bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human liver cytosolic epoxide hydrolases
Authors: SCHLADT Ludwig , THOMAS Helmut , HARTMANN Renate , OESCH Franz 
Published: March 2005

Human liver epoxide hydrolases were characterized by several criteria and a cytosolic cis‐stilbene oxide hydrolase (cEHcso) was purified to apparent homogeneity.Styrene oxide and five phenylmethyloxiranes were tested as substrates for human liver epoxide hydrolases. With microsomes activity was highest with trans‐2‐methylstyrene oxide, followed by styrene 7, 8‐oxide, cis‐2‐With methylstyrene oxide, cis‐1,2‐dimethylstyrene oxide, trans‐1, 2‐dimethylstyrene oxide and 2, 2‐dimethylstyrene oxide. With cytosol the same order was obtained for the first three substrates, whereas activity with 2, 2‐dimethylstyrene oxide was higher than with cis‐1,2‐dimethylstyrene oxide and no hydrolysis occurred with trans‐1, 2‐dimethylstyrene oxide. Generally, activities were lower with cytosol than with microsomes.The isoelectric point for both microsomal styrene 7, 8‐oxide and cis‐stilbene oxide hydrolyzing activity was 7.0, whereas cEHcso had an isoelectric point of 9.2 and cytosolic trans‐stilbene oxide hydrolase (cEHTSO) of 5.7.The cytosolic epoxide hydrolases could be separated by anion‐exchange chromatography and gel filtration. The latter technique revealed a higher molecular mass for cEHcso than for cEHTSO.Both cytosolic epoxide hydrolases showed higher activities at pH 7.4 than at pH 9.0, whereas the opposite was true for microsomal epoxide hydrolase. The effects of ethanol, methanol, tetrahydrofuran, acetonitrile, acetone and dimethylsulfoxide on microsomal epoxide hydrolase depended on the substrate tested, whereas both cytosolic enzymes were not at all, or only slightly, affected by these solvents.Effects of different enzyme modulators on microsomal epoxide hydrolase also depended on the substrates used. Trichloropropene oxide and styrene 7, 8‐oxide strongly inhibited cEHcso whereas cEHTSO was moderately affected by these compounds.Immunochemical investigations revealed a close relationship between cEHcso and rat liver microsomal, but not cytosolic, epoxide hydrolase. Interestingly, cEHTSO has no immunological relationship to rat microsomal, nor to rat cytosolic epoxide hydrolase. cEHTSO from human liver differed also from its counterpart in the rat in that it was only moderately affected by tetrahydrofuran, acetonitrile and trichloropropene oxide.Five steps were necessary to purify cEHcso. The enzyme has a molecular mass (49 kDa) identical to that of rat liver microsomal epoxide hydrolase.
Tetraenoic and pentaenoic mycolic acids from Mycobacterium thamnopheos
Authors: DAFFÉ Mamadou , LANÉELLE Marie‐Antoinette , VALERO GUILLEN Pedro L. 
Published: March 2005

On the basis of the analysis of mycolates, the type strain of Mycobacterium thamnopheos has been considered as a member of the genus Nocardia. In a comparative study conducted on mycobacterial species we found that M. thamnopheos synthesized two types of mycolate having the same mobilities on thin‐layer chromatography as those of mycobacteria, but different from nocardomycolates. Mass spectrometry analyzes showed that the major series of both types consisted of polyunsaturated mycolic acids, ranging from C72 to C78 with four or five double bonds. On pyrolytic mass spectrometry or gas chromatography, the least polar mycolates released mainly mono‐unsaturated C22 esters whereas the other type yielded saturated C20 and C22 esters. These results suggested that M. thamnopheos might be more related to the Aurantiaca taxon than to mycobacteria and Nocardia.The permanganate‐periodate oxidation products of esters obtained by pyrolysis of the least polar mycolates showed that they contained docosen‐4‐oic and docosen‐6‐oic acids. Both types of mycolate esters yielded the same set of long‐chain meroaldehydes on pyrolysis. These meroaldehydes were significantly distinct from those of mycobacterial mycolates in the location of the double bonds.After hydrogenation of the double bond located in the alkyl‐branched chain, the two types of mycolates had the same mobility on thin‐layer chromatography, indicating that the difference of migration was due to the additional double bond found in the least polar mycolates.Based on stereochemical data, the relative configuration of both mycolates was found to be threo, like that established for all mycolates studied so far.
Investigation of protein structure by means of 19F‐NMR
Authors: ADRIAENSENS Peter , BOX Marie Elise , MARTENS Henri I. , ONKELINX Erik , PUT Jos , GELAN Jan 
Published: March 2005

A 19F‐labeled derivative of hen egg‐white lysozyme, in which the six ɛ‐amino groups are trifluoroacetylated (LF,), was prepared by reaction of lysozyme with S‐ethyltrifluorothioacetate. The reaction mixture was fractionated by cation‐exchange chromatography at pH 7.3.A comparison of the circular dichroic spectra and the activity towards Micrococcus lysodeikticus of both LF6 and native lysozyme reveals that the labeling causes no major conformational changes of the polypeptide backbone.Assignment of the six resonances present in the 19F‐NMR spectrum of LF6 was accomplished by using a variety of techniques: specific chemical modifications, the effect of the inhibitor (GlcNAc)3, 19F‐shift/pH information and relaxation parameters.
Tyrosyl‐tRNA synthetase from baker's yeast
Authors: FREIST Wolfgang , STERNBACH Hans 
Published: March 2005

The order of substrate addition to tyrosyl‐tRNA synthetase from baker's yeast was investigated by bisubstrate kinetics, product inhibition and inhibition by dead‐end inhibitors. The kinetic patterns are consistent with a random bi‐uni uni‐bi ping‐pong mechanism.Substrate specificity with regard to ATP analogs shows that the hydroxyl groups of the ribose moiety and the amino group in position 6 of the base are essential for recognition of ATP as substrate.Specificity with regard to amino acids is characterized by discrimination factors D which are calculated from kcat and Km values obtained in aminoacylation of tRNATyr‐C‐C‐A. The lowest values are observed for Cys. Phe. Trp (D= 28000 – 40000), showing that, at the same amino acid concentrations, tyrosine is 28000 – 40000 times more often attached to tRNATyr‐C‐C‐A than the noncognate amino acids. With Gly, Ala and Ser no misacylation could be detected (D > 500000); D values of the other amino acids are in the range of 100000—500000.Lower specificity is observed in aminoacylation of the modified substrate tRNATyr‐C‐C‐A(3'NH2) (D1= 500—55000). From kinetic constants and AMP‐formation stoichiometry observed in aminoacylation of this tRNA species, as well as in acylating tRNATyr‐C‐C‐A hydrolytic proof‐reading factors could be calculated for a pretransfer (II1) and a post‐transfer (II2) proof‐reading step. The observed values of II1= 12 – 280 show that pretransfer proof‐reading is the main correction step whereas post‐transfer proof‐reading is marginal for most amino acids (II2= 1–2).Initial discrimination factors caused by differences in Gibbs free energies of binding between tyrosine and noncognate amino acids are calculated from discrimination and proof‐reading factors. Assuming a two‐step binding process, two factors (I1 and I2) are determined which can be related to hydrophobic interaction forces. The tyrosine side chain is bound by hydrophobic forces and hydrogen bonds formed by its hydroxyl group. A hypothetical model of the amino acid binding site is discussed and compared with results of X‐ray analysis of the enzyme from Bacillus stearothermophilus.
Chemical structure of the lipopolysaccharide of Haemophilus influenzae strain I‐69 Rd−/b+
Authors: HELANDER Ilkka M. , LINDNER Buko , BRADE Helmut , ALTMANN Klaus , LINDBERG Alf A. , RIETSCHEL Ernst Th. , ZÄHRINGER Ulrich 
Published: March 2005

The chemical structure of the lipopolysaccharide of a deep‐rough mutant (strain I‐69 Rd−/b+) of Haemophilus influenzae was investigated. The hydrophilic backbone of lipid A was shown to consist of a β‐(1′,6)‐linked D‐glucosamine disaccharide with phosphate groups at C‐1 of the reducing D‐glucosamine and at C‐4′ of the nonreducing one. Four molecules of (R)‐3‐hydroxytetradecanoic acid were found directly linked to the lipid A backbone, two by amide and two by ester linkage (positions 2, 2′ and 3, 3′, respectively). Laser‐desorption mass spectrometry showed that both 3‐hydroxytetradecanoic acids linked to the non‐reducing glucosamine carry tetradecanoic acid at their 3‐hydroxyl group, so that altogether six molecules of fatty acid are present in lipid A.The lipopolysaccharide was the first described to contain only one sugar unit linked to lipid A. This, sugar in accordance with a previous report [Zamze et al. (1987) Biochem. J. 245, 583–587], was shown to be a dOclA phosphate. The phosphate group was found at position 4, but the analytical procedures employed (permethylation and methanolysis followed by gas‐liquid chromatography/mass spectrometry) also revealed dOclA 5‐phosphate. Since a cyclic 4,5‐phosphate could be ruled out by 31P‐NMR, we conclude that, in this lipopolysaccharide, a mixture of dOclA 4‐ and 5‐phosphate is present. By methylation analysis of the dephosphorylated, deacylated and reduced lipopolysaccharide the attachment site of the dOclA was assigned to position C‐6′ of the nonreducing glucosamine of lipid A. The anomeric linkages present in the lipopolysaccharide were assessed by 1H‐NMR and 13C‐NMR of deacylated lipopolysaccharide. The saccharide backbone of this Haemophilus influenzae lipopolysaccharide possesses the following structure:

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Enzymatic introduction of a fluorescent sialic acid into oligosaccharide chains of glycoproteins
Authors: GROSS Hans Jürgen , BROSSMER Reinhard 
Published: March 2005

Aiming at the introduction of a fluorescent sialic acid into glycoconjugates, 5‐acetamido‐9‐(3‐fluoresceinylthioureido)‐3,5,9‐trideoxy‐2‐nonulosonic acid (9‐fluoresceinyl‐NeuAc) was synthesized which has an intact carbon chain.a) Despite the space‐filling substituent at C‐9 the fluorescent NeuAc analogue was activated to the corresponding CMP‐glycoside by CMP sialic acid synthase from bovine brain. Whereas the km value of the synthase was little affected by the modification (Km= 2.1 mM, for NeuAc Km= 1.4 mM), the V value decreased to 7.5%.b)CMP‐9‐fluoresceinyl‐NeuAc was synthesized on a preparative scale (17% overall yield), and characterized by analytical HPLC, absorption and fluorescence spectra.c) 9‐Fluoresceinyl‐NeuAc was transferred onto asialo‐α1‐acid glycoprotein by both Galβ1, 4GlcNAc α2, 6sialyltransferase and Galβ1,4(3)GlcNAc α2,3sialyltransferase (rat liver), and onto antifreeze glycoprotein by GalNAc α2,6‐sialyltransferase (porcine submaxillary glands). Using analytical HPLC, transfer was confirmed after release of the fluorescent sialic acid by Vibrio cholerae sialidase.d) Initital rate studies indicated a low Km value of Galβ‐1,4GlcNAc α2,6sialyltransferase, and GalNAc α2,6sialyltransferase (specific for O‐linked oligosaccharide chains) for CMP‐9‐fluoresceinyl‐NeuAc.
Temperature‐dependent conformational transition in the head‐rod junctional region of the myosin molecule
Authors: RĘDOWICZ Maria J. , STRZELECKA‐GOLASZEWSKA Hanna 
Published: March 2005

The effects of temperature, Mg2+, ATP, and actin on the conformation of the neck region of the myosin head were studied by limited proteolysis of heavy meromyosin (HMM) and subfragment 1 (S1) preparations obtained by papain digestion of myosin in the presence of Mg2+ (Mg‐S1) or EDTA (EDTA‐S1). The preparations were fluorescently labelled at the SH1 thiol group to enable identification of the COOH‐terminal fragments of the head portion of the heavy chain where this group is located.The results indicate that the head‐rod junctional region of the myosin heavy chain contains at least three different sites readily susceptible to trypsin at 25°C if the light chain LC2 or its LC′2 fragment are absent. The susceptibility of one of these sites dramatically decreases when the temperature is lowered to 0°C, indicating a temperature‐dependent conformational transition in the head‐rod junction. With the method used, this transition is detectable only in LC2/LC′2‐deficient preparations since all three sites are protected, although to different extents, by LC2 and its LC′2 derivative. It is, however, most probable that the effect of the light chain is confined to steric hindrance of trypsin access and that the temperature‐dependent structural transition in the head‐rod junction can occur in the presence of intact LC2 as well and may contribute to the temperature sensitivity of force generation in muscle.
A 15000‐Mr protein proteolytically derived from vitellogenin within oocyte of Perinereis cultrifera (polychaete annelid)
Authors: BAERT Jean‐Luc 
Published: March 2005

It has been shown previously in our laboratory that, in Perinereis cultrifera, the four mature vitellin subunits (Mr 98000, 22000, 20000, 16000) are proteolytically derived within the oocyte from a single extraoocytic precursor, vitellogenin, with an apparent Mr (176000) higher by 20000 than the sum of the Mr of the four end products. In this report, it is shown that a 15000‐Mr protein, designated as P15, not only accumulates in maturing oocytes but also originates from outside these cells similarly to vitellin. Moreover in vivo labelling experiments indicate that the appearance of P15 occurs after vitellogenin enters the oocyte, concurrently with the appearance of the lower‐Mr fragments characteristic of vitellin. From these data, it is concluded that P15 most likely represents a vitellogenin‐derived protein which is generated within the oocytes during the transformation of vitellogenin into vitellin. This conclusion is further supported by the additional finding that P15 immunologically cross‐reacts with vitellogenin but not with mature vitellin.P15 has been purified to homogeneity from the soluble protein fraction of submature oocytes and partially characterized. The 15000‐Mr protein exists in a monomeric form with a pI of about 7.7. Unlike vitellin, P15 does not contain significant amounts of lipid or carbohydrate and has a low absorbance at 280 nm. The amino acid composition of the purified protein is also presented.
Heat‐resistant inhibitors of protein kinase C from bovine brain
Authors: PRIBILLA Iris , KRÜGER Helmut , BUCHNER Klaus , OTTO Henning , SCHIEBLER Werner , TRIPIER Dominique , HUCHO Ferdinand 
Published: March 2005

Bovine brain cytosol is shown to contain two heat‐resistant inhibitors of protein kinase C, with the following characteristics:

One protein kinase C inhibitor can be easily purified to homogeneity. Evidence is presented that this polypeptide of Mr 19 000 is calmodulin. It inhibits protein kinase C with an EC50 of about 2.5 μM and the inhibition is Ca2+‐independent. It inhibits only intact protein kinase C. Removal of the regulatory domain of protein kinase C, by limited proteolysis with trypsin, abolishes the inhibition.
Another protein kinase C inhibitory activity has been partially purified. Its Mr 600–700, as estimated by gel chromatography). It is not digested by proteases, is hydrophilic, acid‐ and alkali‐resistant, acts Ca2+‐independently, and, in contrast to calmodulin, inhibits even the catalytic fragment of protein kinase C after removal of the regulatory domain by limited proteolysis. This inhibition is, at least partially, due to a competition with ATP. Besides protein kinase C, calcium/calmodulin‐dependent protein kinase II is inhibited to a similar extent. cAMP‐dependent protein kinase is not affected.
Involvement of the constituent chains of botulinum neurotoxins A and B in the blockade of neurotransmitter release
Authors: MAISEY E. Anne , WADSWORTH Jonathan D. F. , POULAIN Bernard , SHONE Clifford C. , MELLING Jack , GIBBS Paul , TAUC Ladislav , DOLLY J. Oliver 
Published: March 2005

The abilities of botulinum neurotoxins, types A and B (single and two‐chain forms) to inactivate an intraneuronal component required for transmitter release were quantified in a phrenic‐nerve‐diaphragm preparation, cerebrocortical synaptosomes or the buccal ganglion of Aplysia californica and compared with the mouse toxicity assay.
Homogeneous preparations of the individually renatured polypeptide chains of both toxin types showed low residual toxicity in the whole animal and had no effect on neurotransmission in all three systems, when tested singly.
Mixtures of individually renatured heavy chain, from type A or B, and either light chain proved very effective in blocking the evoked release of acetylcholine when bath‐applied to the buccal ganglion of Aplysia whilst they were relatively inactive on mammalian nerve terminals, indicating a less efficient uptake of the polypeptides in the latter.
When renatured together, the homologous, but not the heterologous, chains of each toxin type yielded toxic, disulphide‐linked two‐chain species.
A role for the heavy chain alone in acceptor recognition and membrane translocation was implicated by the blockade of acetylcholine release produced when light chain was applied to a ganglion of Aplysia previously bathed in heavy chain and washed extensively. No blockade was observed when the order of application of the two chains was reversed.
These findings are discussed in the context of the intracellular requirement for both the constituent toxin chains for toxicity, and in the apparent need for these chains to be linked via a disulphide bond for uptake in rodents but not in Aplysia.
The K+‐translocating Kdp‐ATPase from Escherichia coli
Authors: SIEBERS Annette , ALTENDORF Karlheinz 
Published: March 2005

The Kdp system from Escherichia coli is a derepressible high‐affinity K+ ‐uptake ATPase. Its membrane‐bound ATPase activity was approximately 50 μmol g−1 min−1.The Kdp‐ATPase complex was purified from everted vesicles by solubilization with the nonionic detergent Aminoxid WS 35 followed by DEAE‐Sepharose CL‐6B chromatography at pH 7.5 and pH 6.4 and gel filtration on Fractogel TSK HW‐65. The overall yield of activity was 6.5% and the purity at least 90%. The isolated KdpABC complex had a high affinity for its substrates K+ (Km app. = 10 μM) and Mg2+‐ATP (Km= 80 μM) and a narrow substrate specificity. The ATPase activity was inhibited by vanadate (Ki= 1.5 μM), fluorescein isothiocyanate (Ki= 3.5 μM), N,N′‐dicyclohexylcarbodiimide (Ki= 60 μM) and N‐ethylmaleimide (Ki= 0.1 mM).The purification protocol was likewise applicable to the isolation of a KdpA mutant ATPase which in contrast to the wild‐type enzyme exhibited an increased Km value for K+ of 6 mM and a 10‐fold lowered sensitivity for vanadate.Starting from the purified Kdp complex the single subunits were obtained by gel filtration on Bio‐Gel P‐100 in the presence of SDS.Both the native Kdp‐ATPase and the SDS‐denatured polypeptides were used to raise polyclonal antibodies. The specificity of the antisera was established by immunoblot analysis. In functional inhibition studies the anti‐KdpABC and anti‐KdpB sera impaired ATPase activity in the membrane‐bound as well as in the purified state of the enzyme. In contrast, the anti‐KdpC serum did not inhibit enzyme activity.
Monoclonal antibody recognizes a conformational epitope in a random coil protein
Authors: SAAD Bashar , CORRADIN Gianpietro , BOSSHARD Hans Rudolf 
Published: March 2005

The antigenic determinants for two monoclonal antibodies directed against horse apo‐cytochrome c, a protein of disordered structure, as judged by spectroscopic and hydrodynamic criteria, have been studied by a combination of methods: antigen competition in solution by radio immunoassay and enzyme‐linked immunoassay, and differential acetylation of free and antibody‐bound antigen. In the latter method the accessibility of lysine residues of the antigen in the antigen‐antibody complex is compared to the accessibility in the free antigen.The two antibodies against the heme‐free protein do not recognize intact native cytochrome c, but they crossreact with the heme‐containing peptides 1–38 and 1–65 of cytochrome c. The antigenic determinant recognized by monoclonal antibody SJL 2–4 is conformational and discontiguous, it is composed of residues close to the N‐terminus and around position 25. The other monoclonal antibody, Cyt‐1–59, seems to recognize a contiguous epitope close to the N‐terminus.The present results show that even a seemingly disordered protein which is conventionally classified as a random coil may feature subtle spatial regularities. The presence of ordered conformational elements in apocytochrome c may be important for the enzyme‐catalyzed covalent attachment of the heme and the import of cytochrome c into mitochondria. A discontiguous determinant for SJL 2–4 is particularly interesting because this antibody inhibits the proliferation of a T‐cell clone specific for apo‐cytochrome c [Corradin & Engers (1984) Nature (Lond.) 308, 547–548].
Purification of the yeast mitochondrial methionyl‐tRNA synthetase
Authors: SCHWOB Etienne , SANNI Ambaliou , FASIOLO Franco , MARTIN Robert P. 
Published: March 2005

Yeast‐mitochondrial methionyl‐tRNA synthetase was purified 1060‐fold from mitochondrial matrix proteins of Saccharomyces cerevisiae using a four‐step procedure based on affinity chromatography (heparin‐Ultrogel, tRNAMet‐Sepharose, Agarose‐hexyl‐AMP) to yield a single polypeptide of high specific activity (1800 U/mg). Like the cytoplasmic methionyl‐tRNA synthetase (Mr 85000), the mitochondrial isoenzyme is a monomer, but of significantly smaller polypeptide size (Mr 65000). In contrast, the corresponding enzyme of Escherichia coli is a dimer (Mr 152000) made up of identical subunits. The measured affinity constants of the purified mitochondrial enzyme for methionine and tRNAMet are similar to those of the cytoplasmic isoenzyme. However, the two yeast enzymes exhibit clearly different patterns of aminoacylation of heterologous yeast and E. coli tRNAMet. Furthermore, polyclonal antibodies raised against the two proteins did not show any cross‐reactivity by inhibition of enzymatic activity and by the highly sensitive immunoblotting technique, indicating that the two enzymes share little, if any, common antigenic determinants. Taken together, our results further support the belief that the yeast mitochondrial and cytoplasmic methionyl‐tRNA synthetases are different proteins coded for by two distinct nuclear genes.Like the yeast cytoplasmic aminoacyl‐tRNA synthetases, the mitochondrial enzymes displayed affinity for immobilized heparin. This distinguishes them from the corresponding enzymes of E. coli. Such an unexpected property of the mitochondrial enzymes suggests that they have acquired during evolution a domain for binding to negatively charged cellular components.