bluuebunny/crossref_metadata_2025_split huggingface dataset:
Non‐equilibrium Method for the Radioimmunoassay of Clozapine in the Presence of Metabolites
Authors: ROSENTHALER Joachim , NIMMERFALL Fritz , SIGRIST Robert , MUNZER Hans 
Published: March 2005

Cross‐reactions with metabolites are an ever‐recurring problem encountered in the use of radioimmunoassay techniques to determine active compounds in biological material. Metabolites may interfere with the assay of the parent drug to a variable extent. Taking 8‐chloro‐11‐(4‐methyl‐1‐piperazinyl)‐5H‐dibenzo[b,e][1,4]d iazepine (clozapine) as an example, it was shown that the extent to which the antiserum produced interacts with the parent drug and the metabolites can be estimated by determining the equilibrium constants and the kinetics.In the present case, therefore, it was advantageous to carry out the radioimmunoassay in disequilibrium, i.e, in order to differentiate the metabolites from the parent drug. the sample was incubated with the antiserum for 10 min, after which the labelled antigen was added and the reaction mixture again incubated for a brief, exactly timed interval. It was shown that cross‐reactions did not occur in mixtures of clozapine and its N‐demethyl and N‐oxide metabolites in the proportions 1: 1: 2 over a range of concentration of 1.5–48 ng clozapine per 100 μl human plasma. The equilibrium constants measured with the clozapine goat antiserum were as follows: clozapine 1.2 × 108, M−1, the N‐demethyl metabolite 4.6 × 107 M−1 and the N‐oxide metabolite 3.7 × 107 M−1 (pH 7.5 and 20 °C).
Specificity of Sialyltransferase: Structure of α1‐Acid Glycoprotein Sialylated in vitro
Authors: VAN DEN EIJNDEN Dirk H. , STOFFYN Pierre , STOFFYN Anne , SCHIPHORST Wietske E. C. M. 
Published: March 2005

The terminal galactosyl units of desialylated α1‐acid glycoprotein were selectively labeled with tritium by a galactose oxidase/NaB3H4 procedure. The 3H‐labeled glycoprotein was effective as an acceptor in sialyltransferase reactions catalyzed by rat liver microsomes in vitro with unlabeled CMP‐N‐acetyl‐neuramininic acid as sialic acid donor. Permethylation/hydrolysis of glycopeptides derived from the resialylated 3H‐labeled glycoprotein yielded radioactive 2,3,4‐trimethylgalactose indicating that rat liver microsomes are capable of transferring sialic acid to position C‐6 of the terminal galactosyl units of desialylated α1‐acid glycoprotein. No indication was obtained for transfer of sialic acid to other positions.This result is discussed in view of the multiplicity of positions of attachment of sialic acid to galactosyl residues in native α1‐acid glycoprotein.
Aspects on Microenvironmental Compartmentation
Authors: KOCH‐SCHMIDT Ann‐Christin , MATTIASSON Bo , MOSBACH Klaus 
Published: March 2005

Soluble bifunctional enzyme aggregates have been prepared by cross‐linking the sequential enzymes malate dehydrogenase (EC 1.1.1.37) and citrate synthase (EC 4.1.3.7) using glutaraldehyde. The kinetic behaviour of this two‐enzyme system in its aggregated and non‐aggregated form was studied both in free solution and immobilized on Sepharose beads. This study was undertaken in order to distinguish between the following two factors which may account for the increased efficiency found in general in co‐immobilized consecutive two‐enzyme systems:

closer proximity between the participating enzymes and
establishment of a favourable microenvironment (such as higher local intermediate concentration caused by increased diffusional hindrance in the gel phase).

It was found that in spite of a reduction of the distance between the two enzymes in the aggregated form by an estimated factor of 103, no kinetic advantage (shorter lag phase or higher steady‐state rate) could be detected compared to the corresponding system with the two enzymes not linked to each other. However, both systems immobilized to Sepharose reached the steady‐state rate of citrate formation almost immediately, in contrast to the corresponding free systems which exhibited pronounced lag phase.These results indicate that, at least in the above systems and under the conditions given, diffusional hindrance in the gel phase of the intermediate oxaloacetate, which is present in rate‐limiting concentrations, is the dominant cause of the observed higher efficiency in immobilized systems.
Purification and Properties of Glycogen Synthase I from Human Leukocytes
Authors: SØLLING Henrik , ESMANN Viggo 
Published: March 2005

Glycogen synthase I was purified from human polymorphonuclear leukocytes by a procedure involving affinity chromatography of the glycogen‐enzyme complex, digestion of endogenous glycogen by amylase, starch chromatography and gel filtration. The purified enzyme had a specific activity of 7–11 U/mg protein, or 4–5 U when expressed per mg of residual glycogen. Further purification to 21 U/mg protein could be achieved. The enzyme was inactive in the absence of added glycogen. A subunit molecular weight of 85000 was determined by polyacrylamide electrophoresis in sodium dodecylsulfate. The molecular weight of the native enzyme was estimated to be 390000 (13.2 S) by sucrose gradient centrifugation and 410000 by gel filtration indicating that the native enzyme is a tetramer. The gel filtration behavior was not affected by enzyme concentration, temperature, or the presence of ligands. The energy of activation was estimated to 13500 cal/mol (56.5 kJ/mol), corresponding to a Q10of 2.2. In the presence of glucose 6‐phosphate or Na2SO4, the enzyme showed a broad pH optimum between pH 6.8–9.2. In the absence of these ligands and in particularly in the presence of Mg2+, the enzyme is sensitive to small changes of pH in the interval pH 7.4–8.4.During purification, synthase I requires protection by 0.6 mM dithiothreitol, while high concentrations of mercaptoethanol or dithiothreitol inactivates the enzyme, particularly during freezing. During 24‐h incubations, synthase I undergoes a spontaneous, temperature‐dependent inactivation which is not due to proteolysis, but presumably is caused by irreversible conformational changes. These can be prevented by high concentrations of glucose 6‐phosphate, Na2SO4, inorganic phosphate, UDP and glycogen. Mg2+and traces of ethanol inactivates the enzyme. The lyophilized enzyme is stable for years.
Structure of Seven Oligosaccharides Excreted in the Urine of a Patient with Sandhoff's Disease (GM2 Gangliosidosis‐Variant O)
Authors: STRECKER Gérard , HERLANT‐PEERS Marie‐Claire , FOURNET Bernard , MONTREUIL Jean , DORLAND Lambertus , HAVERKAMP Johan , VLIEGENTHART Johannes F. G. , FARRIAUX Jean‐Pierre 
Published: March 2005

The urine of a patient with Sandhoff's disease (GM2 gangliosidosis‐variant O) contains 10–12 N‐acetylglucosamine‐rich oligosaccharides in high amounts. The structures of seven of these have been determined: β‐GlcNAc‐(1→2)‐α‐Man‐(1→3)‐β‐Man‐(1→4)‐GlcNAc; β‐GlcNAc‐(1→4)‐α‐Man‐(1→3)‐β‐Man‐(1→4)‐GlcNAc; β‐GlcNAc‐(1→2)‐α‐Man‐(1→6)‐β‐Man‐(1→4)‐GlcNAc; β‐GlcNAc‐(1→4)‐α‐Man‐(1→6)‐β‐Man‐(1→4)‐GlcNAc; β‐GlcNAc‐(1→2)‐α‐Man‐(1→3)‐[β‐GlcNAc‐(1→2)‐α‐Man‐(1→6)]β‐Man‐(1→4)‐GlcNAc; β‐GlcNAc‐(1→2)‐α‐Man‐(1→3)[β‐GlcNAc‐(1→2)‐α‐Man‐(1→6)] β‐GlcNAc‐(1→4)]β‐Man‐(1→4)‐GlcNAc; β‐GlcNAc‐(1→2)‐α‐Man(1)‐(1→3) [β‐GlcNAc‐(1→2)‐α‐Man(2)‐(1→6)]β‐Man‐(1→4)‐GlcNAc, with additional β‐GlcNAc‐(1→4) on mannose (1) or (2).An unusual oligosaccharide, with a tri‐branched β‐mannose, has been characterized as the major component excreted in urine.
A 31P‐Nuclear‐Magnetic‐Resonance Study of the Phosphate Groups in Lipopolysaccharide and Lipid A from Salmonella
Authors: MÜHLRADT Peter F. , WRAY Victor , LEHMANN Volker 
Published: March 2005

Untreated and partially deacylated lipopolysaccharides from various P− and P+ strains of Salmonella were studied with 31P nuclear magnetic resonance spectroscopy and by conventional analytical methods.The spectral signals were assigned to various phosphate groups in the lipid A moiety and in the oligosaccharide part. A signal at + 2.3 ppm could be assigned to a phosphodiester linkage formed between 4‐amino‐4‐deoxy‐l‐arabinose linked via the glycosidic hydroxyl group to the 4′‐phosphate group of the glucosamine disaccharide in the lipid A moiety. A strong pyrophosphate signal at + 11 ppm in P− strains was identified as a pyrophosphoryl ethanolamine group at the glycosidic end of this glucosamine disaccharide unit. No evidence was found for phosphodiester or pyrophosphodiester bonds crosslinking lipopolysaccharide ‘subunits’. A revised version of the lipid A structure of Salmonella is presented.By a combination of 31P nuclear magnetic resonance spectroscopy data and conventional analytical methods the extent to which the lipopolysaccharides are substituted by various phosphate groups on the lipid A and the oligosaccharide moiety could be estimated. It was thus shown that substantial heterogeneity, leading to several molecular species of lipopolysaccharides is caused by addition or omission of certain groups. Since changes in substitution were found to be dependent on the growth conditions, it is thought possible that the overall negative surface charge of Salmonella can be modified by addition or omission of neutralising amino groups from ethanolamine and/or 4‐amino‐4‐deoxy‐l‐arabinose, and can thus be adapted to the environment.
Nuclear‐Magnetic‐Resonance Study of the Histidine Residues of S‐Peptide and S‐Protein and Kinetics of 1H‐2H Exchange of Ribonuclease A
Authors: BRADBURY J. Howard , CROMPTON Malcolm W. , TEH Joo Seng 
Published: March 2005

1H NMR spectroscopy at 100 MHz was used to determine the first‐order rate constants for the 1H‐2H exchange of the H‐2 histidine resonances of RNase‐A in 2H2O at 35°C and pH meter readings of 7, 9, 10 and 10.5. Prolonged exposure in 2H2O at 35°C and pH meter reading 11 caused irreversible denaturation of RNase‐A. The rate constants at pH 7 and 9 agreed reasonably well with those obtained in 1H‐3H exchange experiments by Ohe, J., Matsuo, H., Sakiyama, F. and Narita, K. [J. Biochem. (Tokyo) 75, 1197–1200 1974)]. The rate data obtained by various authors is summarised and the reasons for the poor agreement between the data is discussed.The first‐order rate constant for the exchange of His‐48 increases rapidly from near zero at pH 9 (due to its inaccessibility to solvent) with increase of pH to 10.5. The corresponding values for His‐119 show a decrease and those for His‐12 a small increase over the same pH range. These changes are attributed to a conformational change in the hinge region of RNase‐A (probably due to the titration of Tyr‐25) which allows His‐48 to become accessible to solvent. 1H NMR spectra of S‐protein and S‐peptide, and of material partially deuterated at the C‐2 positions of the histidine residues confirm the reassignment of the histidine resonances of RNase‐A [Bradbury, J. H. & Teh, J. S. (1975) Chem. Commun., 936–937]. The chemical shifts of the C‐2 and C‐4 protons of histidine‐12 of S‐peptide are followed as a function of pH and a pK′ value of 6.75 is obtained.The reassignment of the three C‐2 histidine resonances of S‐protein is confirmed by partial deuteration studies. The pK′ values obtained from titration of the H‐2 resonances of His‐48, His‐105 and His‐119 are 5.3, 6.5 and 6.0, respectively. The S‐protein is less stable to acid than RNase‐A since the former, but not the latter, shows evidence of reversible denaturation at pH 3 and 26°C. His‐48 in S‐protein titrates normally and has a lower pK than in RNase‐A probably because of the absence of Asp‐14, which in RNase‐A forms a hydrogen bond with His‐48 and causes it to be inaccessible to solvent, at pH values below 9.
On the Mechanism of Substrate Binding to the Purine‐Transport System of Saccharomyces cerevisiae
Authors: FORÊT Martine , SCHMIDT Rainer , REICHERT Uwe 
Published: March 2005

The yeast Saccharomyces cerevisiae takes up adenine, guanine, hypoxanthine, and cytosine via a common energy‐dependent transport system. The apparent affinity of the transport system to these and other purines and pyrimidines is correlated with their capability to be protonated to the positively charged form. Further organic molecules are competitive inhibitors when they are cationic, e.g. guanidine and octylguanidine in contrast to urea, or hexadecyltrimethylammonium in contrast to dodecylsulfate and Triton X‐100.The influence of the pH on the kinetic constants of hypoxanthine transport points to a stoichiometry of one proton being associated to the transport system together with one substrate molecule.The pKa values of two ionizable groups that are involved in substrate binding are revealed; one of which (pKa= 1.8) may be attributed to the substrate, the other (pKa= 5.1) to an amino acid residue in the recognition site of the transport system. Studies with group‐specific inhibitors indicate that this amino acid residue contains a carboxyl group.The results are in accordance with the assumption that a carboxyl group of the transport system, a proton and a substrate molecule arrange to an uncharged ternary complex.
The Structure of Human‐Plasma Low‐Density Lipoprotein B
Authors: MÜLLER Karl , LAGGNER Peter , GLATTER Otto , KOSTNER Gerhard 
Published: March 2005

The X‐ray small‐angle scattering of human plasma lipoprotein B of the low‐density fraction (ϱ= 1.016−1.060 g · cm−3) has been recorded to high precision at different electron density contrasts.
The overall structure of the particles is characterized by a quasi‐spherical shape and radial symmetry. A maximum diameter of 23 nm and a molecular weight of 2.4 × 106 have been determined.
The internal structure is described in terms of a model consisting of spherical layers with different electron densities indicating that the neutral lipids are arranged in the core of the molecule up to a radius of about 8 nm surrounded by a monolayer of free cholesterol, phospholipids and protein. The neutral lipids are shown to be in an ordered, liquid crystalline state at 4°C and to undergo a thermotropic transition into a disordered state at higher temperatures.
A New Method for the Purification of RNA Polymerase II (or B) from the Lower Eukaryote Physarum polycephalum
Authors: SMITH Steven S. , BRAUN Richard 
Published: March 2005

The DNA‐dependent RNA polymerase II or B from the lower eukaryote Physarum polycephalum has been purified to apparent homogeneity by a new method employing poly(ethylene imine) precipitation and elution, and heparin‐Sepharose affinity chromatography. The method is readily scaled up or down and affords a purification of over 5000‐fold with a yield of 35–45%.The procedure is easy to perform and can be carried out in less than three days even on a large scale. Furthermore, it gives enzyme of higher purity and in at least 10‐fold greater yield than previously published procedures for its purification from this organism. These improvements have allowed the detection of a series of subforms of the enzyme.The combination of precipitation using poly(ethylene imine) with chromatography on heparin‐Sepharose may prove useful in the preparation of other proteins which interact with nucleic acids.
Conformations of Purine Ribosyl 5′‐Nucleotides Bound to Glycogen Phosphorylase b
Authors: CHACHATY Claude , FORCHIONI Alain , MORANGE Michel , BUC Henri 
Published: March 2005

The effects of the phosphate ion, glucose 1‐phosphate and glycogen on the binding of several nucleotides to phosphorylase b have been investigated by 1H and 2H linewidth measurements and by electron spin resonance. A graphical method is proposed to determine the respective contribution of the strong and weak nucleotide sites of the enzyme to the linewidth of H‐8, H‐2 and H‐1′.The contribution of the strong site to the linewidth is governed by the residence time of nucleotides in the case of AMP. clAMP and IMP ternary complexes with phosphorylase b and HPO42‐ and by the transverse relaxation time in that of the GMP ternary complex. It is shown that in this latter complex the GMP takes an anti conformation instead of a syn conformation in its binary complex with phosphorylase b. This change is related to an enhanced activity of the complex. The reorientation correlation time of binary and ternary complexes estimated from 2H‐8 linewidth is found of the order of 10−7 s in agreement with our previous 1H linewidth measurements.The dependence of the binding of a nucleotide on the concentrations of substrates is studied by the electron spin resonance of N6‐(2,2,6,6‐tetramethyl‐piperidine‐4‐yl‐1‐oxy)‐adenosine 5′‐monophosphate. The proton relaxation induced by the nitroxide group of this compound indicates a distance of the order of 1–2 nm between the two nucleotide strong sites of phosphorylase b.
Freeze‐Fracture Appearance and Disposition of Band 3 Protein from the Human Erythrocyte Membrane in Lipid Vesicles
Authors: GERRITSEN Willem J. , VERKLEY Arie J. , ZWAAL Robert F. A. , VAN DEENEN Laurens L. M. 
Published: March 2005

Single bilayer lipid vesicles were formed by removal of Triton X‐100 with Bio Beads SM‐2 from a mixture of egg lecithin and a Triton X‐100 extract of human erythrocyte ghosts. Upon freeze‐fracturing, these vesicles showed intramembrane particles, similar to those seen in the erythrocyte membrane. Similar particles were also observed when a partially purified band 3 preparation was used instead of the crude Triton X‐100 extract.In the reconstituted vesicles an equal distribution of the intramembrane particles between the two fracture faces was observed. This is in contrast to the unequal distribution of the particles in the erythrocyte membrane, which did not seem to be altered by removal of the extrinsic proteins.From digestion studies with trypsin and chymotrypsin of vesicles, reconstituted from the crude X‐100 extract, it is concluded that band 3 protein in the vesicle bilayer has a similar orientation as in the native membrane.
Interaction of Adenylosuccinate Synthetase with F‐Actin
Authors: OGAWA Hirofumi , SHIRAKI Hiroshi , MATSUDA Yoshihiro , NAKAGAWA Hachiro 
Published: March 2005

Both crude and purified preparations of adenylosuccinate synthetase from muscle were found to combine with, and dissociate from, muscle debris precipitated from a homogenate of the muscle with water. The binding and dissociation depended on ionic strength. Further study showed that the muscle enzyme was adsorbed to F‐actin, but not to G‐actin or myosin. The muscle‐type enzyme from the liver also associated with F‐actin, but the liver‐type enzyme from the liver did not. In the absence of KCl the molar ratio of adenylosuccinate synthetase from skeletal muscle to actin monomer in F‐actin in the complex formed was 1 to 4. From a Scatchard plot the dissociation constant was calculated to be 0.72 μM. The binding was maximal at pH 5.5–7 in 30 mM potassium phosphate buffer. The complex was completely dissociated in the presence of 0.21 M KCl.The physiological significance of this binding is discussed on the basis of these findings.
Uptake of Proteoglycans and Sulfated Glycosaminoglycans by Cultured Skin Fibroblasts
Authors: TRUPPE Wolfgang , KRESSE Hans 
Published: March 2005

Pinocytosis and degradation of [35S]proteoglycans [35S]glycosaminoglycan chains were studied in cultured skin fibroblasts. Proteoglycans were obtained from fibroblast secretions, glycosaminoglycan chains either by β‐elimination of proteoglycans or from secretions of fibroblasts grown in the presence of d‐xylose or P‐nitrophenyl‐β‐d‐xyloside, respectively. The following results were obtained.

The uptake of proteoglycans as well as of glycosaminoglycan chains is dose‐dependent and follows saturation kinetics. The maximal rate of pinocytosis og glycosaminoglycan chains induced by xylose and P‐nitrophenyl‐β‐d‐xyloside is only about 25% of that of proteoglycans as determined on the basis of the sulfate content. On a molar basis the maximal uptake rate for proteoglycans is 10‐fold lower than for free glycosaminoglycan chains, but proteoglycans have a higher affinity than chains for the proposed receptors.
Uptake is followed by degradation. Its relative proportion is inversely related to the amount of endocytosed material.
Unlabeled proteoglycans compete for the uptake of [35S]proteoglycans, but protein‐free proteoglycan chains are inefficient competitors at the doses used.
In case of [35S]glycosaminoglycans induced by xylose and P‐nitrophenyl‐β‐d‐xyloside, competition for uptake occurs by the addition of unlabeled glycosaminoglycan chains and of hybrid chondroitin‐sulfate‐dermatan‐sulfate decasaccharides, but not by the addition of smaller saccharide fragments.
Isolation of Phosphoglycerate Kinases by Affinity Chromatography
Authors: KUNTZ Günter W. K. , EBER Stefan , KESSLER Waltraud , KRIETSCH Helgard , KRIETSCH Wolfgang K. G. 
Published: March 2005

A variety of Sepharose derivatives containing dl‐O‐phosphorylserine or adenosine nucleotides with different points of attachment, has been synthesized and tested for affinity to phosphoglycerate kinase. The most effective gels contained periodate‐oxidized ATP or ADP bound via the ribose by hydrazone formation to adipoly‐dihydrazo‐Sepharose.The effect of pH, magnesium and buffer ions on the binding capacity of the ATP derivative of Sepharose has been examined. Optimal elution of phosphoglycerate kinase was investigated using different combinations of adenosine nucleotides, 3‐phosphoglycerate and magnesium ions.A method is presented giving conditions for the purification of phosphoglycerate kinase from different sources (spinach, human erythrocytes, human, rabbit and trout muscle). It includes extract preparation, affinity chromatography and gel filtration. The method is greatly superior to known isolation procedures by virtue of its technical simplicity, excellent yield (85–100%) and reproducability. The capacity of the ATP‐ribosyl‐adipoly‐dihydrazo‐Sepharose was 5 mg phosphoglycerate kinase per 1 g of matrix. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate indicated that the final products are homogenous.The phosphoglycerate kinases from different sources appear to have the same affinity for this ATP derivative of Sepharose, the same molecular weight and the same specific activity.
Isolation of the ADP/ATP Translocator from Beef Heart Mitochondria as the Bongkrekate‐Protein Complex
Authors: AQUILA Heinrich , EIERMANN Wolfgang , BABEL Wilfried , KLINGENBERG Martin 
Published: March 2005

The isolation of the ADP/ATP translocator from beef heart mitochondria as the bongkrekate‐protein complex is described, using hydroxyapatite chromatography and gel filtration in Triton X‐100 solution.
The inhibitor is bound to the protein prior to solubilization with detergent for protection against denaturation. Only the intact bongkrekate‐protein passes easily through the hydroxyapatite column. Bongkrekate shields the protein in contrast to carboxyatractylate only partially against proteinases present in the crude extract.
The isolated bongkrekate protein shows the same molecular weights in dodecylsulfate and Triton X‐100, the same amino acid composition and the same isoelectric point as the earlier isolated carboxyatractylate‐protein complex. It differs by its higher sensitivity against trypsin and thermolysin.
The identity of both proteins is demonstrated by interconversion of the bongkrekate‐protein into the carboxyatractylate‐protein. The process requires the catalysis by ADP or ATP, the natural substrates of the protein.
The formation of the extractable [3H]bongkrekate‐protein complex in mitochondria requires the presence of ADP or ATP.
These data, the immunological studies presented earlier, and the differences in the reactivity of –SH groups of the isolated bongkrekate and carboxyatractylate complexes (to be published) indicate that both proteins represent different conformational states of the translocator protein (m‐state and c‐state).