bluuebunny/crossref_metadata_2025_split huggingface dataset:
13C and proton NMR studies of horse cytochrome c
Authors: SANTOS Helena , TURNER David L. 
Published: March 2005

The CHn groups in the aliphatic side chains of horse cytochrome c have been characterized according to the chemical shifts of both 13C‐NMR and 1H‐NMR signals, their temperature dependence and the number of attached protons, n. The primary assignments of resonances from the 55 side‐chain methyl and the 27 methine groups were obtained directly for the oxidised and the reduced forms. Specific assignments of the 13C resonances were obtained through shift‐correlation experiments and comparison with earlier 1H‐NMR studies, by further measurements of proton‐proton interactions, or by elimination.Comparison of the paramagnetic shifts of carbon and protons indicates a small redox‐related change of conformation in the vicinity of Trp59 and a significant expansion of the protein over 30–50°C.
The legumin precursor from white lupin seed
Authors: DURANTI Marcello , GUERRIERI Nicoletta , CERLETTI Paolo , VECCHIO Giuseppe 
Published: March 2005

The precursors of the legumin‐like storage protein from developing white lupin seeds (35 days after flowering) are trimers composed of protomers of Mr, 72 000 or 67 000. Some subunits of these oligomers contain processed precursor polypeptides, namely α polypeptides of either 52 000 or 44 000 linked through disulphide bonds to a β polypeptide of 21 000, typical of the mature legumin. The prolegumin is glycosylated.Legumin oligomers purified from the same seeds are both trimers and hexamers; some of their subunits are still made of precursor polypeptides. The hexamer contains less precursor polypeptide than the trimer. A low level or absence of precursor appears to be a condition of hexamer assembly. The heterogenous prolegumin and legumin oligomers represent intermediates in the processing of the prolegumin to mature legumin.Hydrophobic‐interaction chromatography on TSK‐phenyl‐5PW and titration with the hydro‐phobic probe 8‐anilino‐1‐naphthalenesulphonate indicate that the legumin is less hydrophobic than the prolegumin. This is attributed to structural rearrangements at processing of the propolypeptide, made evident by the behaviour in CD and by the second‐derivative ultraviolet spectra of the two proteins.The total protein extract of developing cotyledons at 40 days after flowering contains endopeptidases, similar to those existing in the resting seeds, which cause a limited cascade degradation of the prolegumin and legumin.
Complete down‐regulation of low‐density‐lipoprotein‐receptor activity in the human hepatoma cell line Hep G2 by β‐migrating very‐low‐density lipoprotein and non‐lipoprotein cholesterol
Authors: KAMPS Jan A. A. M. , van BERKEL Theo J. C. 
Published: March 2005

Regulation of low‐density‐lipoprotein‐receptor activity by low‐density lipoprotein (LDL), cholesteryl‐ester‐rich β‐migrating very‐low‐density lipoprotein (β‐VLDL) and non‐lipoprotein cholesterol was investigated in the human hepatoma cell line Hep G2.Competition studies indicate that LDL and β‐VLDL are bound to the same recognition site, tentatively the LDL receptor. The regulatory response of the LDL receptor upon prolonged incubation with LDL or β‐VLDL was, however, markedly different. 22 h preincubation of Hep G2 cells with excess LDL caused a partial down regulation to 31% of the initial level of the high‐affinity association of LDL and 26% of the high‐affinity degradation of LDL, while with β‐VLDL a complete down regulation of the LDL‐receptor activity is observed. Preincubation of Hep G2 cells with β‐VLDL for 22 h led to a fourfold increase in intracellular cholesterol esters and a twofold increase in acylcoA:cholesterol acyltransferase activity. With LDL, the amount of intracellular cholesterol esters is increased 1.6‐fold. The more effective down regulation of LDL receptors by β‐VLDL as compared to LDL can be explained by the more effective intracellular cholesterol delivery with β‐VLDL than with LDL. Preincubation of Hep G2 cells for 22 h with acetylated LDL hardly influenced the LDL‐receptor activity. Non‐lipoprotein cholesterol, however, caused a complete down regulation of LDL‐receptor activity at even lower extracellular cholesterol concentrations than with β‐VLDL. The complete down regulation of LDL receptors by non‐lipoprotein cholesterol is not accompanied by a significant increase in acyl‐coA:cholesterol acyltransferase activity, while the intracellular cholesterol ester concentration is only increased 1.6‐fold. It is suggested that the effectiveness of non‐lipoprotein cholesterol to regulate LDL receptors is caused by its efficiency to reach the sterol regulatory site. The inability of LDL to down regulate its receptor completely can thus be explained by the inability of LDL to deliver cholesterol adequately at the intracellular regulatory site of the LDL receptor.The observed complete down regulation of the LDL receptor by β‐VLDL may be responsible for the cholesterol‐rich‐diet induced, complete down regulation of LDL‐receptor‐mediated clearance of LDL in vivo.
Structural studies by proton‐NMR spectroscopy of plant horseradish peroxidase C, the wild‐type recombinant protein from Escherichia coli and two protein variants, Phe41 → Val and Arg38 → Lys
Authors: VEITCH Nigel C. , WILLIAMS Robert J. P. , BRAY Robert C. , BURKE Julian F. , SANDERS Stephen A. , THORNELEY Roger N.F. , SMITH Andrew T. 
Published: March 2005

Wild‐type recombinant horseradish peroxidase isoenzyme C and two protein variants, Phe41 → Val anfd Arg38 → Lys, have been characterised using both one‐ and two‐dimensional NMR spectroscopy. Proton NMR spectra recorded in both resting and cyanide‐ligated states of the proteins were compared with those of the corresponding plant peroxidase. The latter contains 18% carbohydrate in eight N‐linked oligosaccharide side chains whereas the recombinant proteins are expressed in non‐glycosylated form. The spectra of the plant enzyme and refolded recombinant protein are essentially identical with the exception of carbohydreate‐linked resonances in the former indicating that their solution structures are highly similar. This comparison also identifies classes of carbohydrate resonances in the plant enzymes which provides new information on the local environment and mobility of the oligosaccharide side chains.Comparison of the spectra of the Cyanide‐ligated states of the two variants and those of plant horseradish peroxidase C indicated that there were significant differences with respect to haem and haem‐linked resonances. These could not be rationalised simply on the basis of the local perturbation expected from a single‐site substitution. The two substititions made to residues on the distal side of the haem apparently influenced the degree of imidazolate character of the proximal His170 imidazole ring thus perturbing the magnetic environment of the haem group. Inspection of the spectra of the Phe41 → Val varient also showed that the resonances of a phenylalanine residue in the haem pocket had been incorrectly made for the Phe41 → Val varient were also a basis to investigate the structure of the complex formed with the aromatic donor molecule, benzhydroxamic acid.
Detailed structural analysis of asparagine‐linked oligosaccharides of the nicotinic acetylcholine receptor from Torpedo californica
Authors: SHOJI Hiroki , TAKAHASHI Noriko , NOMOTO Hiroshi , ISHIKAWA Masami , SHIMADA Ichio , ARATA Yoji , HAYASHI Kyozo 
Published: March 2005

The structures of the major oligosaccharide moieties of the nicotinic acetylcholine receptor (AcChoR) protein from Torpedo californica have been reported [Nomoto, H., Takahashi, N., Nagaki, Y., Endo, S., Arata, Y. and Hayashi, K. (1986) Eur. J. Biochem. 157, 233–242] to be high‐mannose types. Here we report detialed analysis of the structure of the remaining oligosaccharides in this receptor.The sialylated oligosaccharides released by glycopeptidase (almond) digestion were separated according to the number of sialic acid residues using high‐performance anion‐exchange chromatography with pulsed amperometric detection. After removal of sialic acid from each fraction, the resulting neutral oligosaccharides were separately pyridylaminated and were analyzed by a combination of sequential exoglycosidase digestion and HPLC, then identified on a two‐dimensional sugar map. The structures of two desialylated pyridylamino‐oligosaccharides were further analyzed by high‐resolution proton NMR.Each oligosaccharide was composed of species containing varying numbers of sialic acids. The desialylated complex‐type oligosaccharides of AcChoR consisted of ten, eight and one different biantennary, triantennary tetraantennary oligosaccharide, respectively. The biantennary oligosaccharides were divided into two groups; oligosaccharides with fucose at the proximal N‐acetylglucosamine (six varities) and oligosaccharides without fucose (four varieties). Each group consisted of species differing in the number of terminal galactose residues. The major component of the biantennary oligosaccharides had two galactose residues at the non‐reducing termini. The terminal α‐galactose residue(s) linked to C3 of β‐galactose were found in the fucose‐containing biantennary oligosaccharides (two varieties). The triantennary oligosaccharides were also divided into two groups; oligosaccharides with (four varieties) and without (four varieties) besecting N‐acetylglucosamine. These groups were composed of species differing in the number of terminal galactose residues. The major component of the triantennary oligosaccharides was fully galactosylated with three galactose residues. An unusual group, Galβ1‐3GlcNAc, was present in low levels in the triantennary oligosaccharides. In contrast, the tetraantennary oligosaccharide was composed of only species, which is fully galactosylated with four galactose residues.
Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A
Authors: PEEK Keith , DANIEL Roy M. , MONK Colin , PARKER Lynne , COOLBEAR Tim 
Published: March 2005

Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25–10.5) which can be exploited in purification by using cation‐exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half‐cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0–10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0–10.0 and maximum activity, in a 10‐min assay, was observed at 90°C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70°C and the half‐lives at 80°C and 90°C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60°C and the half‐life at 70°C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents.Rt41A proteinase cleaved a number of synthetic amino acid p‐nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p‐nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1′ amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1′ position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1′ sites.
A method for the generation of monoclonal antibodies against rare cell‐surface molecules
Authors: KAYYEM Jon F. , ROMAN Janet M. , VON BOXBERG Ysander , SCHWARZ Uli , DREYER William J. 
Published: March 2005

Monoclonal antibodies provide a powerful tool for the identification and analysis of novel cell‐surface molecules. We present here a method for antigen preparation and an immunization protocol that facilitates generation of mAb reactive with cell‐surface molecules of low abundance and/or low antigenicity. The procedure involves isolation and extensive fractionation of cell‐surface and detergent‐soluble extracellular‐matrix molecules prior to immunization. Cell‐surface proteins on intact tissue are biotin‐labeled using a reagent that does not penetrate cells. Avidin affinity chromatography is then used to purify these biotinylated molecules. Size‐exclusion HPLC is used to separate these surface molecules on the basis of apparent molecular mass. Finally, immunization with antigen coupled to keyhole‐limpet hemocyanin is combined with long‐term booster immunizations to generate a hyperimmune response resulting in high‐affinity IgG.A test application of this approach was aimed at the generation of mAb against cell‐surface molecules of approximately 135 kDa in the developing chicken retinotectal system. Immunochemical analyses using antibodies produced by this approach which showed restricted patterns of tissue staining reveal that mAb were generated against all previously identified immunoglobulin superfamily molecules of this size in this system. Furthermore, we produced many additional antibodies that labeled retinotectal tissue in novel staining patterns. In the two cases analyzed in detail, these new patterns reflect the distributions of previously uncharacterized members of the immunoglobulin superfamily. The success of this initial study suggests that this method may represent a broadly applicable approach towards the preparation of extensive libraries of antibodies against cell‐surface molecules expressed on cells from numerous sources.
Modulation of cell‐surface transferrin receptor by the imino sugar N‐butyldeoxynojirimycin
Authors: PLATT Frances M. , KARLSSON Gunilla B. , JACOB Gary S. 
Published: March 2005

The imino sugar, N‐butyldeoxynojirimycin, is an inhibitor of the glycoprotein‐processing enzyme glucosidase I and exhibits anti‐(human immunodeficiency virus) activity in vitro. We have investigated the effect(s) of this compound on cell‐surface glycoproteins by flow cytometry. We observed selective modulation of the transferrin receptor in response to treatment with 0.5 mM N‐butyldeoxynojirimycin resulting in reduced cell‐surface transferrin‐receptor expression. The receptor modulation was dose dependent, resulted in reduced 59Fe uptake by treated cells and was fully reversible within 24 h of culture in the absence of the compound.Pulse/chase analysis in conjunction with endoglycosidase‐H digestion demonstrated that transferrin‐receptor glycosylation was altered following N‐butyldeoxynojirimycin treatment, which is compatible with glucosidase inhibition. In addition, modulation of transferrin receptor in response to N‐butyldeoxynojirimycin was not confined to a single cell line, but was also observed with certain human lymphoid and myeloid cell lines. Mechanism(s) of action of the imino sugar resulting in reduced cell‐surface transferrin‐receptor expression are discussed.
Intermediates of peroxisomal β‐oxidation of [U‐14C]hexadecanedionoate
Authors: POURFARZAM Morteza , BARTLETT Kim 
Published: March 2005

1. The oxidation of [U‐14C]hexadecanedionoyl‐mono‐CoA was stimulated by CoA, by carnitine in the absence of CoA and by the presence of an NAD+‐regenerating system.2. Substrate inhibition was observed with respect to [U‐14C]hexadecanedionoyl‐mono‐CoA at concentrations greater than 35 μM.3. Acetyl‐CoA and the dicarboxyl‐CoA esters of chain length C6–16 were detected by HPLC under standard incubation conditions.4. In the absence of the NAD+‐regenerating system, 2‐enoyl‐CoA and 3‐hydroxyacyl‐CoA esters were detected.5. In general, the peroxisomal β‐oxidation of dicarboxylates is very similar to that of monocarboxylates [Bartlett, K., Hovik, R., Eaton, S., Watmough, N.J. & Osmundsen, H. (1990) Biochem. J. 270, 175–180] except that chain shortening does not proceed beyond C6.6. We conclude that the peroxisomal β‐oxidation of dicarboxylates is regulated by the redox state of the peroxisomal matrix and CoA availability.
Point mutation in cytochrome b of yeast ubihydroquinone:cytochrome‐c oxidoreductase causing myxothiazol resistance and facilitated dissociation of the iron‐sulfur subunit
Authors: GEIER Birgitta M. , SCHÄGGER Hermann , BRANDT Ulrich , COLSON Anne‐Marie , VON JAGOW Gebhard 
Published: March 2005

Cytochrome‐c reductase was isolated from Saccharomyces cerevisiae GM50‐3C. A tenth subunit was detected with molecular mass 8.5 kDa on SDS/PAGE.Two yeast mutants selected for resistance to myxothiazol, an inhibitor of the Q0 center (Q, ubiquinone) of cytochrome‐c reductase, were analysed. The single amino acid substitution in the cytochrome‐b subunit, N256Y in the mutant Myx‐119 and G137R in the mutant Myx‐118, caused a general resistance to all methoxyacrylate inhibitors to about fivefold higher concentrations.The kinetic measurements with the substrate analogue nonylbenzohydroquinone revealed a decrease in the Km by fivefold and of the maximal turnover number by fourfold in the N256Y mutant. The Km of the G137R mutant was not affected and the Vmax was 50% higher.Cytochrome‐c reductase was isolated from mutants to allow determination of the Kd values of methoxyacrylate‐stilbene and myxothiazol by means of fluorescence‐quench and red‐shift titration. Changes in the structure of the multisubunit complex due to a single amino acid exchange became obvious during the purification procedure. SDS/PAGE of the purified enzyme revealed that the substitution N256Y in cytochrome b led to a loss of the iron‐sulfur protein and the fifth small subunit with no change in the pattern of the remaining eight subunits. The subunit pattern of the G137R mutant was identical to the wild type.This is the first report of a single amino acid exchange in the catalytic subunit of cytochrome b, greatly affecting the iron‐sulfur protein, the second important catalytic subunit of the Q0 center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron‐sulfur subunit.
Degradation of gangliosides by the lysosomal sialidase requires an activator protein
Authors: FINGERHUT Ralph , VAN DER HORST Gijsbertus T. J. , VERHEIJEN Frans W. , CONZELMANN Ernst 
Published: March 2005

Lysosomal sialidase, which was formerly believed to degrade only water‐soluble substrates but not glycolipids, cleaves ganglioside substrates II3NeuNAc‐LacCer, IV3NeuNAc, II3NeuNAc‐GgOse4Cer, IV3 NeuNAc, II3(NeuNAc)2‐GgOse4Cer when these are dispersed either with an appropriate detergent (taurodeoxycholate) or with the sulfatide activator protein, a physiologic lipid solubilizer required for the lysosomal hydrolysis of other glycolipids by water‐soluble hydrolases. In the presence of the activator protein, time and protein dependence were linear within wide limits, while the detergent rapidly inactivated the enzyme.The disialo group of the b‐series gangliosides was only poorly attacked by the enzyme when the lipids were dispersed with the activator protein, whereas in the presence of the detergent, they were hydrolyzed as fast as terminal sialic acid residues.With the appropriate assay method, significant ganglioside sialidase activity could be demonstrated in the secondary lysosome fraction of normal skin fibroblasts but not of sialidosis fibroblasts.Our results support the notion that there is only one lysosomal sialidase, which degrades both the water‐soluble and the membrane‐bound sialyl glycoconjugates.
Purification and characterization of vanillyl‐alcohol oxidase from Penicillium simplicissimum
Authors: DE JONG Ed , VAN BERKEL Willem J. H. , VAN DER ZWAN Rick P. , DE BONT Jan A. M. 
Published: March 2005

Vanillyl‐alcohol oxidase was purified 32‐fold from Penicillium simplicissimum, grown on veratryl alcohol as its sole source of carbon and energy. SDS/PAGE of the purified enzyme reveals a single fluorescent band of 65 kDa. Gel filtration and sedimentation‐velocity experiments indicate that the purified enzyme exists in solution as an octamer, containing 1 molecule flavin/subunit. The covalently bound prosthetic group of the enzyme was identified as 8α‐(N3‐histidyl)‐FAD from pH‐dependent fluorescence quenching (pKa= 4.85) and no decrease in fluorescence upon reduction with sodium borohydride.The enzyme shows a narrow substrate specificity, only vanillyl alcohol and 4‐hydroxybenzyl alcohol are substrates for the enzyme. Cinnamyl alcohol is a strong competitive inhibitor of vanillylalcohol oxidation.The visible absorption spectrum of the oxidized enzyme shows maxima at 354 nm and 439 nm, and shoulders at 370, 417 and 461 nm. Under anaerobic conditions, the enzyme is easily reduced by vanillyl alcohol to the two‐electron reduced form. Upon mixing with air, rapid reoxidation of the flavin occurs. Both with dithionite reduction and photoreduction in the presence of EDTA and 5‐deazaflavin the red semiquinone flavin radical is transiently stabilized. Opposite to most flavoprotein oxidases, vanillyl‐alcohol oxidase does not form a flavin N5‐sulfite adduct. Photoreduction of the enzyme in the presence of the competitive inhibitor cinnamyl alcohol gives rise to a complete, irreversible bleaching of the flavin spectrum.
Induction of the three peroxisomal β‐oxidation enzymes is synergistically regulated by dexamethasone and fatty acids, and counteracted by insulin in Morris 7800C1 hepatoma cells in culture
Authors: SØRENSEN Hilde Nebb , GAUTIK Kaare M. , BREMER Jon , SPYDEVOLD Øystein 
Published: March 2005

This work describes the molecular mechanism of hormonal modulation of fatty‐acid peroxisomal β oxidation in liver. Morris 7800C1 hepatoma cells and isolated hepatocytes were cultured in the presence of myristic acid (1mM) and tetradecylthioacetic acid, a 3‐thia fatty acid (50 μM), separately or in combination with dexamethasone (0.25 μM) or insulin (0.4 μM).Myristic acid stimulated acyl‐CoA oxidase and a synergistic action was observed with dexamethasone. Parallel changes were recognized in enzyme protein and mRNA levels as quantified from immunoblots and Northern analyses. Myristic acid and tetradecylthioacetic acid had similar effects on this enzyme, while insulin inhibited the basal activity and blocked all inductions by the fatty acids and dexamethasone.Parallel mRNA and immunoblot analyses of the subsequent enzymes in the peroxisomal β‐oxidation pathway, enoyl‐CoA hydratase/3‐hydroxyacyl‐CoA dehydrogenase/Δ3,Δ2‐enoyl‐CoA isomerase and 3‐oxoacyl‐CoA thiolase, showed an even stronger induction by tetradecylthioacetic acid and dexamethasone, while the counteraction by insulin was maintained in both 7800C1 hepatoma cells and hepatocytes. In hepatoma cells, the thiolase always showed the most pronounced induction (about 40‐fold) after 14 days, with parallel changes in protein and mRNA levels.The results suggest that the changes in peroxisomal β‐oxidation enzymes in 7800C1 hepatoma cells are due to a major effect on steady‐state mRNA levels giving rise to corresponding alterations in enzyme protein. These results may be explained by regulation at the level of transcription of corresponding genes, but mRNA stability changes and/or translational effects may also be of importance.