bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proton NMR studies of bovine serum albumin
Authors: SADLER Peter J. , TUCKER Alan 
Published: March 2005

A variety of one‐ and two‐dimensional 1H‐NMR methods have been applied to the study of defatted 66.5‐kDa bovine serum albumin in solution.

The majority of the protons gave rise to broad unresolved resonances and spectral enhancement methods for one‐dimensional spectra were investigated in detail. A combination of exponential and sine‐bell functions was particularly effective.
The presence of contaminating glycoproteins in some commercial samples of bovine serum albumin was readily detectable from their N‐acetyl resonances at about 2.1 ppm.
The release of bound Cys (from mixed disulphide at Cys34) was observed after addition of dithiothreitol.
Through the use of two‐dimensional shift‐correlated spectroscopy, assignments of some 80 spin systems to amino acid type were made.
The pKa of the N‐terminal Asp was measured as 7.8 (0.1 M phosphate buffer. 310 K).
1H NMR spectra of bovine, human, porcine and rat serum albumins have been compared. Using sequence comparisons, specific assignments have been made for the N‐terminal residues of bovine (Asp‐Thr‐His), human (Asp‐Ala‐His), porcine (Asp‐Thr‐Tyr) and rat (Glu‐Ala‐His) albumins, and for Thr189, Tyr155 and His59/377 of bovine albumin.
These NMR data suggest that certain local regions of bovine serum albumin are highly mobile yet structured in solution, and demonstrate that the application of both one‐ and and two‐dimensional NMR methods will allow more detailed investigations of structural transitions in serum albumins induced by, for example, pH, drug and metal binding.
Biochemical properties of recombinant single‐chain urokinase‐type plasminogen activator mutants with deletion of Asn2 through Phe157 and/or substitution of Cys279 with Ala
Authors: LIJNEN Henri R. , LI Xian‐Kui , NELLES Luc , HU Mei‐Hao , COLLEN Désiré 
Published: March 2005

The contribution of the NH2‐terminal polypeptide chain and of the Cys148–Cys279 interchain disulphide bond to the enzyme activity of urokinase‐type plasminogen activator (u‐PA) was studied using site‐specific mutagenesis. Recombinant single‐chain u‐PA (rscu‐PA) variants were produced by transfecting Chinese hamster ovary cells with cDNA encoding des(Asn2–Phe157)rscu‐PA (rscu‐PA with deletion of Asn2–Phe157), [Ala279]rscu‐PA (rscu‐PA with Cys279→Ala mutation) or des(Asn2–Phe157)[Ala279]rscu‐PA [des(Asn2–Phe157)rscu‐PA with Cys279→Ala mutation].Des(Asn2–Phe157)rscu‐PA, [Ala279]rscu‐PA and des(Asn2–Phe157)[Ala279]rscu‐PA, purified from conditioned cell culture medium, were obtained as nearly homogeneous single‐chain molecules with Mr approximately 30000, 54000 and 30000, and specific fibrinolytic activities on fibrin plates of (mean ± SD; n= 3) 860 → 150 IU/mg, 43.0 → 2.5 IU/μg and 240 → 20 IU/mg, respectively, compared to 69.0 → 4.3 IU/μg for wild‐type rscu‐PA obtained in the same expression system. The plasminogen activating potential in a buffer milieu of [Ala279]rscu‐PA was somewhat lower than that of rscu‐PA, but that of both deletion mutants was virtually abolished. In a human plasma milieu in vitro, consisting of a radiolabelled human plasma clot submerged in plasma, 50% clot lysis in 2 h required 6.5 μg/ml [Ala279]rscu‐PA or 3.4 μg/ml rscu‐PA, whereas with both deletion mutants no significant clot lysis was observed with up to 16 μg/ml. Treatment of [Ala279]rscu‐PA or rscu‐PA with plasmin resulted in quantitative conversion to two‐chain molecules and was associated with an increase in specific amidolytic activity from about 600 IU/mg to 62.5 IU/μg for [Ala279]rscu‐PA as compared to an increase from about 0.3 IU/μg to 75.0 IU/μg for rscu‐PA. In contrast, no significant amidolytic activity could be generated by treatment of des(Asn2–Phe157)rscu‐PA or des(Asn2–Phe157)[Ala279]rscu‐PA with plasmin.The u‐PA B‐chain, isolated from plasmin‐treated [Ala279]rscu‐PA, had enzymic properties which were comparable to those of rtcu‐PA, with respect to specific fibrinolytic activity, amidolytic activity, kinetics of plasminogen activation and clot‐lysis activity in a human plasma milieu in vitro. Following bolus injection into hamsters, the plasma clearances were comparable (0.7–1.1 ml/min) for wild‐type rscu‐PA and for the three truncated rscu‐PA mutants.These results indicate that (a) deletion of residues Asn2–Phe157 results in abolition of the enzyme activity of rscu‐PA, (b) the interchain disulphide bond in u‐PA is not required for the enzymic activity of scu‐PA, (c) all the determinants required for the enzymic activity of two‐chain u‐PA are contained within the B‐chain, and (d) the region comprising residues Asn2–Phe157 of u‐PA is not required for the rapid in vivo clearance.
Human hemi‐myeloperoxidase
Authors: ZUURBIER Karin W. M. , van den BERG Joop D. , Van GELDER Bob F. , MUIJSERS Anton O. 
Published: March 2005

Human neutrophilic myeloperoxidase (MPO) is involved in the defence mechanism of the body against micro‐organisms. The enzyme catalyses the generation of the strong oxidant hypochlorous acid (HOCI) from hydrogen peroxide and chloride ions. In normal neutrophils MPO is present in the dimeric form (140 kDa). The disulphide‐linked protomers each consist of a heavy subunit and a light one. Reductive alkylation converts the dimeric enzyme into two protomers, ‘hemi‐myeloperoxidase’.We studied the initial activities of human dimeric MPO and hemi‐MPO at the physiological pH of 7.2 and found no significant differences in chlorinating activity. These results indicate that, at least at neutral pH, the protomers of MPO function independently. The absorption spectra of MPO compounds II and III, both inactive forms concerning HOCI generation, and the rate constants of their formation were the same for dimeric MPO and hemi‐MPO, but hemi‐MPO required a slightly larger excess of H2O2 for complete conversion. Hemi‐MPO was less stable at a high temperature (80°C) as compared to the dimeric enzyme. Furthermore, the resistance of the chlorinating activity of hemi‐MPO against its oxidative product hypochlorous acid was somewhat lower (IC50= 32 μM HOCI) compared to dimeric MPO (IC50= 50 μM HOCI). The higher stability of dimeric MPO in the presence of its oxidative product compared to that of monomeric MPO might be the reason for the occurrence of MPO as a dimer.
Characterization of the chylomicron‐remnant‐recognition sites on parenchymal and Kupffer cells of rat liver Selective inhibition of parenchymal cell recognition by lactoferrin
Authors: van DIJK Marc C. M. , ZIERE Gijsbertus J. , van BERKEL Theo J. C. 
Published: March 2005

Upon injection of chylomicrons into rats, chylomicron remnants are predominantly taken up by parchenymal cells, with a limited contribution (8.6% of the injected dose) by Kupffer cells. In vitro storage of partially processed chylomicron remnants for only 24 h leads, after in vivo injection, to an avid recognition by Kupffer cells (uptake up to 80% of the total liver‐associated radioactivity). Lactoferrin greatly reduces the liver uptake of chylomicron remnants, which appears to be the consequence of a specific inhibition of the uptake by parenchymal cells. Kupffer‐cell uptake is not influenced by lactoferrin. In vitro studies with isolated parenchymal and Kupffer cells show that both contain a specific recognition site for chylomicron remnants. The Kupffer‐cell recognition site differs in several ways from the recognition site on parenchymal cells as follows. (a) The maximum level of binding is 3.7‐fold higher/mg cell protein than with parenchymal cells. (b) Binding of chylomicron remnants is partially dependent on the presence of calcium, while binding to parenchymal cells is not. (c) β‐Migrating very‐low‐density lipoprotein is a less effective competitor for chylomicron‐remnant binding to Kupffer cells compared to parenchymal cells. (d) Lactoferrin leaves Kupffer‐cell binding uninfluenced, while it greatly reduces binding of chylomicron remnants to parenchymal cells. The properties of chylomicron‐remnant recognition by parenchymal cells are consistent with apolipoprotein E being the determinant for recognition.It can be concluded that the chylomicron‐remnant recognition site on Kupffer cells possesses properties which are distinct from the recognition site on parenchymal cells. It might be suggested that partially processed chylomicron remnants are specifically sensitive to a modification, which induces an avid interaction with the Kupffer cells. The recognition site for (modified) chylomicron remnants on Kupffer cells might function as a protection system against the occurrence of these potential atherogenic chylomicron‐remnant particles in the blood.
The carbohydrate chains of the β subunit of human chorionic gonadotropin produced by the choriocarcinoma cell line BeWo
Authors: HÄRRD Karl , DAMM Jan B. L. , SPRUIJT Mark P. N. , BERGWERFF Aldert A. , KAMERLING Johannis P. , VAN DEDEM Gijs W. K. , VLIEGENTHART Johannes F. G. 
Published: March 2005

The N‐linked carbohydrate chains of the β subunit of human chorionic gonadotropin (hCG‐β) isolated from the culture fluid of the choriocarcinoma cell line BeWo were released enzymatically by peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F. Subsequently, the O‐linked oligosaccharides were split off from the N‐deglycosylated protein by mild alkaline borohydride treatment. The carbohydrate chains were purified in their intact sialylated forms by FPLC anion‐exchange chromatography on Mono Q, HPLC on Lichrosorb‐NH2, and high‐pH anion‐exchange chromatography on CarboPac PA1. 1H‐NMR spectroscopic analysis of the major fractions demonstrates the occurrence of the following sialylated diantennary and triantennary N‐linked oligosaccharides. Residues not written in bold letters are variably present.

image

The incidence of triantennary carbohydrate chains is much higher than in normal urinary hCG‐β (26% vs 2%). The same holds for the α1–6‐fucosylation of the asparagine‐bound GlcNAc (95% vs 42%). The presence of a bisecting GlcNAc and the occurrence of α2–6‐linked Neu5Ac in the most abundant N‐glycans, are new features for hCG‐β.The major O‐linked carbohydrate chains identified are the tetrasaccharide Neu5Acα2–3Galβ1‐3(Neu5Acα2–6)GalNAc‐ol and the hexasaccharide Neu5Acα2–3Galβ1–4GlcNAcβ1–6(Neu5‐Acα2–3Galβ1–3)GalNAc‐ol, both also found in normal urinary hCG. In addition, two novel O‐glycans were characterized:

image
Novel oligosaccharide constituents of the cellulase complex of Bacteroides cellulosolvens
Authors: GERWIG Gerrit J. , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G. , MORAG Ely , LAMED Raphael , BAYER Edward A. 
Published: March 2005

The multiple cellulase‐containing protein complex, isolated from the cellulolytic bacterium Bacteroides cellulosolvens, contains oligosaccharides which are O‐linked mainly to a 230‐kDa subunit. The oligosaccharide chains were liberated by alkaline‐borohydride treatment and fractionated as oligosaccharide alditols via gel‐permeation chromatography and HPLC. The fractions were investigated by one‐ and two‐dimensional (correlation, homonuclear Hartmann‐Hahn, rotating‐frame nuclear Overhauser enhancement) 500‐MHz 1H‐NMR spectroscopy in combination with monosaccharide and methylation analyses and with fast‐atom‐bombardment mass spectrometry. The following carbohydrate structures could be established:

image

image

image

image

image

and

image
.The results indicate an interesting similarity between the oligosaccharide moieties of the cellulase complex of B. cellulosolvens and of Clostridium thermocellum [Gerwig, G. J., Kamerling, J. P., Vliegenthart, J. F. G., Morag (Morgenstern), E., Lamed, R. & Bayer, E. A. (1991) Eur. J. Biochem. 196, 115–122], having 3, 5 and 6 as common elements.The furanose form of a terminal α‐d‐galactose residue demonstrated an inhibitory effect on the interaction of Griffonia simplicifolia I isolectin B4 with the cellulosome‐like entity of B. cellulosolvens.
13C‐NMR study of autotrophic CO2 fixation pathways in the sulfur‐reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus
Authors: STRAUSS Gerhard , EISENREICH Wolfgang , BACHER Adelbert , FUCHS Georg 
Published: March 2005

The unresolved autotrophic CO2 fixation pathways in the sulfur‐reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus have been investigated. Autotrophically growing cultures were labelled with [1,4‐13C1]succinate, and the 13C pattern in cell constituents was determined by 1H‐ and 13C‐NMR spectroscopy of purified amino acids and other cell constituents.In both organisms succinate contributed to <10% of cell carbon, the major part of carbon originated from CO2. All cell constituents became 13C‐labelled, but different patterns were observed in the two organisms. This proves that two different cyclic CO2 fixation pathways are operating in autotrophic carbon assimilation in both of which succinate is an intermediate.The 13C‐labelling pattern in T. neutrophilus is consistent with the operation of a reductive citric acid cycle and rules out any other known autotrophic CO2 fixation pathway. Surprisingly, the proffered [1,4‐13C1]succinate was partially converted to double‐labelled [3,4‐13C2]glutamate, but not to double‐labelled aspartate. These findings suggest that the conversion of citrate to 2‐oxoglutarate is readily reversible under the growth conditions used, and a reversible citrate cleavage reaction is proposed.The 13C‐labelling pattern in C. aurantiacus disagrees with any of the established CO2 fixation pathways; it therefore demands a novel autotrophic CO2 fixation cycle in which 3‐hydroxypropionate and succinate are likely intermediates. The bacterium excreted substantial amounts of 3‐hydroxypropionate (5 mM) and succinate (0.5 mM) at the end of autotrophic growth. Autotrophically grown Chloroflexus cells contained acetyl‐CoA carboxylase and propionyl‐CoA carboxylase activity. These enzymes are proposed to be the main CO2‐fixing enzymes resulting in malonyl‐CoA and methylmalonyl‐CoA formation; from these carboxylation products 3‐hydroxypropionate and succinate, respectively, can be formed.
An investigation of large inhibitors binding to phosphoglycerate kinase and their effect on anion activation
Authors: JOAO Heidi C. , WILLIAMS Robert J. P. , LITTLECHILD Jennifer A. , NAGASUMA R. , WATSON Herman C. 
Published: March 2005

This study extends, to a series of larger anions, our earlier investigation of the interaction of the trypanocidal drug suramin and other small negatively charged molecules with yeast phosphoglycerate kinase. 1H‐NMR structural studies of phosphoglycerate kinase in the presence of varying concentrations of these large molecules (designed to mimic, at one end, the anionic charge distribution in the substrate 3‐phosphoglycerate, while possibly being able to interact across the cleft of the enzyme) including inositol 1,4,5‐triphosphate, 4‐amino‐6‐trichloroethenyl‐l,3‐benzenedisulphonamide, gallic acid and sulphasalazine are described. The anion activation and/or inhibition of the enzyme by these molecules are also reported.Evidence that binding to the general anion site in the ‘basic patch’ region of the protein may be responsible for either the activating or inhibiting effects, while binding at the hydrophobic (catalytic) site leads to inhibition only is presented. A reaction scheme which explains these observations is given.
Evolutionary optimization of the catalytic efficiency of enzymes
Authors: PETTERSSON Gösta 
Published: March 2005

The rate equation for a generalized Michaelian type of enzymic reaction mechanism has been analyzed in order to establish how the mechanism should be kinetically designed in order to optimize the catalytic efficiency of the enzyme for a given average magnitude of true and apparent first‐order rate constants in the mechanism at given concentrations of enzyme, substrate and product.
As long as on‐velocity constants for substrate and product binding to the enzyme have not reached the limiting value for a diffusion‐controlled association process, the optimal state of enzyme operation will be characterized by forward (true and apparent) first‐order rate constants of equal magnitude and reverse rate constants of equal magnitude. The drop in free energy driving the catalysed reaction will occur to an equal extent for each reaction step in the mechanism. All internal equilibrium constants will be of equal magnitude and reflect only the closeness of the catalysed reaction to equilibrium conditions.
When magnitudes of on‐velocity constants for substrate and product binding have reached their upper limits, the optimal kinetic design of the reaction mechanism becomes more complex and has to be established by numerical methods. Numerical solutions, calculated for triosephosphate isomerase, indicate that this particular enzyme may or may not be considered to exhibit close to maximal efficiency, depending on what value is assigned to the upper limit for a ligand association rate constant.
Arguments are presented to show that no useful information on the evolutionary optimization of the catalytic efficiency of enzymes can be obtained by previously taken approaches that are based on the application of linear free‐energy relationships for rate and equilibrium constants in the reaction mechanism.
The closely related homomeric and heterodimeric mannose‐binding lectins from garlic are encoded by one‐domain and two‐domain lectin genes, respectively
Authors: VAN DAMME Els J. M. , SMEETS Koen , TORREKENS Sophie , VAN LEUVEN Fred , GOLDSTEIN Irwin J. , PEUMANS Willy J. 
Published: March 2005

Lectin cDNA clones for two different lectins from garlic (Allium sativum L.) bulbs, ASAI and ASAII (ASA, Allium sativum agglutinin), were isolated and characterized. the first lectin, ASAI, is a heterodimer composed of two different subunits of 11.5 kDa and 12.5 kDa. It is translated from an mRNA of 1400 nucleotides encoding a polypeptide of 306 amino acids with two very similar domains. N‐terminal sequenceing of the two polypeptides of the mature lectin confirmed that both subunits are derived from the same precursor and that each corresponds to one of the two domains in the sequence.In contrast to ASAI, the second garlic lectin, ASAII, is a homodimer of two identical 12‐kDa subunits. It is translated from an mRNA of approximately 800 nucleotides encoding a polypeptide of 154 amino acids. Interestingly, the coding region of the ASII cDNA clones is almost identical to that of the second domain of the ASAI cDNA clones.
NAD‐linked, factor‐dependent formaldehyde dehydrogenase or trimeric, zinc‐containing, long‐chain alcohol dehydrogenase from Amycolatopsis methanolica
Authors: van OPHEM Peter W. , Van BEEUMEN Jozef , DUINE Johannis A. 
Published: March 2005

NAD‐linked, factor‐dependent formaldehyde dehydrogenase (FD‐FA1DH) of the Gram‐positive methylotrophic bacterium, Amycolatopsis methanolica, was purified to homogeneity. It is a trimeric enzyme with identical subunits (molecular mass 40 kDa) containing 6 atoms Zn/enzyme molecule. The factor is a heat‐stable, low‐molecular‐mass compound, which showed retention on an Aminex HPX‐87H column. Inactivation of the factor occurred during manipulation, but activity could be restored by incubation with dithiothreitol. The identity of the factor is still unknown. It could not be replaced by thiol compounds or cofactors known to be involved in metabolism of C1 compounds. Of the aldehydes tested, only formaldehyde was a substrate. However, the enzyme showed also activity with higher aliphatic alcohols and the presence of the factor was not required for this reaction. Methanol was not a substrate, but high concentrations of it could replace the factor in the conversion of formaldehyde. Presumably, a hemiacetal of formaldehyde is the genuine substrate, which, in the case of methanol, acts as a factor leading to methylformate as the product. This view is supported by the fact that formate could only be detected in the reaction mixture after acidification. Inhibition studies revealed that the enzyme contains a reactive thiol group, being protected by the binding of NAD against attack by heavy‐metal ions and aldehydes. Studies on the effect of the order of addition of coenzyme and substrate suggested that optimal catalysis required NAD as the first binding component. Substrate specificity and the induction pattern clearly indicate a role of the enzyme in formaldehyde oxidation. However, since FD‐FA1DH was also found in A. methanolica grown on n‐butanol, but not on ethanol, it may have a role in the oxidation of higher aliphatic alcohols as well. FD‐FA1DH and the factor from A. methanolica are very similar to a combination already described for Rhodococcus erythropolis [Eggeling, L. & Sahm, H. (1985) Eur. J. Biochem. 150, 129–134].NAD‐linked, glutathione‐dependent formaldehyde dehydrogenase (GD‐FA1DH) resembles FD‐FA1DH in many respects. Since glutathione has so far not been detected in Gram‐positive bacteria, FD‐FA1DH could be the counterpart of this enzyme in Gram‐positive bacteria. Alignment of the N‐terminal sequence (31 residues) of FD‐FA1DH with that of GD‐FA1DH from rat liver indeed showed similarity (30% identical positions). However, comparable similarity was found with class I alcohol dehydrogenase from this organism and with cytosolic alcohol dehydrogenase from Saccharomyces cerevisiae, isozyme 1. Therefore, it is concluded that the trimeric FD‐FA1DH is related to the dimeric/tetrameric, zinc‐containing, long‐chain alcohol dehydrogenases.
Complete cDNA sequence of chicken vigilin, a novel protein with amplified and evolutionary conserved domains
Authors: SCHMIDT Cornelia , HENKEL Barbara , PÖSCHL Ernst , ZORBAS Haralabos , PURSCHKE Werner G. , GLOE Torsten R. , MÜLLER Peter K. 
Published: March 2005

The complete cDNA (4375 bp), coding for a new protein called vigilin, was isolated from chicken chondrocytes. The cDNA shows an open reading frame of 1270 amino acids which are organized in 14 tandemly repeated homologous domains. Each domain consists of two subdomains, one with a conserved sequence motif of 35 amino acids (subdomain A) and another one with a presumptive α‐helical structure of 21–33 amino acids (subdomain B). 149 amino acids at the N‐terminus and 71 amino acids at the C‐terminus of vigilin do not show the characteristic domain structure. No sequence characteristic of a signal peptide has been found, which argues for an intracellular localisation of vigilin. Vigilin is highly expressed in freshly isolated chicken chondrocytes but little in chondrocytes after prolonged time in culture. Vigilin mRNA exists in two size species, 4.4 kb and 6.5 kb in length due to the usage of different polyadenylation sites.Comparison of the vigilin sequence with data bases showed a remarkable similarity to protein HX from Saccharomyces cerevisiae [Delahodde, A., Becam, A. M., Perea, J. & Jacq, C. (1986) Nucleic Acids Res. 14, 9213–9214]. The yeast protein consists of eight homologous domains with 11 conserved amino acid residues within a set of 35 amino acids. The N‐terminal and C‐terminal regions of vigilin and protein HX do not reveal any sequence similarity. These results, together with the demonstration of the characteristic vigilin sequence motif in a human cDNA clone, suggest that the repeats represent evolutionary conserved autonomous domains within a family of proteins found in yeast, chicken and man.