bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dissociation of outer‐sphere water is rate‐limiting for the binding of ligands in the active site of horse liver alcohol dehydrogenase
Authors: SARTORIUS Christian , ZEPPEZAUER Michael 
Published: March 2005

The association of imidazole and auramine O to native horse‐liver alcohol dehydrogenase [Zn(II)LADH] and active‐site specifically cobalt(II)‐substituted horse‐liver alcohol dehydrogenase [Co(II)LADH], respectively, has been investigated. In all cases [except imidazole binding to Zn(II)LADH in the presence of auramine O] the association rates approached an upper limit (kmax). The kmax values were compared for the metal ligands imidazole (monodentate), 1,10‐phenanthroline and 2,2′‐bipyridine (bidentate; see also the preceding paper), and for auramine O which does not coordinate to the catalytic metal ion. Independent of the large differences in their structure and metal‐bonding capability, all these compounds exhibit common, maximum, limiting rate constants of about 60 s−1 and 200 s−1 for Co(II)LADH and Zn(II)LADH, respectively. These results demonstrate that kmax is strongly dependent on the catalytic metal ion but not on the ligand. The absence of spectral changes in the d‐d transitions of the catalytic Co(II) ion upon auramine O binding to Co(II)LADH indicates that the rate‐limiting step is not accompanied by a major conformational change. Finally, it is concluded that reactions in the inner coordination sphere of the catalytic metal ion (i.e. the metal‐bound water molecule) are not responsible for the step characterized by kmax.We propose the rate‐limiting step to consist of the dissociation of one or several water molecules from the second coordination sphere of the catalytic metal ion in the active site of LADH in its open conformation.
Relationship between the tyrosination state of tubulin and the activities of tubulin: tyrosine ligase and tubulin carboxypeptidase in rat muscle during development
Authors: ALONSO Alejandra del C. , ARCE Carlos A. , BARRA Héctor S. 
Published: March 2005

Tubulin can be post‐translationally modified by the incorporation or the release of a tyrosine residue at the COOH‐terminus of the α subunit. The present study demonstrates that rat muscle soluble preparations contain tubulin carboxypeptidase besides tubulin:tyrosine ligase.The state of tyrosination of tubulin and the activities of both the ligase and the carboxypeptidase were examined in rat muscle during development. The proportion of tyrosinated tubulin with respect to tyrosinable tubulin (tyrosinated plus detyrosinated tubulin) decreased from 83% (new‐born rats) to 28% (adult rats) with the corresponding increase in detyrosinated tubulin. The activities of the enzymes decreased continuously and in a near parallel fashion during development. These results indicate that the changes in the tyrosination state of tubulin can not be explained merely by changes in the enzyme activities.We also compared the ability of rat muscle and brain [14C]tyrosinated tubulin to act as substrate of the carboxypeptidase. Muscle tubulin was found to be a less efficient substrate than brain tubulin.
Structural and functional characterization of mutants of recombinant single‐chain urokinase‐type plasminogen activator obtained by site‐specific mutagenesis of Lys158, Ile159 and Ile160
Authors: LIJNEN H. Roger , NELLES Luc , VAN HOEF Berthe , DEMARSIN Eddy , COLLEN Désiré 
Published: March 2005

Single‐chain urokinase‐type plasminogen activator (scu‐PA) is converted to urokinase by hydrolysis of the Lys158‐Ile159 peptide bond. Site‐directed mutagenesis of Lys158 to Gly or Glu yields plasmin‐resistant mutants with a 10–20‐fold reduced catalytic efficiency for the activation of plasminogen [Nelles et al. (1987) J. Biol. Chem. 262, 5682–5689]. In the present study, we have further evaluated the enzymatic properties of derivatives of recombinant scu‐PA (rscu‐PA), produced by site‐directed mutagenesis of Lys158, Ile159 or Ile160, in order to obtain additional information on the structure/function relations underlying the enzymatic properties of the single‐and two‐chain u‐PA moieties.[Arg158]rscu‐PA (rscu‐PA with Lys158 substituted with Arg) appeared to be indistinguishable from wild‐type rscu‐PA with respect to plasminogen‐activating potential (catalytic efficiency k2/Km=0.21 mM−1 s−1 versus 0.64 mM−1 s−1), conversion to active two‐chain urokinase by plasmin (k2/Km=0.13 μM−1 s−1 versus 0.28 μM−1 s−1), as well as its specific activity (48000 IU/mg as compared to 60000 IU/mg) and its fibrinolytic potential in a plasma medium (50% lysis in 2 h with 2.8 μg/ml versus 2.1 μg/ml). [Pro159]rscu‐PA (Ile159 substituted with Pro) and [Gly159]rscu‐PA (Ile159 converted to Gly) are virtually inactive towards plasminogen (k2/Km less than 0.004 mM−1 s−1). They are however converted to inactive two‐chain derivatives by plasmin following cleavage of the Arg156‐Phe157 peptide bond in [Pro159]rscu‐PA and of the Lys158‐Gly159 peptide bond in [Gly159]rscu‐PA. [Gly158,Lys160]rscu‐PA (with Lys158 converted to Gly and Ile160 to Lys) has a low catalytic efficiency towards plasminogen both as a single‐chain form (k2/Km=0.012 mM−1 s−1) and as the two‐chain derivative (k2/Km=0.13 mM−1 s−1) generated by cleavage of both the Arg156‐Phe157 and/or the Lys160‐Gly161 peptide bonds by plasmin.These findings suggest that the enzymatic properties of rscu‐PA are critically dependent on the amino acids in position 158 (requirement for Arg or Lys) and position 159 (requirement for Ile). Conversion of the basic amino acid in position 158 results in a 10–20‐fold reduction of the catalytic efficiency of the single‐chain molecule but yields a fully active two‐chain derivative. The presence of Ile in position 159 is not only a primary determinant for the activity of the two‐chain derivative, but also of the single‐chain precursor. Cleavage of the Arg156‐Phe157 or the Lys160‐Gly161 peptide bonds by plasmin yields inactive two‐chain derivatives.
Hormone‐sensitive adenylate cyclase of prolactin‐producing rat pituitary adenoma (GH4C1) cells: molecular organization
Authors: GORDELADZE Jan O. , SLETHOLT Kjersti , THORN Niels A. , GAUTVIK Kaare M. 
Published: March 2005

Hormonal activation and inhibition of the GH4Cl1 cell adenylate cyclase complex is delineated. In the presence of the guanyl nucleotide GTP, enzyme activity was enhanced twofold by thyroliberin, sixfold by vasoactive intestinal peptide (VIP), twofold by prostaglandin E2 and twofold by isoproterenol. The diterpene, forskolin, increased, the activity 14‐fold. In the presence of high GTP (400 μM) and NaCl (150 mM) concentrations, somatostatin inhibited (ED50= 0.5 μM) the cyclase activity by 40%.In the presence of 10 μM somatostatin, the ED50 values (5 nM) for thyroliberin‐ and VIP‐stimulated adenylate cyclase activities were shifted to 20 nM. Forskolin‐elicited activation was, however, not affected by somatostatin. Cholera‐toxin and pertussis‐toxin pretreatment of the enzyme brought about some 20‐fold and twofold activation, respectively. Inhibition by somatostatin was abolished upon pre‐exposure to pertussis toxin.Mild alkylation by N‐ethylmaleimide increased basal and hormone‐activated adenylate cyclase while somatostatin again failed to express its inhibitory potential. Further alkylation caused a gradual decline and convergence of hormone‐modulated cyclase activities towards zero. The N‐ethylmaleimide‐induced attenuation of thyroliberin‐elicited activity was paralleled by a decrease in [3H]thyroliberin binding.Trifluoperazine and an anti‐calmodulin serum reduced basal and net thyroliberin‐, VIP‐ and forskolin‐enhanced cyclase activities by some 30%, 100%, 70% and 80%, respectively. The Vmax of basal and thyroliberin‐stimulated adenylate cyclase was diminished by 65%, leaving the apparent Km values (7.2 mM and 2.6 mM, respectively) for Mg2+ unaltered. Finally, the phorbol ester 12‐O‐tetra‐decanoyl‐phorbol 13‐acetate (TPA) doubled the activity. This effect was counteracted by the protein kinase C inhibitor, polymyxin B, while thyroliberin‐enhanced adenylate cyclase remained unaffected.In summary, we have described an adenylate cyclase with stimulatory (Rs) and inhibitory (Ri) receptors coupled to a calmodulin‐sensitive holoenzyme through the Gs and Gi type of GTP‐binding proteins. The ratio of the Gs to Gi is high. It appears that the GH4C1 cell adenylate cyclase is also activated by protein kinase C by interference with Gi. Apparently, thyroliberin activates the cyclase both directly through Gs and indirectly via protein kinase C stimulation.
Spectroscopic characterization of a high‐potential monohaem cytochrome from Wolinella succinogenes, a nitrate‐respiring organism
Authors: MOURA Isabel , LIU Ming Y. , COSTA Cristina , LIU Ming C. , PAI Govind , XAVIER António V. , LeGALL Jean , PAYNE William J. , MOURA José J. G. 
Published: March 2005

When purified, a high‐potential c‐type monohaem cytochrome from the nitrate‐respiring organism, Wollinella succinogenes (VPI 10659), displayed a minimum molecular mass of 8.2 kDa and 0.9 mol iron and 0.95 mol haem groups/mol protein. Visible light spectroscopy suggested the presence of an equilibrium between‐two ligand arrangements around the haem, i.e. an absorption band at 695 nm characteristic of haem‐methionine coordination (low‐spin form) coexisting with a high‐spin form revealed by a band at 619 nm and a shoulder at 498 nm. The mid‐point redox potential measured by visible redox titration of the low‐spin form was approximately +100 mV. Binding cyanide (Ka= 5 × 105 M−1) resulted in the displacement of the methionyl axial residue, and full conversion to a low‐spin, cyanide‐bound form.Structural features were studied by 300‐MHz 1H‐NMR spectroscopy. In the oxidized state, the pH dependence of the haem methyl resonances (pH range 5–10) and the magnetic susceptibility measurements (using an NMR method) were consistent with the visible light spectroscopic data for the presence of a high‐spin/low‐spin equilibrium with a transition pKa of 7.3. The spin equilibrium was fast on the NMR time scale. The haem methyl resonances presented large downfield chemical shifts. An unusually broad methyl resonance at around 35 ppm (pH = 7.5, 25°C) was extremely temperature‐dependent [δ(323K) –δ(273K) = 7.2 ppm] and was assigned to the S‐CH3 group of the axial methionine. In the ferrous state only a low‐spin form is present. The haem meso protons, the methyl group and the methylene protons from the axial methionine were identified in the reduced form. The resonances from the aromatic residues (three tyrosines and one phenylalanine) were also assigned.Detailed monitoring of the NMR‐redox pattern of the monohaem cytochrome from the fully reduced up to the fully oxidized state revealed that the rate of the intermolecular electronic exchange process was approximately 6 × 106 M−1 s−1 at 303 K and pH = 6.31.A dihaem cytochrome also present in the crude cell extract and purified to a homogeneous state, exhibited a molecular mass of 11 kDa and contained 2.43 mol iron and 1.89 mol haem c moieties/mol cytochrome. The absorption spectrum in the visible region exhibited no band at 695 nm, suggesting that methione is not a ligand for either of the two haems. Recovery of only small amounts of this protein prevented more detailed structural analyzes.
Three‐dimensional structure of proteinase K at 0.15‐nm resolution
Authors: BETZEL Christian , PAL Gour P. , SAENGER Wolfram 
Published: March 2005

The crystal and molecular structure of proteinase K was determined by X‐ray diffraction data to 0.15‐nm resolution. The enzyme belongs to the subtilisin family with an active‐site catalytic triad Asp39–His69–Ser224 but is a representative of a subgroup with a free Cys73 close to and ‘below' the active His69. Besides this Cys72, proteinase K has two disulfide bonds, Cys34–Cys123 and Cys178–Cys249, which contribute to the stability of the tertiary structure consisting of an extended central parallel β‐sheet decorated by six α‐helices, three short antiparallel β‐sheets, 18 β‐turns and involving several internal, structurally important water molecules.Proteinase K exhibits two Ca2+‐binding sites, one very strong and the other weak, which were the sites of the heavy atoms (Pb2+, Sm3+) used to solve the crystal structure. The weak binding site is liganded to the N and C termini, Thr16 and Asp260, and is only incompletely coordinated by oxygen ligands. The strong binding site is coordinated in the form of a pentagonal bipyramid with the side chain carboxylate of Asp200 and the C = O of Pro175 as apex, and C = O of Val177 and four water molecules in the equatorial plane. Upon removal of this Ca2+, proteinase K loses activity which is interpreted in terms of a local structural deformation involving the substrate‐recognition site (Ser132–Gly136), probably associated with a cis → trans isomerization of cis Pro171.Several water molecules are located in the active site. One, W335, is positioned in the ‘oxyanion hole' and is displaced by the C=O of the scissile peptide bond of the substrate, as indicated by crystallographic studies with peptide chloromethane inhibitors. Based on these experiments, a reaction mechanism is proposed where the peptide substrate forms a three‐stranded antiparallel pleated sheet with the recognition site of proteinase K consisting of Ser132–Leu133–Gly134 on one side and Gly100–Ser101 on the other, followed by expulsion of the oxyanion hole water W335 and hydrolytic cleavage by the Asp39–His69–Serr224 triad. These latter residues display low thermal motion corresponding to well‐defined geometry and are hardly accessible to solvent molecules. whereas the recognition‐site amino acids are more flexible and partially exposed to solvent.
Differences in the binding of H1 variants to DNA
Authors: CLARK David J. , THOMAS Jean O. 
Published: March 2005

A study of the complexes formed between short linear DNA and three H1 variants, a typical somatic H1, and the extreme variants H5, from chicken erythrocytes, and spH1 from sea urchin sperm, has revealed differences between H1, H5 and spH1 that have implications for chromatin structure and folding.

All three histones bind cooperatively to DNA in 35 mM NaCl forming similar, but not identical, rod‐like complexes. With sufficiently long DNA the complexes may be circular, circles forming more easily with H5 and spH1 than with H1.
The binding of H5 and spH1 to DNA is cooperative even in 5 mM NaCl, resulting in well‐defined thin filaments that appear to contain two DNA molecules bridged by histone molecules. In contrast, H1 binds distributively over all the DNA molecules in 5 mM NaCl, but forms short stretches similar in appearance to the thin filaments formed with H5 and spH1. Rods appear to arise from the intertwining of regular thin filaments containing cooperatively bound histone molecules on raising the NaCl concentration to 35 mM.
The compositions of the rods correspond to one histone molecule for about every 47 bp (H1), 81 bp (H5) and 112 bp (spH1), suggesting average spacings of 24 bp (H1), 41 bp (H5) and 56 bp (spH1) in the component thin (double) filaments. Strikingly, these values are proportional to the linker lengths of the chromatins in which the particular H1 variant is the main or sole H1.
Involvement of a serine protease in tumour‐necrosis‐factor‐mediated cytotoxicity
Authors: SUFFYS Philip , BEYAERT Rudi , VAN ROY Frans , FIERS Walter 
Published: March 2005

We investigated the effect of various protease inhibitors on the anti‐proliferative and cytotoxic action of tumour necrosis factor (TNF) on mouse L929 fibrosarcoma cells.

The following serine‐type protease inhibitors led to inhibition of TNF action: phenylmethylsulfonyl fluoride, Nα‐p‐tosyl‐L‐lysine chloromethane, Nα‐p‐tosyl‐L‐phenylalanylchloromethane, Nα‐p‐tosyl‐L‐arginine methyl ester, L‐leucine methyl ester, DL‐phenylalanine methyl ester, N‐acetyl‐DL‐phenylalanine‐β‐naphthyl ester, p‐nitrophenyl p′‐guanidino‐benzoate and antipain. We could not detect an effect of inhibitors specific for thiol protease on TNF.
Inhibition of TNF‐mediated cytotoxicity was evident in both the presence and absence of actinomycin D or cycloheximide.
TNF itself was not found to be a protease, as it had no proteolytic activity in a sensitive colorimetric assay. [1, 3‐3H]Diisopropyl fluorophosphate, an effective irreversible inhibitor of serine proteases, did not bind to TNF. Pretreatment of TNF with Nα‐p‐tosyl‐L‐lysine chloromethane did not influence its biological activity.
The addition of protease inhibitor to the cells at various times after TNF administration led to a gradual loss of protection, suggesting that the protease acts at a rather late stage.
Protease inhibitors did not influence TNF binding, internalization or metabolization.
No increase in supernatant protease activity or in cell‐associated protease activity could be detected after treatment of L929 cells with TNF.

Our results document the involvement of protease activity, acting quite late during the cytolytic and growth‐inhibiting processes induced by TNF.
Heme may not be a positive regulator of cytochrome‐P450 gene expression
Authors: SRIVASTAVA Gopesh , BAWDEN Michael J. , HANSEN Antony J. , MAY Brian K. 
Published: March 2005

It has been proposed that transcription of cytochrome‐P450 genes is positively regulated by heme, the prosthetic group of cytochrome‐P450 proteins. We have investigated this proposal in rats treated with succinylacetone, a known specific inhibitor of the heme biosynthetic pathway. While 2‐allyl‐2‐isopropylacetamide, phenobarbitone, dexamethasone, β‐naphthoflavone and clofibrate induced specific cytochrome‐P450‐mRNA species in rat liver, the levels of these induced mRNAs were not affected by succinylacetone administration. Synthesis of the first enzyme of the heme biosynthetic pathway, 5‐aminolevulinate synthase, is known to be regulated by the end‐product heme, with heme inhibiting 5‐aminolevulinate‐synthase‐gene transcription. Hepatic 5‐aminolevulinate‐synthase mRNA was induced by drugs and the level increased further by succinylacetone. Furthermore, treatment of rats with succinylacetone alone resulted in elevated levels of 5‐aminolevulinate‐synthase mRNA but did not affect cytochrome‐P450‐mRNA levels.The results show that while lowered heme levels lead to an increase in 5‐aminolevulinate‐synthase‐mRNA synthesis there is no effect on cytochrome‐P450‐mRNA levels, implying that heme is not required for the cytochrome‐P450‐gene transcription.
Characterization and application of a thermostable primary transport system: Cytochrome‐C oxidase from Bacillus stearothermophilus
Authors: DE VRIJ Wim , HEYNE Rene I. R. , KONINGS Wil N. 
Published: March 2005

Cytochrome‐c oxidase from Bacillus stearothermophilus has been purified to homogeneity by detergent extraction followed by DEAE‐cellulose, hydroxyapatite‐ and gel‐filtration chromatography. The enzyme is a typical cytochrome‐aa3‐type oxidase which binds carbon monoxide and is sensitive to classical oxidase inhibitors like cyanide and azide. The purified enzyme is composed of three different subunits (57, 37 and 22 kDa). The subunit with intermediate molecular mass contains a covalently attached heme‐c moiety. The enzyme appeared to be extremely thermostable (inactivation temperature = 81°C). Highest turnover rates of the reconstituted enzyme were obtained with Saccharomyces cerevisiae cytochrome c or reduced forms of non‐physiological electron donors like N,N,N′,N′‐tetramethyl‐p‐phenylenediamine and phenazine methosulphate. The reconstituted enzyme can generate a proton‐motive force consisting of a high membrane potential and trans‐membrane pH gradient. The high electro‐motive force of the enzyme (Δp=–180 to –200 mV) indicates that this enzyme functions as a high‐capacity electrogenic proton pump.Liposomes containing the purified thermostable and thermoactive cytochrome‐c oxidase were fused with membranes from the fermentative bacterium Clostridium acetobutylicum. In the hybrid system a high protonmotive force can be generated upon oxidation of reduced N,N,N′,N′‐tetramethyl‐p‐phenylenediamine by the incorporated oxidase which subsequently can be used to drive secondary transport of amino acids. This demonstrates the applicability of the cytochrome‐c oxidase to study solute transport in membranes of fermentative bacteria.
The Aspergillus toxin restrictocin is a suitable cytotoxic agent for generation of immunoconjugates with monoclonal antibodies directed against human carcinoma cells
Authors: CONDE Francisco P. , ORLANDI Rosaria , CANEVARI Silvana , MEZZANZANICA Delia , RIPAMONTI Marina , MUÑOZ Saturnino M. , JORGE Pablo , COLNAGHI Maria I. 
Published: March 2005

The protein toxin restrictocin, isolated from the mould Aspergillus restrictus, inactivates protein synthesis in eukaryotic cells by blocking the ribosome elongation cycle. This protein acts as a specific nuclease that cuts off a small fragment from the 28‐S rRNA. Biochemical and biological characterization of this toxin indicated that it is a non‐glycosylated polypeptide of Mr 16836, exhibiting in cell‐free systems a protein synthesis inhibition capacity similar to that of the ricin A chain. This polypeptide seemed unable to penetrate most of the cancer cell lines tested, as measured by its low in vitro cytotoxicity. In addition in vivo studies in BALB/c mice demonstrated that restrictocin toxicity was very low and that in rabbits, after intravenous injection 15% of the toxin was still present in the blood stream 24 h later.After derivatization with N‐succinimidyl 3‐(2‐pyridyldithio)propionate and reduction by dithiothreitol, the restrictocin maintained its protein synthesis inhibitory activity, as assayed in a cell‐free system. This derivatized toxin was then coupled to monoclonal antibodies (MBr1, MLuC1, MLuC2, MOv17, MOv18, MOv19) which exhibited a restricted spectrum of reactivity against human carcinomas. The biochemical and biological characterization of the immunoconjugates indicated that (a) when restrictocin was coupled to monoclonal antibodies with an average molar ratio of about 2, the immunoconjugates maintained the binding activity of the antibody and protein synthesis inhibition activity of the toxin; (b) four immunoconjugates were tested for cytotoxicity and three of them obtained with the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited a good level of cytotoxicity for relevant target cells and low or no toxicity for the irrelevant cell lines. The MLuC2 monoclonal antibody which gave rise to a completely ineffective immunoconjugate, induced internalization of less than one tenth of the antigenic sites whereas the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited about one third of the antigenic sites internalized. From these data it is concluded that, providing an appropriate target antigen and coupling procedure are selected, restrictocin can be considered a suitable toxin for immunoconjugate generation.