, as well as the lack of enzymatic induction after benzo[a]pyrene treatment of DBA/2 mice, which display a high level of 4‐S protein, do not support the implication of this binding component in the positive control of cytochrome P‐450 induction.
Attachment of human placental‐type alkaline phosphatase via phosphatidylinositol to syncytiotrophoblast and tumour cell plasma membranes
Authors: WEBB Paul D. , TODD Janet
Published: March 2005

Phosphatidylinositol anchors human placental‐type alkaline phosphatase (PLAP) to both syncytiotrophoblast and tumour cell plasma membranes. PLAP activity was released from isolated human placental syncytiotrophoblast plasma membranes and the surface of tumour cells with a phospholipase C from Bacillus cereus. This was a specific event, not the result of proteolysis or membrane perturbation, but the action of a phosphatidylinositol‐specific phospholipase C in the preparation. Soluble PLAP, released with B. cereus phospholipase C and purified by immunoaffinity chromatography, ran on SDS‐PAGE as a 66‐kDa band. This corresponded to intact PLAP molecules. The protease bromelain cleaved lower‐molecular‐mass PLAP (64 kDa) from the membranes. Flow cytometry demonstrated that B. cereus phospholipase C released human tumour cell membrane PLAP in preference to other cell‐surface molecules. This was in contrast to the non‐specific proteolytic action of bromelain or Clostridium perfringens phospholipase C, which had no effect on membrane PLAP expression. Radiolabelling of tumour cells with fatty acids indicated PLAP to be labelled with both [3H]myristic and [3H]palmitic acid. This fatty‐acid –PLAP bond was sensitive to pH 10 hydroxylamine treatment indicating an O‐ester linkage.
Complete amino acid sequences of the ribosomal proteins L25, L29 and L31 from the archaebacterium Halobacterium marismortui
Authors: HATAKEYAMA Tomomitsu , KIMURA Makoto
Published: March 2005

Ribosomal proteins were extracted from 50s ribosomal subunits of the archaebacterium Halobacterium marismortui by decreasing the concentration of Mg2+ and K+, and the proteins were separated and purified by ion‐exchange column chromatography on DEAE‐cellulose. Ten proteins were purified to homogeneity and three of these proteins were subjected to sequence analysis. The complete amino acid sequences of the ribosomal proteins L25, L29 and L31 were established by analyses of the peptides obtained by enzymatic digestion with trypsin, Staphylococcus aureus protease, chymotrypsin and lysylendopeptidase. Proteins L25, L29 and L31 consist of 84, 115 and 95 amino acid residues with the molecular masses of 9472 Da, 12293 Da and 10418 Da respectively. A comparison of their sequences with those of other large‐ribosomal‐subunit proteins from other organisms revealed that protein L25 from H. marismortui is homologous to protein L23 from Escherichia coli (34.6%), Bacillus stearothermophilus (41.8%), and tobacco chloroplasts (16.3%) as well as to protein L25 from yeast (38.0%). Proteins L29 and L31 do not appear to be homologous to any other ribosomal proteins whose structures are so far known.
Improper expression of insulin receptors on fibroblasts from a leprechaun patient
Authors: MAASSEN J. Antonie , KLINKHAMER Marc P. , ODINK Roel J. H. , SIPS Hetty , VAN DER ZON Gerard C. M. , WIERINGA Tjalling , KRANS H. Michiel J. , MÖLLER Wim
Published: March 2005

Leprechaunism is an inherited human disorder characterized by severe insulin resistance. We have examined the properties of the insulin receptor in fibroblasts from a leprechaun patient. In vitro, severe insulin resistance is reflected by a low level of insulin binding to the patients fibroblasts and impaired insulin‐mediated uptake of 2‐deoxyglucose. Quantification of the receptor in detergent‐solubilized total glycoprotein indicates a normal receptor number, in agreement with the observed normal level of insulin receptor mRNA on northern blots. The insulin‐stimulated autophosphorylation of the patient's receptor shows a normal profile. The insulin receptor is present on the plasma membrane as indicated by cell‐surface iodination experiments. No abnormalities in the molecular masses of the receptor's α and β chains were observed. The results indicate that an apparently normal receptor is synthesized in sufficient amounts but functional expression of the receptor on the plasma membrane is impaired.
Comparison of the in vivo and in vitro activity of the δ‐endotoxin of Bacillus thuringiensis var. morrisoni (HD‐12) and two of its constituent proteins after cloning and expression in Escherichia coli
Authors: GRANUM Per Einar , PINNAVAIA Sandra M. , ELLAR David J.
Published: March 2005

The insecticidal crystal δ‐endotoxin of Bacillus thuringiensis var. morrisoni HD‐12 contains at least five polypeptides in the range 126–140 kDa. Immune blotting revealed that individual proteins in this complex share homology with a range of other B. thuringiensisδ‐endotoxins. In vivo the native HD‐12 crystal killed a lepidopteran larva (Pieris brassicae) and a dipteran larva (Anopheles gambiae), but not the related dipteran Aedes aegypti. In vitro the solubilized activated crystal lysed Choristoneura fumiferana cells (lepidopteran) and dipteran cells derived from Anopheles gambiae and Culex quinquefasciatus but not those from Aedes aegypti. An intragenic probe derived from a B. thuringiensis var. sotto lepidoptera‐specific δ‐endotoxin gene hybridized with one of six plasmids extracted from HD‐12. When cloned into pUC18 two HindIII fragments from this plasmid (PEG1 and pEG2) were shown to encode polypeptides cross‐reacting with HD‐12 antiserum. Escherichia coli lysates containing pEG2 were toxic in vivo to lepidoptera and diptera larvae and in vitro to a broader range of insect cell lines than the native crystal. E. coli cells containing pEG3, a subclone derived from pEG1, synthesised large amounts of a 140‐kDa protein in the cytoplasm as inclusion bodies. The cytotoxicity of the protein encoded by pEG3 was restricted to C. fumiferana and A. gambiae cell lines.
The role of MgATP hydrolysis in nitrogenase catalysis
Authors: CORDEWENER Jan , KRÜSE‐WOLTERS Margreet , WASSINK Hans , HAAKER Huub , VEEGER Cees
Published: March 2005

Kinetic studies on MgATP hydrolysis by nitrogenase of Azotobacter vinelandii were performed in the presence and in the absence of reducing equivalents. By measuring the ATPase activity of dye‐oxidized nitrogenase proteins it can be excluded that reductant‐independent ATPase activity is the result of futile cycling of electrons. The turnover rates of MoFe protein during reductant‐dependent and reductant‐independent ATPase activity, when measured with excess Fe protein, have approximately the same value, i.e. 5 s−1 at pH 7.4 and 22°C, assuming the hydrolysis of four molecules of MgATP per turnover of MoFe protein. For Fe protein on the other hand, the maximum turnover rate during reductant‐independent ATPase activity is only about 6% of that of reductant‐dependent ATPase activity. While the reductant‐dependent ATPase activity shows a sigmoidal dependence on the concentration of MgATP, the reductant‐independent ATPase activity yields hyperbolic saturation curves.

To account for these results it is proposed that the rate‐limiting step during MgATP hydrolysis by oxidized nitrogenase is the rate of regeneration of active Fe protein. In the presence of reductant, the regeneration of active Fe protein is stimulated, explaining the higher ATPase activity of nitrogenase during substrate reduction.
Free sphingoid bases in normal murine tissues
Authors: KOBAYASHI Takuro , MITSUO Kunihiko , GOTO Ikuo
Published: March 2005

Free sphingoid bases, which have been considered not to occur naturally, were detected in murine tissues by derivatization with o‐phthalaldehyde and the use of high‐performance liquid chromatography. The concentrations were 10–30 pmol/mg tissue. The lung contained the largest amounts of sphingoid bases. In the molecular species of sphingoid bases, the most abundant was C18‐sphingenine followed by C18‐sphinganine, 4‐hydroxysphinganine and C20‐sphingenine, in that order. The central nervous tissues contained relatively high amounts of C20‐sphingenine and there was a high concentration of 4‐hydroxysphinganine in the kidney. In addition, galacto‐sylsphingenine was detected simultaneously in the spinal cord and sciatic nerve. Sphingoid bases were purified from normal murine lungs using lipid‐extraction, cation‐exchange and silicic acid column chromatographies, alkaline saponification and preparative thin‐layer chromatography. In the purified sphingoid bases, erythro‐C18‐sphingenine and erythro‐C18‐sphinganine were identified using thin‐layer chromatography, high‐performance liquid chromatography and fast‐atom‐bombardment mass spectrometry. Free sphingoid bases occurring in normal tissues may be metabolic intermediates required for the synthesis or be products of degradation of the sphingolipids and function to regulate cellular metabolism.
Isoleucyl‐tRNA synthetase from baker's yeast and from Escherichia coli MRE 600
Authors: FREIST Wolfgang , STERNBACH Hans , CRAMER Friedrich
Published: March 2005

For discrimination between isoleucine and 19 other amino acids by isoleucyl‐tRNA synthetase from baker's yeast and from Escherichia coli MRE 600, discrimination factors D have been determined from kcat and Km values in amino‐acylation of cognate tRNAIle‐C‐C‐A. Factors D are also products of initial discrimination factors I′ and proof‐reading factors II′; D=I′ II′. Factors II′ were calculated from AMP formation stoichiometries and factors I′ as quotients of D and II′; I′=D/II′. II′ is considered as a product of a pre‐and post‐transfer proof‐reading factor (II′=II1II2), I′ as a product of initial discrimination factors I1 and I2 which are due to two steps of initial discrimination.

With the yeast enzyme the highest accuracy is achieved in discrimination between isoleucine and valine (D= 38000); other D values in a high range (10000–20000) are observed for Gly, Ser, Thr, Leu and Met; the lowest factors D belong to Cys, Asp, Asn and Trp (300–700); the remaining amino acids are discriminated with medium D values (1000–10000). Discrimination factors D observed for isoleucyl‐tRNA synthetase from E. coli are on average 2–3 times higher than for the yeast enzyme. Highest values were found in discrimination against Gly, Ala and Val (60000–72000), the lowest values for Cys, Arg and Trp (600–3000); the other amino acids exhibit D values between 20000 and 50000.

Initial discrimination factors can be related to hydrophobic interaction forces between the substrates and the enzyme; a hypothetical model of the amino acid binding site is discussed.
Human cellular retinol‐binding protein gene organization and chromosomal location
Authors: NILSSON Magnus H. L. , SPURR Nigel K. , LUNDVALL Joakim , RASK Lars , PETERSON Per A.
Published: March 2005

The gene encoding the human cellular retinol‐binding protein (CRBP) has been isolated from genomic libraries and its structure determined. Only one copy of the gene is present in the human genome. We have located the CRBP gene to segment 3p11–3qter on human chromosome 3 using hybridizations to mouse‐human, rat‐human and hamster‐human cell hybrids. The gene harbors four exons encoding 24, 59, 33, and 16 amino acid residues respectively. The second intervening sequence alone occupies 19 kb of the 21 kb of the CRBP gene. The nucleotide sequence of the gene has been determined with the exception of the second intron. The positions of the introns agree with those in the rat CRBPII, the rat liver fatty‐acid‐binding protein and the mouse adipose P2 protein genes encoding molecules belonging to the same protein family as CRBP. In contrast to the other sequenced members of this family the promoter of the CRBP gene resembles those found in the ‘housekeeping’ genes in that it is (G + C)‐rich, contains multiple copies of the CCGCCC sequence and lacks TATA box. A 9‐bp homology containing the core sequence of the simian virus 40 enhancer repeat was found in the 5′ upstream region. A genomic Southern blot probed with CRBP cDNA revealed hybridizing bands in restricted chicken and frog DNA.
The N‐and O‐linked carbohydrate chains of human, bovine and porcine plasminogen
Authors: MARTI Thomas , SCHALLER Johann , RICKLI Egon E. , SCHMID Karl , KAMERLING Johannis P. , GERWIG Gerrit J. , van HALBEEK Herman , VLIEGENTHART Johannes F. G.
Published: March 2005

The structures of the N‐and O‐glycans of human, bovine and porcine plasminogen were determined by 500‐MHz 1H‐NMR spectroscopy. The N‐glycans of all three species proved to be of the N‐acetyllactosamine type differing from one another with respect to the sialylation and fucosylation patterns. In the N‐glycan of human plasminogen the two antennae are sialylated with N‐acetylneuraminic acid (NeuAc), whereas in the bovine counterpart both branches carry significant amounts of N‐glycolylneuraminic acid (NeuGc). In porcine plasminogen the sialic acid is mainly NeuAc; the Manα1→6 branch, however, is only partially sialylated. In addition, the porcine N‐glycan is fucosylated to about 80% in α1→6 linkage to the GlcNAc‐1 residue. The O‐glycans of the three species possess an identical Galβ1→3GalNAc core which is α2→3 sialylated with NeuAc at Gal. The disialylated form, which is also present in all three species, has an additional NeuAc residue in α2→6 linkage to GalNAc. Mono‐and disialylated forms occur in different molar ratios in the different plasminogens: 80:20 in human, 70:30 in bovine and 50:50 in porcine. This study on the carbohydrate moiety of these three plasminogens reveals species specificity in terms of various types of microheterogeneities.
Protein stabilization via hydrophilization
Authors: MOZHAEV Vadim V. , ŠIKŠNIS Virginius A. , MELIK‐NUBAROV Nikolay S. , GALKANTAITE Nida Z. , DENIS Gervydas J. , BUTKUS Eugenius P. , ZASLAVSKY Boris Yu. , MESTECHKINA Nataliya M. , MARTINEK Karel
Published: March 2005

This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach.

The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two‐step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one.

α‐Chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxylic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of α‐chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll‐400/Dextran‐70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65 – 98°C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date.
A binding‐site‐deficient, catalytically active, core protein of endoglucanase III from the culture filtrate of Trichoderma reesei
Authors: STÅHLBERG Jerry , JOHANSSON Gunnar , PETTERSSON Göran
Published: March 2005

From the culture filtrate of Trichoderma reesei we have isolated a novel endoglucanase (38 kDa) which was shown to be identical to endoglucanase III (E III, 50 kDa), but lacking the first 61 N‐terminal amino acids. This core protein, designated E III core, is fully active against soluble substrates, such as carboxymethylcellulose, whereas both activity against and adsorption to microcrystalline cellulose (Avicel) is markedly decreased. Sedimentation velocity experiments revealed that the intact E III enzyme has much higher assymmetry than the E III core protein, suggesting that the N‐terminal region split off constitutes a protruding part of the native enzyme. These results lead to the proposal that native E III consists of two functionally separated domains: a catalytically active core and a protruding N‐terminal domain which acts in the binding to insoluble cellulose. The N‐terminal peptide missing in E III core corresponds to the heavily glycosylated common structural element found also in the N‐terminus of cellobiohydrolase II and in the C‐termini of cellobiohydrolase I and endoglucanase I. A similar bifunctional organization could thus be the rule for Trichoderma cellulases, endoglucanases as well as cellobiohydrolases.
Cysteine‐proteinase‐inhibiting function of T kininogen and of its proteolytic fragments
Authors: MOREAU Thierry , ESNARD Fréderic , GUTMAN Ninette , DEGAND Pierre , GAUTHIER Francis
Published: March 2005

Previous attempts to liberate T kinin from T kininogen [Moreau et al. (1986) Eur. J. Biochem. 159, 341–346; Gutman et al. (1988) Eur. J. Biochem. 171, 577–582] have shown that complete fragmentation of the precursor molecule into inhibitory peptides was achieved before any vasoactive peptide was released, suggesting a possible physiological significance for this phenomenon. In this study, cysteine‐proteinase‐inhibiting properties of rat T kininogen and of its proteolytic fragments issuing from trypsin and submaxillary gland endopeptidase k hydrolysis, have been investigated using rat lysosomal cathepsins B, H and L, papain and bovine calpains I and II. All three lysosomal cathepsins were inhibited by T kininogen but tighter interactions were observed with cathepsin L and papain. Though higher K1 values were obtained for cathepsins B and H, rate constants for association were found to have high and almost similar values (in the 106 M−1 s−1 range) whathever the enzyme used. Proteolytic fragments also inhibited cathepsin L and papain very strongly and even better than the entire molecule for some of them, but no significant inhibition of cathepsins B and H was observed. Bovine calpains were not inhibited by T kininogen nor by its proteolytic fragments. From the results of this kinetic analysis, which indicates that both the association and the dissociation of lysosomal cysteine proteinases with T kininogen may occur rapidly, an hypothesis has been put forward on the possible in vivo functioning of T kininogen as a proteinase inhibitor.
The error in the cryptic stereospecificity of methylmalonyl‐CoA mutase
Authors: HULL William E. , MICHENFELDER Martha , RÉTEY János
Published: March 2005

A preparation containing 80.0 ± 0.5% (2RS)‐methylmalonyl‐carba‐(dethia)‐CoA and 20.0 ± 0.5% propionylcarba‐(dethia)‐CoA was reacted in buffered deuterium oxide with catalytic amounts of coenzyme B12, methylmalonyl‐CoA mutase and methylmalonyl‐CoA epimerase. The rearrangement of the methylmalonyl‐carba‐(dethia)‐CoA to succinyl‐carba‐(dethia)‐CoA was monitored by recording 500‐MHz 1H‐NMR spectra in short time intervals. After reaching equilibrium (∼ 28 min) the products showed chemical stability for about 17 h, i.e. succinyl species did not undergo the spontaneous hydrolysis encountered with normal succinyl‐CoA. In the pre‐equilibrium stage only about 66% of the produced succinyl‐CH2CoA was the expected monodeuterated species. The remainder was 15.5% unlabelled and 18.3% 3,3‐dideuterated.

After reaching equilibrium a continuous deuterium incorporation (washing‐in) from the solvent to the products was observed and quantified. The time course of the appearance of unlabelled, mono‐, di‐ and trideuterated succinyl‐CH2CoA species was determined by assigning and integrating the isotope‐shifted 1H signals from the various species. Furthermore, mutase catalyses slow deuterium incorporation into first the methylene and then the methyl group of propionyl‐CH2CoA.

On the basis of these data it was concluded that methylmalonyl‐CoA mutase and epimerase are responsible for continuous deuterium incorporation and multiple incorporation occurs when the backward reaction (succinyl‐CH2CoA → methylmalonyl‐CH2CoA) becomes important. To account for all of the results obtained with dethia and natural substrates we propose a new mutase mechanism whereby the enzyme can retain full stereospecificity at C‐3 of succinyl while an internal 1,2‐H shift to give a C‐2 succinyl radical is responsible for partial scrambling of diastereotopic protons at C‐3. This mechanism successfully predicts the observed deuterium disproportionation in succinyl species and the order of appearance of di‐ and trideuterated products via the washing‐in process.
Studies of the solution structure of the bleomycin‐A2– zinc complex by means of two‐dimensional NMR spectroscopy and distance geometry calculations
Authors: AKKERMAN Marcel A. J. , HAASNOOT Cornelis A. G. , HILBERS Cornelis W.
Published: March 2005

Application of various two‐dimensional NMR techniques (SECSY, COSY and NOESY) enabled the complete assignment of the 1H‐NMR spectrum of the bleomycin‐A2–zinc complex in H2O and D2O at pH 6.7. The spectra were interpreted at 277 K as well as at 300 K. Identification of the resonances permitted a vicinal coupling constant analysis which revealed that the conformation around the Cα and Cβ bond of the β‐aminoalanine and β‐hydroxyhistidine residues is fixed. From this finding it was concluded that both amino functions of the β‐aminoalanine fragment and the amide and imidazole groups of the β‐hydroxyhistidine moiety are involved in zinc coordination. Also, for the mannose carbamoyl group and the pyrimidine ring active participation in zinc coordination could be established.

NOE data together with the six coordination sites proposed above were used as interpoint distance constraints in distance geometry calculations for the bleomycin‐A2–zinc complex in H2O. Sets often structures, randomly chosen within the distance constraints, were calculated (with and without the zinc ion). The calculated structures were very similar but in case that the zinc ion was omitted some flexibility was observed, within the distance constraints, in the pyrimidine‐aminoalanine region. Because of the great overall similarity between the structures, a reliable representation of the solution conformation of the bleomycin‐zinc complex was reached. Surprisingly, no regular symmetry around the zinc ion was found to be present in the generated structures.
Purification and characterization of two lysozymes from rainbow trout (Salmo gairdneri)
Authors: GRINDE Bjørn , JOLLÈS Jacqueline , JOLLÈS Pierre
Published: March 2005

Two different lysozymes, designated I and II, were purified from the kidney of rainbow trout. The enzymes had isoelectric points of approximately 9.5 and 9.65, and differed in their binding characteristics to a cation exchanger. Lysozyme II had the highest specific activity against Micrococcus luteus.

By sodium dodecyl sulphate gel electrophoresis, a molecular mass of 14.4 kDa was established for the two lysozymes.

For both type I and II enzymes, optimum pH under the present conditions was 5.5 and optimum temperature (at pH 6.2) around 45°C.

N‐terminal amino acid sequence determination indicated that the two trout lysozymes were c‐type lysozymes.

The fish lysozymes had a much higher rate of diffusion in agar than did the other lysozymes tested (possibly due to less interaction with agarose). This implies that a foreign lysozyme, such as hen egg white lysozyme, should not be used as a standard when assaying lysozyme activity with the lysoplate technique.
Active‐site‐specific zinc‐depleted and reconstituted cobalt(II) human‐liver alcohol dehydrogenase
Authors: SCHNEIDER‐BERNLÖHR Helga , FORMICKA‐KOZŁOWSKA Grażyna , BÜHLER Rolf , von WARTBURG Jean‐Pierre , ZEPPEZAUER Michael
Published: March 2005

The active‐site zinc atom of the β1β1 isozyme of class I alcohol dehydrogenase (EC 1.1.1.1) from human liver was specifically removed by the chelating agent dipicolinic acid. From β1γ1 and γ1γ1 isozyme the active‐site zinc is extracted much more slowly than from β1β1 isozyme. Only partially active‐site metal‐depleted enzyme species were obtained from these isozymes.

The active‐site‐specific reconstituted cobalt(II) derivative of the β1β1 isozyme shows spectroscopic properties comparable to those of the active‐site‐specific reconstituted cobalt(II) horse liver alcohol dehydrogenase. The coenzyme‐induced conformational change of the protein leads to a red shift of the d‐d band from 648 nm to 673 nm.

The chromophoric substrate trans‐4‐(N,N‐dimethylamino)‐cinnamaldehyde forms ternary complexes with NADH and the different isozymes, in close analogy to horse liver alcohol dehydrogenase. The differences in the active sites between β1 and γ1 subunits (threonine‐48 instead of serine‐48) or between zinc and cobalt(II) are reflected in the visible absorption spectra of the metal‐bound chromophoric substrate.
H8Zn(c)2 and Zn(c)2Co(n)2 human liver alcohol dehydrogenase
Authors: FORMICKA‐KOZŁOWSKA Grażyna , SCHNEIDER‐BERNLÖHR Helga , von WARTBURG Jean‐Pierre , ZEPPEZAUER Michael
Published: March 2005

The zinc ion in the noncatalytic site of human β1β1 and β1γ1 isozymes of class I alcohol dehydrogenases (EC 1.1.1.1) was specifically replaced by Co(II) ion. The absorption and CD spectra prove that these derivatives contain cobalt bound at the noncatalytic site to the same ligands and in the same coordination geometry as in the corresponding species obtained from the horse liver EE isozyme. These Zn(c)2Co(n)2 human liver alcohol dehydrogenases could be obtained in two ways: (a) by exchange dialysis, (b) by removal of the noncatalytic zinc and subsequent insertion of cobalt(II) ion into the empty site.

The human isozymes differ from the horse liver EE enzyme in the possibility of forming stable species lacking the noncatalytic zinc ion. This difference in chemical reactivity of the noncatalytic zinc atom may be related to amino acid changes in the human isozymes, compared to horse liver alcohol dehydrogenase.
Purification and carbohydrate structure of natural murine interferon‐β
Authors: CIVAS AHMET , FOURNET Bernard , COULOMBEL Colette , LE ROSCOUET Danièle , HONVAULT Anne , PETEK Fahrettin , MONTREUIL Jean , DOLY Janine
Published: March 2005

Mouse interferon‐β (Mu‐IFN‐β) induced in C‐243 cells with Newcastle disease virus was purified in four steps including ammonium sulfate fractionation, DEAE‐cellulose, monoclonal Mu‐IFN‐β antibody affinity and Mono‐S cation‐exchange chromatographies. Specific activity of the purified Mu‐IFN‐β ranged over 1.1–1.4 × 109 NIH units/mg protein. This preparation was submitted to pronase digestion and gel filtration on Fractogel TSK HW‐40. The permethylated and acetylated glycopeptide fraction was analyzed by chemical‐ionization (ammonia) mass spectrometry. The major glycopeptide is composed of Gal, Man, GlcNAc and NeuAc with a molar ratio of 2.0:3.6:3.4:0.5. The GLC pattern of methyl derivatives obtained by methanolysis and acetylation of fully methylated glycopeptide identified 2,3,4,6‐tetra‐O‐methylgalactose; 3,4,6‐tri‐O‐methyl‐mannose; 2,3,4‐and 2,4,6‐tri‐O‐methyl galactose; 2,4,di‐O‐methyl mannose and 3,6‐di‐O‐methylglucosamine.

These results when compared with data on N‐glycans suggest the following structure for the carbohydrate moiety of Mu‐IFN‐β:

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One‐ and two‐dimensional NMR investigations of the heme pocket in free α(CO) chains from human hemoglobin
Authors: SCHAEFFER Corinne , CRAESCU Constantin T. , MISPELTER Joël , GAREL Marie‐Claude , ROSA Jean , LHOSTE Jean‐Marc
Published: March 2005

Two‐dimensional nuclear magnetic resonance techniques were used to assign resonances corresponding to heme pocket residues of the isolated α(CO) subunits of the human adult hemoglobin (HbA). The assignment procedure was based on the partial identification of the amino acid spin system from the J‐correlated (COSY) spectrum and on the nuclear Overhauser effect connectivities (from NOESY spectra) with the heme substituents. We present here partial assignments corresponding to five amino acid residues: Leu‐86, Leu‐91, Val‐93, Leu‐101 and Leu‐136.

Starting from the known crystallographic structure of the α subunit in the hemoglobin tetramer, we applied a dipolar model to compute the ring‐current shift of the protons from fifteen amino acid residues in the heme pocket. Comparison of the predicted and observed chemical shifts suggests that there is a very close similarity between the heme pocket tertiary structure of the α(CO) subunits in crystals of HbA(CO) and of the free α(CO) chains.

The one‐dimensional NMR spectra were used to monitor the pH‐induced structural changes, the effects of chemical modification and of ligand substitution. Upon increasing the pH from 5.6 to 9.0 the structure of the heme environment appears to be invariant with the exception of some residues in the CD corner. The structure is also largely conserved when p‐chloromercuribenzoate is bound to Cys‐104. In contrast, the substitution of CO by O2 as ligand induces many large changes in the heme cavity which can be partially characterized by NMR spectroscopy.
Control of pyruvate carboxylase activity by the pyridine‐nucleotide redox state in mitochondria from rat liver
Authors: SIESS Elmar A. , BANIK Eva , NEUGEBAUER Susanne
Published: March 2005

Pyruvate carboxylation by isolated mitochondria from rat liver is inhibited by t‐butylhydroperoxide in a fully reversible manner. The rate of malate formation at 10 mM pyruvate was decreased by some 80% by 30 μM t‐butylhydroperoxide. The effective peroxide concentration was dependent on the mitochondrial hydrogen supply, being increased to about 120 μM in the presence of 50 μM palmitoylcarnitine. Regarding the mechanism(s) of the t‐butylhydroperoxide action, pyruvate transport and intramitochondrial energy or activator supply are unlikely involved, because the effect also took place with alanine as the substrate and was not accompanied by a change in the intramitochondrial levels of adenine nucleotides and acetyl‐CoA respectively. However, t‐butylhydroperoxide caused a rapid fall in the 3‐hydroxybutyrate/acetoacetate ratio and a marked increase in the oxidized glutathione content. Therefore, experiments were designed to disclose the participation of the respective redox couples in the expression of pyruvate carboxylase activity. From measurements of NADPH, NADH, oxidized and reduced glutathione contents of mitochondria incubated under a variety of conditions, evidence has been obtained indicating that the mitochondrial NADH supply represents an important factor in the regulation of pyruvate carboxylase activity. The results presented seemingly provide a new basis for the understanding of the functional relationship between β‐oxidation and pyruvate carboxylation.
Purification of the human O6‐methylguanine‐DNA methyltransferase and uracil‐DNA glycosylase, the latter to apparent homogeneity
Authors: MYRNES Bjørnar , WITTWER Christian Urs
Published: March 2005

Uracil‐DNA glycosylase, the enzyme that catalyzes the release of free uracil from single‐stranded and double‐stranded DNA, has been purified 26 600‐fold from HeLa S3 cell extracts. The enzyme preparation was essentially homogeneous as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme is a small monomeric protein of molecular mass 29 kDa. A minor uracil‐DNA glycosylase preparation was also obtained in the final chromatographic step. This preparation is homogeneous with a molecular mass of 29 kDa and may represent the mitochondrial enzyme.

This report also presents a 700‐fold purification of HeLa S3 cell O6‐methylguanine‐DNA methyltransferase. The glycosylase and methyltransferase showed very similar chromatographic properties. The report indicates that the lability of the methyltransferase upon purification may be a consequence of the total separation of the two DNA repair enzymes or of the possibility that some other stabilizing factor is involved.
Characterization and specific assay for a galactoside β‐3‐galactosyltransferase of human kidney
Authors: BAILLY Pascal , Friedrich PILLER  , CARTRON Jean‐Pierre
Published: March 2005

Two galactosyltransferases identified as UDP‐galactose: lactose (lactosylceramide) α‐4‐ and β‐3‐galactosyltransferases [Bailly P. et al. (1986) Biochem. Biophys. Res. Commun. 141, 84–91] have been characterized in human kidney microsomes. Using methyl β‐d‐galactoside as acceptor substrate, we have determined the experimental conditions (pH 5.0, 4 mM Cd2+) in which only the β‐3‐galactosyltransferase activity is detectable. The reaction product has been characterized by chemical methods and glycosidase studies. Under these experimental conditions, some of the enzyme properties have been further investigated. Apparent Km values are for UDP‐galactose, 0.170 mM; for lactose, 242 mM; and for lactosylceramide, 2.5 mM.

Acceptor specificity studies suggest that the β‐3‐galactosyltransferase is specific for terminal Galβ1‐4Glc(NAc) residues and responsible for elongation of oligosaccharide chains in glycolipids. Competition studies with lactose and N‐acetylgalactosamine as acceptor substrates indicate that the transferase described here can be distinguished from the UDP‐galactose: 2‐acetamide‐2‐deoxy‐d‐galactose β‐3‐galactosyltransferase and therefore represents a novel enzyme capable of synthesizing unusual carbohydrate structures similar to those which accumulate in certain neurological diseases.
Subunits of Panulirus japonicus hemocyanin
Authors: MAKINO Nobuo
Published: March 2005

Hexameric hemocyanin from a spiny lobster, Panulirus japonicus, comprises three major subunits (Ib, II and III) and one minor subunit (Ia), as reported in the preceding paper in this journal. It has previously been shown that the O2 equilibria of Panulirus hemocyanin can be described by a concerted model extended to three affinity states [Makino, N. (1986) Eur. J. Biochem. 154, 49–55].

In this study the equilibrium binding of O2 to the reassociated subunits (Ib, II and III) was examined at various pH in the presence or absence of Ca2+ in order to test the applicability of the three‐state model to the homogeneous hexamers. The hexameric structure of the reassembled subunits was less stable than that of the native protein under the conditions examined. The model could be fitted to the O2‐binding isotherms of the homohexamers composed of the subunits II or III, if the molecular dissociation of the protein was taken into account. It was postulated that the monomeric hemocyanin has the same ligand affinity as that of the hexamer in the intermediate‐affinity state (S).

The fitting of the model to the O2 binding of the subunit I was unsuccessful mainly because of the low cooperativity of the assembled subunits.
A functional five‐enzyme complex of chloroplasts involved in the Calvin cycle
Authors: GONTERO Brigitte , CÁRDENAS María Luz , RICARD Jacques
Published: March 2005

A complex of five different enzymes: ribose‐phosphate isomerase, phosphoribulokinase, ribulose‐bisphosphate carboxylase/oxygenase, phosphoglycerate kinase and glyceraldehyde‐phosphate dehydrogenase, has been purified from spinach chloroplasts. These enzymes catalyse five consecutive reactions of the Calvin cycle, of which the two reactions catalysed by phosphoribulokinase and ribulose‐bisphosphate carboxylase/oxygenase are unique to this cycle. The five‐enzyme complex has been purified by successive chromatographies on DEAE‐Trisacryl, Sephadex G‐200 and hydroxyapatite. The homogeneity of the complex has been tested by analytical centrifugation and electrophoresis. Depending on the technique used to estimate its molecular mass, the value obtained varies between 520 kDa and 536 kDa. In addition to the five enzymes mentioned above, the complex contains a 65‐kDa polypeptide. The quaternary structure of these enzymes in the complex appears to be different from what has been described for the individual enzymes in the ‘noncomplexed state’.

The five‐enzyme complex is functional as glyceraldehyde 3‐phosphate is formed from ribose 5‐phosphate. Preliminary experiments suggest that channelling of reaction intermediates occurs within the complex.