bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fine tuning of the catalytic properties of carbonic anhydrase
Authors: BEHRAVAN Gity , JONSSON Bengt‐Harald , LINDSKOG Sven 
Published: March 2005

The active sites of carbonic anhydrases I contain a unique histidine residue at sequence position 200. To test the hypothesis that His200 is essential for the isoenzyme‐specific catalytic and inhibitor‐binding properties of carbonic anhydrases I, a variant of human carbonic anhydrase II, having His200 for Thr200, was prepared by oligonucleotide‐directed mutagenesis. The variant has a circular dichroic spectrum that is indistinguishable from that of the parent enzyme. The kinetics of CO2 hydration and HCO−3 dehydration has been investigated. The results show that the amino acid substitution has led to changes of catalytic parameters as well as Ki values for anion inhibition in the expected directions towards the values for isoenzyme I. However, the maximal 4‐nitrophenyl acetate hydrolase activity of the variant is higher than for any naturally occurring carbonic anhydrase studied so far.A detailed analysis of the kinetic observations suggests that the modification has resulted in a change of the step that limits the maximal rate of CO2 hydration at saturating buffer concentrations. This rate‐limiting step is an intramolecular proton transfer in unmodified isoenzyme II and, presumably, HCO−3 dissociation in the variant and in human isoenzyme I. A free‐energy profile for the dominating pathway of CO2 hydration at high pH was constructed. The results suggest that the major effect of His200 is a stabilization of the enzyme–HCO−3 complex by about 7.5 kJ/mol (variant) and 6.1 kJ/mol (human isoenzyme I) relative to unmodified isoenzyme II, while proton transfer between the metal site and the reaction medium is only marginally affected by the amino acid replacement.
Structure of human myeloma IgG3 Kuc
Authors: RYAZANTSEV Sergey , TISHCHENKO Vladimir , VASILIEV Victor , ZAV'YALOV Vladimir , ABRAMOV Vyacheslav 
Published: March 2005

An electron microscopy study of human myeloma IgG3 Kuc has shown that the hinge region in an intact molecule is in a compact state. The subunits are not fixed rigidly and are very mobile. These data are supported by results of ultracentrifugation and microcalorimetry.Non‐extremal denaturating effects (pH 4.0, 20°C or pH 7.8, 65°C) lead to ‘unfolding’ of the hinge region which has a rod‐like shape in electron micrographs.
Thermodynamic parameters for binding of fatty acids to human serum albumin
Authors: PEDERSEN Anders Overgaard , HONORÉ Bent , BRODERSEN Rolf 
Published: March 2005

Binding of laurate and myristate anions to human serum albumin has been studied over a range of temperatures, 5–37°C, at pH 7.4. The binding curves indicate that the strength of binding of the first few molecules of fatty acid to albumin (r < 5) decreases with increasing temperature, whereas binding of the following molecules seems to proceed independently of temperature.Binding data were analyzed according to the general binding equation yielding several sets of acceptable binding constants within a probability limit of 0.75. From the temperature dependence of the first step constant, it was possible to calculate values for the changes in enthalpy and entropy during the initial binding step. For the medium‐chain fatty acids, laurate and myristate, binding of the first molecule to albumin appeared to be enthalpic, with a tendency to an increasing contribution of entropy to binding energy with increasing chain length of the fatty acid.
Sites phosphorylated in bovine cardiac troponin T and I
Authors: SWIDEREK Kristine , JAQUET Kornelia , MEYER Helmut E. , SCHÄCHTELE Christoph , HOFMANN Franz , HEILMEYER Ludwig M. G. 
Published: March 2005

Bovine cardiac troponin isolated in a highly phosphorylated form shows four 31P‐NMR signals [Beier, N., Jaquet, K., Schnackerz, K. & Heilmeyer, L. M. G. Jr (1988) Eur. J. Biochem. 176, 327–334]. Troponin I, which contains phosphate covalently linked to serine‐23 and/or‐24 [Swiderek, K., Jaquet, K., Meyer, H. E. & Heilmeyer, L. M. G. Jr (1988) Eur. J. Biochem. 176, 335–342], shows three resonances. Mg2+‐saturation of holotroponin shifts these troponin I resonances to higher fields. Direct binding of Mg2+ to the phosphate groups can be excluded. Both these serine residues of troponin I, 23 and 24, are substrates for cAMP‐ and cGMP‐dependent protein kinases as well as for protein kinase C.Isolated bovine cardiac troponin T contains 1.5 mol phosphoserine/mol protein, indicating that minimally two serine residues are phosphorylated. One phosphoserine residue is located at the N‐terminus. An additional phosphoserine is located in the C‐terminal cyanogen bromide fragment, CN4, which contains covalently bound phosphate. Protein kinase C phosphorylates serine‐194, thus demonstrating exposure of this residue on the surface of holotroponin.
The molybdoenzyme formylmethanofuran dehydrogenase from Methanosarcina barkeri contains a pterin cofactor
Authors: KARRASCH Marion , BÖRNER Gerhard , ENSSLE Marion , THAUER Rudolf K. 
Published: March 2005

Recently formylmethanofuran dehydrogenase from the archaebacterium Methanosarcina barkeri has been shown to be a novel molybdo‐iron‐sulfur protein. We report here that the enzyme contains one mol of a bound pterin cofactor/mol molybdenum, similar but not identical to the molybdopterin of milk xanthine oxidase. The two pterins, after oxidation with I2 at pH 2.5, showed identical fluorescence spectra and, after oxidation with permanganate at pH 13, yielded pterin 6‐carboxylic acid. They differed, however, in their apparent molecular mass: the pterin of formylmethanofuran dehydrogenase was 400 Da larger than that of milk xanthine oxidase, a property also exhibited by the pterin cofactor of eubacterial molybdoenzymes.A homogeneous formylmethanofuran dehydrogenase preparation was used for these investigations. The enzyme, with a molecular mass of 220 kDa, contained 0.5–0.8 mol molybdenum, 0.6–0.9 mol pterin, 28 ± 2 mol non‐heme iron and 28 ± 2 mol acid‐labile sulfur/mol based on a protein determination with bicinchoninic acid. The specific activity was 175 μmol · min−1· mg−1 (kcat= 640 s−1) assayed with methylviologen (app. Km= 0.02 mM) as artificial electron acceptor. The apparent Km for formylmethanofuran was 0.02 mM.
Analysis of the membrane potential of rat‐ and mouse‐liver mitochondria by flow cytometry and possible applications
Authors: PETIT Patrice X. , O'CONNOR José E. , GRUNWALD Didier , BROWN Spencer C. 
Published: March 2005

Washed and purified rat‐ or mouse‐liver mitochondria exhibiting high membrane integrity and metabolic activity were studied by flow cytometry. The electrophoretic accumulation/redistribution of cationic lipophilic probes, rhodamine 123, safranine O and a cyanine derivative, 3,3′‐dihexyloxadicarbocyanine iodide, during the energization process was studied and was consistent with the generation of a negative internal membrane potential. An exception to this was nonylacridine orange which spontaneously bound to the mitochondrial membrane by hydrophobic interactions via its hydrocarbon chain. Energized purified mitochondria stained with potentiometric dyes exhibited both higher fluorescence and population homogeneity than the non‐energized or deenergized (nigericin plus valinomycin) mitochondria. By contrast, under non‐energized or deenergized conditions, the mitochondrial population exhibited fluorescence intensity heterogeneity related to the residual membrane potential; two subpopulations were evident, one of low fluorescence which may be related to the autofluorescence of the mitochondria (plus non‐specific dye binding) and a second population which exhibited high fluorescence.Flow cytometry of the unpurified, simply washed, rat‐liver mitochondria stained with rhodamine 123, a classically used dye, provided evidence of their heterogeneity in terms of light‐scattering properties and membrane‐potential‐related fluorescence. One third of the washed mitochondria were found to be non‐functional by such assays.The fluorescence of purified rat‐liver mitochondria due to the membrane potential built up by endogenous substrates indicates heterogeneity of the mitochondrial population with respect to levels of endogeneous substrates.The low‐angle light scattering increases upon energization and provides some original information about the shape and modification of the inner mitochondrial conformation accompanying the energization.The heterogeneity of the rat liver mitochondrial population, from a structural, metabolic (existence of endogenous substrates) and functional (active and non‐active mitochondrial population dispersion) point of view could thus be demonstrated by flow‐cytometry analysis.Two animal models were examined with regard to the alteration of the mitochondrial membrane potential under the effects of drugs (rat‐liver mitochondria), and the effects of ammonium toxicity (mouse‐liver mitochondria). These results are promising and open new perspectives in the study of mitochondriopathies.
Characteristics of sinusoidal uptake and biliary excretion of cysteinyl leukotrienes in perfused rat liver
Authors: WETTSTEIN Matthias , GEROK Wolfgang , HÄUSSINGER Dieter 
Published: March 2005

In single‐pass perfused rat liver, the sinusoidal uptake of infused 3H‐labelled leukotriene (LT) C4 (10nmol · 1−1) was inhibited by sulfobromophthalein. Inhibition was half‐maximal at sulfobromophthalein concentrations of approximately 1.2 μmol · 1−1 in the influent perfusate and leukotriene uptake was inhibited by maximally 34% Sulfobromophthalein (20 μmol · 1−1) also decreased the uptake of infused [3H]LTE4 (10 nmol · 1−1) by 31%. Indocyanine green (10 μmol · 1−1) inhibited the sinusoidal [3H]LTC4 uptake by 19%.Replacement of sodium in the perfusion medium by choline decreased the uptake of infused [3H]LTC4 (10 nmol · 1−1) by 56%, but was without effect on the uptake of sulfobromophthalein.The canalicular excretion of LTC4, LTD4 and N‐acetyl‐LTE4 was inhibited by sulfobromophthalein. In contrast, the proportion of polar ω‐oxidation metabolites recovered in bile following the infusion of [3H]LTC4 was increased. Taurocholate, which had no effect on the sinusoidal leukotriene uptake, increased bile flow and also the biliary elimination of the radioactivity taken up. With increasing taurocholate additions, the amount of LTD4 recovered in bile increased at the expense of LTC4.Following the infusion of [3H]LTD4 (10 nmol · 1−1), a major biliary metabolite was LTC4 indicating a reconversion of LTD4 to LTC4. In the presence of taurocholate (40 μmol · 1−1), however, this reconversion was completely inhibited.The findings suggest the involvement of different transport systems in the sinusoidal uptake of cysteinyl leukotrienes. LTC4 uptake is not affected by bile acids and has a sodium‐dependent and a sodium‐independent component, the latter probably being shared with organic dyes. Sulfobromophthalein also interferes with the canalicular transport of LTC4, LTD4 and N‐acetyl‐LTE4, but not with the excretion of ω‐oxidized cysteinyl leukotrienes. The data may be relevant for the understanding of hepatic leukotriene processing in conditions like hyperbilirubinemia or cholestasis.
Molecular cloning of the gene for the E1α subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae
Authors: STEENSMA H. Yde , HOLTERMAN Lennart , DEKKER Iris , van SLUIS Cees A. , WENZEL Thibaut J. 
Published: March 2005

The E1α and E1β subunits of the pyruvate dehydrogenase complex from the yeast Saccharomyces cerevisiae were purified. Antibodies raised against these subunits were used to clone the corresponding genes from a genomic yeast DNA library in the expression vector λgt11.The gene encoding the E1α subunit was unique and localized on a 1.7‐kb HindIII fragment from chromosome V. The identity of the gene was confirmed in two ways. (a) Expression of the gene in Escherichia coli produced a protein that reacted with the anti‐E1α serum. (b) Gene replacement at the 1.7‐kb HindIII fragment abolished both pyruvate dehydrogenase activity and the production of proteins reacting with anti‐E1α serum in haploid cells. In addition, the 1.7‐kb HindIII fragment hybridized to a set of oligonucleotides derived from amino acid sequences from the N‐terminal and central regions of the human E1α peptide.We propose to call the gene encoding the E1α subunit of the yeast pyruvate dehydrogenase complex PDA1. Screening of the λgt11 library using the anti‐E1β serum resulted in the reisolation of the RAP1 gene, which was located on chromosome XIV.
A critical crosslink region in human‐bone‐derived collagen type I
Authors: BÄTGE Boris , NOTBOHM Holger , DIEBOLD Joachim , LEHMANN Hartwig , BODO Michael , DEUTZMANN Rainer , MÜLLER Peter K. 
Published: March 2005

Collagen was extracted from human adult bone by limited pepsin digestion and collagen types were purified by consecutive salt precipitation first under neutral and then under acid conditions. In SDS/PAGE, all collagen type I preparations showed a protein band [α1s(I)] migrating between α(I) and α2(I) as well as a band [α2s(I)] migrating in front of α2(I). The collagenous nature of the pepsin‐stable α1s(I) protein was clearly demonstrated by digestion with human‐leucocyte‐derived collagenase, immunoblotting with antibodies against collagen type I and amino acid analysis. Partial amino acid sequencing of α1(I) and α1s(I) identified α1s(I) as a shortened α1(I) chain due to a specific cleavage site between residues Leu95 and Asp96 which is in close vicinity to the hydroxylysine‐derived crosslink at position 87. In circular dichroism, the proportion of thermally labile collagen molecules was proportional to the amount of shortened α1(I) and α2(I) chains, respectively. The melting temperature was found to be 36 ± 0.5°C as judged from circular dichroism and susceptibility to proteolysis.Our data provide clear evidence that a shortened α1‐derived collagen chain can be extracted from human adult bone whereas it is hardly found in human skin. The unique cleavage site might provide important information about the collagen I molecule embedded in the calcified matrix of human bone.
Pyridoxal phosphate inhibits the DNA‐binding activity of the ecdysteroid receptor
Authors: OŻYHAR Andrzej , KILTZ Hans‐Hermann , PONGS Olaf 
Published: March 2005

The effect of pyridoxal 5′‐phosphate on the binding of the ecdysteroid receptor from a nuclear extract of Drosophila melanogaster to DNA‐cellulose was studied. The binding of hormone‐receptor complexes to DNA‐cellulose was completely blocked after a 30‐min incubation with 3 mM pyridoxal 5′‐phosphate at 0–4°C. The effect was specific for pyridoxal 5′‐phosphate since related compounds (pyridoxal, pyridoxamine 5′‐phosphate and pyridoxamine) were not effective or gave only 17% inhibition (pyridoxal). Under standard conditions, none of the compounds tested exerted a significant effect on the stability of [3H](20R,22R)‐2β,3β,14α,20,22‐pentahydroxy‐5β‐cholest‐7‐en‐6‐one ([3H]ponasterone A)‐receptor complexes. The loss of DNA‐binding activity caused by pyridoxal 5′‐phosphate is accompanied by changes in the molecular properties of [3H]ponasterone‐A–receptor complexes. A shift of [3H]ponasterone‐A binding was observed from the 8.0–8.5 S to the 4.5–5.0 S region, when [3H]ponasterone‐A–receptor complexes were exposed to pyridoxal 5′‐phosphate during sucrosegradient centrifugation.The inhibition of DNA‐cellulose binding by pyridoxal 5′‐phosphate can be reversed. Probably, pyridoxal 5′‐phosphate forms a Schiff base with a critical lysine group of the ecdysteroid receptor, presumably at its DNA‐binding site. The hormone‐receptor complexes obtained after removal of pyridoxal 5′‐phosphate had the same affinity for DNA‐cellulose as ‘native’ complexes.DNA–cellulose‐bound [3H]ponasterone‐A complexes were efficiently eluted from DNA‐cellulose with pyridoxal 5′‐phosphate in 0.1 M K Cl resulting in a 104‐fold purification of the ecdysteroid receptor.The results reflect possible structural similarities between ecdysteroid and vertebrate steroid receptors.
Structural features of the lipopeptidophosphoglycan from Trypanosoma cruzi common with the glycophosphatidylinositol anchors
Authors: de LEDERKREMER Rosa M. , LIMA Carlos , RAMIREZ María I. , CASAL Olga L. 
Published: March 2005

The lipopeptidophosphoglycan (LPPG) from Trypanosoma cruzi, a major constituent of the plasma membrane of epimastigote forms, has been now extracted with butanol/water from delipidated cells and purified by hydrophobic chromatography. We have found that the LPPG undergoes two reactions, characteristic of the glycosylphosphatidylinositol anchors: (a) cleavage of the ceramide by phosphatidylinositol‐specific phospholipase C(PtdIns‐specific phospholipase C) from Bacillus thuringiensis, (b) nitrous acid deamination of the non‐N‐acylated glucosamine. Palmitoylsphinganine, palmitoylsphingosine, lignoceroylsphinganine and, as minor components, the stearoylceramides were identified by gas liquid chromatography/mass spectrometry.The presence of cross reacting determinant (CRD) epitopes in the glycophosphoinositol released by PtdIns‐specific phospholipase C was investigated by direct and inhibition ELISA. A sample of glycophosphoinositol containing 5 μg carbohydrate caused 60% inhibition of the binding of anti‐CRD antibodies raised against the soluble form of variant surface glycoprotein.
Control of respiration and oxidative phosphorylation in isolated rat liver cells
Authors: BROWN Guy C. , LAKIN‐THOMAS Patricia L. , BRAND Martin D. 
Published: March 2005

NAD(P)H fluorescence, mitochondrial membrane potential and respiration rate were measured and manipulated in isolated liver cells from fed and starved rats in order to characterize control of mitochondrial respiration and phosphorylation. Increased mitochondrial NADH supply stimulated respiration and this accounted for most of the stimulation of respiration by vasopressin and extracellular ATP. From the response of respiration to NADH it was estimated that the control coefficient over respiration of the processes that supply mitochondrial NADH was about 0.15–0.3 in cells from fed rats.Inhibition of the ATP synthase with oligomycin increased the mitochondrial membrane potential and decreased respiration in cells from fed rats, while the uncoupler carbonyl cyanide p‐trifluoromethoxyphenylhydrazone had the opposite effect. There was a unique relationship between respiration and membrane potential irrespective of the ATP content of the cells indicating that phosphorylation potential controls respiration solely via phosphorylation (rather than by controlling NADH supply).From the response of respiration to the mitochondrial membrane potential (ΔΨM) it was estimated that the control coefficients over respiration rate in cells from fed rats were: 0.29 by the processes that generate ΔΨM, 0.49 by the process of ATP synthesis, transport and consumption, and 0.22 by the processes that cycle protons across the inner mitochondrial membrane other than via ATP synthesis (e.g. the passive proton leak). Control coefficients over the rate of mitochondrial ATP synthesis were 0.23, 0.84 and −0.07, respectively, by the same processes. The control distribution in cells from starved rats was similar.
Hepatic processing and biliary secretion of the cholesteryl esters from β very‐low‐density lipoproteins in the rat
Authors: De WATER Ruud , HESSELS Esther M. A. J. , BAKKEREN Hille F. , VAN BERKEL Theo J. C. 
Published: March 2005

β‐Migrating very‐low‐density lipoproteins (β‐VLDL) are cholesteryl‐ester‐enriched lipoproteins which accumulate in the serum of cholesterol‐fed animals or patients with type III hyperlipoproteinemia. In the rat, β‐VLDL are rapidly cleared by the liver and parenchymal liver cells form the major site for uptake. In this investigation, β‐VLDL were labeled with [3H]cholesteryl esters and the hepatic intracellular transport of these esters was followed. 2 min after injection, the major part of the [3H]cholesteryl esters is already associated with the liver and a significant proportion is recovered in endosomes. Up to 25 min after injection, an increase in radioactivity in the lysosomal compartment is noticed. This radioactivity initially represents cholesteryl esters, while from 25 min onward, radioactivity is mainly present in unesterified cholesterol. Between 45 min and 90 min after β‐VLDL injection, specific transfer of unesterified [3H]cholesterol to the endoplasmic reticulum is observed, while by 3 h the majority is located in this fraction. The appearance of radioactivity in the bile was rather slow as compared to the rapid initial uptake and processing, and up to 5 h after injection only 10% of the injected dose had reached the bile (mainly as bile acids). 72 h after injection, the amount of the injected radioactivity recovered in the bile had increased to 50%. Chloroquine treatment of the rats inhibited the hydrolysis of the cholesteryl esters and the appearance of radioactivity in the bile was retarded.It is concluded that β‐VLDL are rapidly processed by parenchymal liver cells and that the cholesteryl esters from β‐VLDL are hydrolyzed in the lysosomal compartment. Unesterified cholesterol remains associated with the endoplasmic reticulum for a prolonged time, although ultimately the majority will be secreted into the bile as bile acids. The effective operation of this pathway will prevent extrahepatic accumulation of cholesteryl esters from β‐VLDL, while the prolonged residence time of unesterified cholesterol in the endoplasmic reticulum might be important for regulation of low‐density lipoprotein (LDL) receptors in liver and thus for LDL levels in the blood.
The bovine plasma retinol‐binding protein
Authors: BERNI Rodolfo , STOPPINI Monica , ZAPPONI Maria Carla , MELONI Maria Laura , MONACO Hugo L. , ZANOTTI Giuseppe 
Published: March 2005

The primary structure of bovine plasma retinol‐binding protein (RBP) has been determined and found to be more than 90% identical to human and rabbit RBPs, and more than 80% identical to rat RBP. Main changes in amino acid sequence are observed in two regions on the surface of the protein molecule (residues 138–148 and 169–183).
The interactions of bovine RBP with bovine and human transthyretins were investigated using the technique of fluorescence polarization. Bovine RBP was able to form high affinity complexes (K′d= 0.34 ± 0.02 μM) with both bovine and human transthyretins, displaying a stoichiometry of approximately 2 molecules RBP/molecule transthyretin in both cases. The sites that participate in protein‐protein interactions are thus very similar, and this tends to exclude the involvement of the superficial regions more significantly substituted in mammalian RBPs (residues 138–151 and 167–183) in the protein–protein recognition.
Bovine RBP has been crystallized (space group P212121, with a= 4.61 nm, b= 4.91 nm, c= 7.61 nm) and the crystals are suitable for high‐resolution X‐ray diffraction studies.