bluuebunny/crossref_metadata_2025_split huggingface dataset:
A study on the regulation of N‐glycoloylneuraminic acid biosynthesis and utilization in rat and mouse liver
Authors: LEPERS Anne , SHAW Lee , SCHNECKENBURGER Petra , Cacan René , VERBERT André , SCHAUER Roland 
Published: March 2005

The relative contribution of N‐glycoloyl‐β‐d‐neuraminic acid (Neu5Gc) to total sialic acids expressed in mouse and rat liver glycoconjugates was found to be 95% and 11%, respectively. This considerable difference in sialic acid composition made these two tissues suitable modles for a comparative investigation into the regulation of Neu5Gc biosynthesis and utilization.An examination of the CMP‐glycoside specificity of Golgi‐associated sialyltransferases using CMP‐N‐acetyl‐β‐d‐neuraminic acid (CMP‐Neu5Ac) and CMP‐Neu5Gc revealed no significant tissue‐dependent differences. The Golgi membrane CMP–sialic acid transport system from rat liver did, however, exhibit a slightly higher internalisation rate for CMP‐Neu5Ac, though no preferential affinity for this sugar nucleotide over CMP‐Neu5Gc was observed.In experiments, where Golgi membrane preparations were incubated with an equimolar mixture of labelled CMP‐Neu5Ac and CMP‐Neu5Gc, no significant tissue‐dependent differences in [14C]sialic acid composition were observed, either in the luminal soluble sialic acid fraction or in the precipitable sialic acid fraction, results which are consistent with the above observations. From this experiment, evidence was also obtained for the presence of a Golgi‐lumen‐associated CMP–sialic acid hydrolase which exhibited no apparent specificity for either CMP‐Neu5Ac or CMP‐Neu5Gc.The specific activity of the CMP‐Neu5Ac hydroxylase, the enzyme responsible for the biosynthesis of Neu5Gc, was found to be 28‐fold greater in high‐speed supernatants of mouse liver than of rat liver. No hydroxylase activity was detected in the Golgi membrane preparations. It is therefore proposed that the cytoplasmic ratio of CMP‐Neu5Ac and CMP‐Neu5Gc produced by the hydroxylase, remains largely unmodified after CMP‐glycoside uptake into the Golgi apparatus and transfer on to growing glycoconjugate glycan chains. The close relationship between the total sialic acid composition and the sialic acid pattern in the CMP‐glycoside pools of the tissues lends considerable weight to this hypothesis.
Analysis of wheat‐germ RNA polymerase II by trypsin cleavage
Authors: TEISSERE Marcel , SERGI Isabelle , JOB Claudette , JOB Dominique 
Published: March 2005

When wheat‐germ RNA polymerase II is subjected to mild proteolytic attack in the presence of trypsin, the resulting form of the enzyme migrates as a single species on electrophoresis in native polyacrylamide gels, with an apparent Mr significantly smaller than that of the native enzyme. Analysis by denaturing gel electrophoresis of the truncated eukaryotic polymerase revealed that the two largest subunits of the native enzyme, i.e. the 220 000‐Mr and 140000‐Mr subunits, were cleaved, giving rise to shorter polypeptide chains of Mr 172800, 155000, 143000, 133800, 125000 and 115000. The use of affinity‐purified antibodies directed against each of the two large subunits of the native enzyme allowed us to probe for possible precursor/product relationships between the 220000‐Mr and 140000‐Mr subunits of wheat‐germ RNA polymerase II and their breakdown products generated in the presence of trypsin.None of the smaller subunits of the plant RNA polymerase II appeared to be sensitive to trypsin attack. The results indicate that the truncated RNA polymerase retained a multimeric structure, and therefore that the proteolyzed largest subunits of the enzyme remained associated with the smaller ones.Furthermore, in transcription of a poly[d(A‐T)] template, the catalytic activity of the proteolyzed form of wheat‐germ RNA polymerase II was identical to that of the native enzyme. Therefore, the protein domains that can be deleted by the action of trypsin from the two large subunits of the plant transcriptase are not involved in DNA binding and/or nucleotide binding, and do not play an important role in template‐directed catalysis of phosphodiester bond formation.
Interaction of hemoglobin with chloride and 2,3‐bisphosphoglycerate
Authors: GIARDINA Bruno , BRIX Ole , COLOSIMO Alfredo , PETRUZZELLI Raffaele , CERRONI Loredana , CONDO Saverio G. 
Published: March 2005

The equilibrium oxygen‐binding properties of hemoglobins from reindeer (Rangifer tarandus tarandus), musk ox (Ovibos muschatos) and a bat (Rousettus aegyptiacus) have been investigated with special reference to the effect of heterotrophic ligands such as chloride and 2,3‐bisphosphoglycerate [Gri(2,3)P2].The results obtained with hemoglobins from reindeer and musk ox indicate that their low oxygen affinity and their insensitivity to Gri(2,3)P2 are not only an intrinsic property of the molecule, as proposed in the case of ruminant hemoglobins, but also the results of the interplay between chloride and Gri(2,3)P2 interactions. In other words, insensitivity of reindeer and musk ox hemoglobins to Gri(2,3)P2 is mainly due to a decreased affinity constant for this cofactor and to an increased affinity constant for chloride anions; this renders more effective the competition of chloride for the anion‐binding site.On the other hand bat hemoglobin behaves in a completely different way and could be regarded as a type case of low‐affinity hemoglobin since its functional properties are modulated neither by chloride nor by Gri(2,3)P2.The results are discussed in the light of the amino acid residues which are known to be involved in the binding of organic phosphates.
Human liver manganese superoxide dismutase
Authors: MATSUDA Yukihiko , HIGASHIYAMA Shigeki , KIJIMA Yoshiyuki , SUZUKI Keiichiro , KAWANO Kiyoshi , AKIYAMA Morio , KAWATA Sumio , TARUI Seiichiro , DEUTSCH Harold F. , TANIGUCHI Naoyuki 
Published: March 2005

Manganese superoxide dismutase (Mn‐SOD) has been purified with a high yield (320 mg) from human liver (2 kg) and crystallized. Low‐angle laser light scattering of the enzyme has shown that native enzyme is a tetrametic form.Four of the eight cysteine residues in the tetramer reacted with−5,5′dithiobis(2‐nitrobenzoic acid) or with iodoacetamide. The others were only reactive in protein heated with SDS or urea after reduction with dithiothreitol or 2‐mercaptoethanol. The reactive sulfhydryl group was found to be located at Cys196 by amino acid sequence analysis of Nbs2‐reactive peptides isolated by activated thiol‐Sepharose covalent chromatography. Incubation of Mn‐SOD in 1% SDS for 2 or 3 days at 25°C or 5 min at 100°C gave material showing two prominent components on polyacrylamide gel electrophoresis in the presence of 0.1% SDS. The major component had a molecular mass of 23 kDa; the other, 25 kDa. Reduction of the protein by dithiothreitol or 2‐mercaptoethanol heated in SDS produced only the 25‐kDa monomer species. Essentially, no thiol groups were detected in the 23‐kDa form, in which two cysteine residues appear to have been oxidized to form an intrasubunit disulfide. This indicates that Cys196 has a reactive sulfhydryl and appears to be a likely candidate for a mixed disulfide formation in vivo.
Purification and characterization of lipoamide dehydrogenase from Trypanosoma cruzi
Authors: LOHRER Helmut , KRAUTH‐SIEGEL R. Luise 
Published: March 2005

From Trypanosoma cruzi, the causative agent of Chagas' disease, a lipoamide dehydrogenase was isolated. The enzyme, an FAD —cystine oxidoreductase, shares many physical and chemical properties with T. cruzi trypanothione reductase, the key enzyme of the parasite's thiol metabolism.

From 60 g epimastigotic T. cruzi cells, 2.7 mg lipoamide dehydrogenase was extracted. The flavoenzyme was purified 3000‐fold to homogeneity with an overall yield of 26%.
The enzyme is a dimer with a subunit Mr of 55000. With 1 mM lipoamide (Km∼ 5 mM) and 100 μM NADH (Km= 23 μM), the specific activity at pH 7.0 is 297 U/mg.
With excess NADH, the enzyme is reduced to the EH2· NADH complex and, by addition of lipoamide, it is reoxidized, indicating that it can cycle between the oxidized state E and the two‐electron‐reduced state, EH2.
As shown by N‐terminal sequencing of the enzyme, 21 out of 30 positions are identical with those of pig heart and human liver lipoamide dehydrogenase. The sequenced section comprises the GGGPGG stretch, which represents the binding site for the pyrophosphate moiety of FAD.
After reduction of Eox to the two‐electron‐reduced state, the enzyme is specifically inhibited by the nitro‐sourea drug 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea (Carmustine), presumably by carbamoylation at one of the nascent active‐site thiols.
Polyclonal rabbit antibodies raised against T. cruzi lipoamide dehydrogenase and trypanothione reductase are specific for the respective enzyme, as shown by immunoblots of the pure proteins and of cell extracts.
Analysis of inhibitor binding to the mitochondrial cytochrome c reductase by fluorescence quench titration
Authors: BRANDT Ulrich , von JAGOW Gebhard 
Published: March 2005

The binding characteristics of inhibitors of the mitochondrial cytochrome c reductase were studied by fluorescence quench titration. Based on the standard binding equation, the applied numerical method allowed the online recorded titration curves to be interpreted by fitting the Kd, the number of binding sites, and the specific fluorescence of the free and the bound inhibitor. For the Qi, center, 2‐n‐nonyl‐4‐hydroxyquinoline N‐oxide and for the Qo center (E)‐β‐methoxyacrylate‐stilbene (MOA‐stilbene) were used as fluorescing inhibitors. The experiments could be extended to other, non‐fluorescing inhibitors by competition analysis. Using this method we were able to compare the binding behaviour of Qi and Qo center inhibitors under different redox states of the enzyme using the same experimental set up.We studied the competition between inhibitors of the cytochrome c reductase representative for all subgroups and demonstrated that at least three inhibitor binding sites exist, two located in the Qo center, one located in the Qi center.Determination of the dissociation constants of the oxidized, the partially reduced and the fully reduced enzyme showed that inhibitor binding at the Qi center is not redox‐dependent. In contrast, the binding of MOA‐stilbene to the Qo center is decreased after reduction of the iron‐sulfur center and cytochrome c1 whereas this redox change increases the affinity for a Qo center inhibitor of the hydroxynaphthoquinone type, 3‐n‐undecyl‐2‐hydroxy‐naphthoquinone.From these results, aware of the fact that the inhibitory mechanism at the Qo center is a non‐competitive one, we made the hypothesis of a ‘catalytic switch’ to explain both the bifurcation of electron flow and the inhibition at the Qo center. A steric blockage of one of two conformational states could serve as a cogent explanation for the great structural variability of the inhibitors and differential effects on the redox centers exerted by the inhibitors. Moreover, the proposed ‘switch’ gives some insight into other experimental results which are difficult to explain with the ubiquinone cycle as currently formulated.
Submicromolar manganese dependence of Golgi vesicular galactosyltransferase (lactose synthetase)
Authors: KUHN Nicholas J. , WARD Simon , LEONG Weng S. 
Published: March 2005

Manganese(II) buffers were set up with inorganic triphosphate, trimethylenediaminetetraacetate and tetramethylenediaminetetraacetate to study the Mn dependence of β, 4‐galactosyltransferase (lactose synthetase) in preparations of rat mammary gland.
In intact particulate preparations, treated with the calcium ionophore A23187, lactose synthesis was abolished by chelators and restored by bivalent transition metal ions in a manner characteristic of activation site I of the pure enzyme. Ni(II) also activated, as did Mg at high concentration.
Only Mn(II) could restore endogenous rates, giving an apparent Km of 0.1–0.2 μM, and eliciting about 70% full activity without addition of a site II activator.
In purified Golgi membrane vesicles, Mn gave an apparent Km of 0.4 μM. This increased sharply to about 10 μM on permeabilization with filipin, lysis with detergents, solubilization with Triton X‐100, or in the pure enzyme. Preparations of chemically undamaged Golgi vesicles, known to include a proportion of the enzyme on exposed membranes, exhibited both low‐Km and high‐Km components.
The response of particulate galactosyltransferase to apparently physiological concentrations of free Mn(II) ion is interpreted as either due to a sensitizing factor within the Golgi lumen, or to the accumulation of Mn at elevated concentrations. Alternatively, the high Km of the soluble enzyme may reflect proteolytic damage.
Kinetics of the conformational transition of the spinach chloroplast fructose‐1,6‐bisphosphatase induced by fructose 2,6‐bisphosphate
Authors: SOULIÉ Jean‐Michel , RIVIÈRE Mireille , BALDET Pierre , RICARD Jacques 
Published: March 2005

The activation of oxidized chloroplast fructose‐1,6‐bisphosphatase by fructose 2,6‐bisphosphate and magnesium previously described at pH 7.5 [Soulié et al. (1988) Eur. J. Biochem. 176, 111 – 117] has now been studied at pH 8, the pH which prevails under light conditions in the chloroplast stroma. The process obeys a hysteretic mechanism but the rate of activation is considerably increased with half‐times down to 50 s and the apparent dissociation constant of fructose 2,6‐bisphosphate from the enzyme is lowered from 1 mM at pH 7.5 to 3.3 μM at pH 8. The process is strictly metal‐dependent with a half‐saturation concentration of 2.54 mM for magnesium.The conformational transition postulated in our hysteretic model has been investigated through both the spectrophometric and chemical modification approaches. The activation of the enzyme by fructose 2,6‐bisphosphate in the presence of magnesium results in a slow modification of the ultraviolet absorption spectrum of the enzyme with an overall increase of 3% at 290 nm. The same treatment leads to the protection of two free sulfhydryls and an increased reactivity of one sulfhydryl group/enzyme monomer to modification by 5,5′‐dithiobis(2‐nitrobenzoic acid). The titration of the exposed cysteinyl residue prevents the relaxation of enzyme species induced by fructose 2,6‐bisphosphate to the native form.The activation of chloroplast fructose‐1,6‐bisphosphatase by fructose 2,6‐bisphosphate is discussed both with respect to the understanding of the overall regulation properties of the enzyme and to a possible physiological significance of this process.
Investigations on microbial sulfur respiration
Authors: ZÖPHEL Andreas , KENNEDY M. Claire , BEINERT Helmut , KRONECK Peter M. H. 
Published: March 2005

The sulfur‐reducing bacterium Spirillum 5175 was investigated with regard to membrane constituents that might be part of the sulfur oxidoreductase which converts elemental sulfur to hydrogen sulfide. Regardless of the electron acceptor used for cultivation of the bacteria, i.e. elemental sulfur, fumarate, or nitrate (Sp. 5175S,F,N), the qualitative pattern of cytochromes and Fe‐S proteins did not change significantly, as documented by ultraviolet/visible and electron paramagnetic resonance spectroscopy of oxidized (as isolated) and reduced (dithionite) samples. With elemental sulfur the prominent cytochrome exhibited absorption maxima at 553, 522.5 and 426 nm in the reduced state. In fumarate‐grown cells two prominent cytochromes were found with maxima at 561, 551, 530, 521 and 430 nm. Two b‐type cytochromes with Em at −198 mV and −20 mV vs the standard hydrogen electrode were identified in the membrane fraction of Sp. 5175F.A yellow pigment was extracted and identified as a flexirubin‐type pigment. Although present in large quantities, it seemed not to be involved in the reduction of elemental sulfur.Menaquinone, MK 6 (Mr 580) was the prominent quinone identified in Sp. 5175. Characterization of a second quinone was not attempted because of its much lower concentration.The membrane constituents of Sp. 5175 were solubilized by a variety of detergents and detergent mixtures. A colorimetric procedure with photochemically reduced phenosafranin as the electron donor and cysteamine trisulfide (RS‐S‐SR, R =−CH2CH2NH2) as the electron acceptor was used to detect sulfur oxidoreductase activity.Three membrane proteins of Sp. 5175 were purified: (1) an [NiFe] hydrogenase, homogeneous by SDS/polyacrylamide gel electrophoresis, with electron paramagnetic resonance signals as isolated at gx,y,z= 2.01, 2.16, 2.33 (100 K), and a strong signal at g= 2.02 below 20 K; (2) a cytochrome b, Fe‐S‐dependent fumarate reductase, and (3) a protein apparently linked to the sulfur oxidoreductase activity. In contrast to fumarate reductase, no b‐type cytochrome was present in the fractions exhibiting sulfur oxidoreductase activity. The presence of Fe‐S centers was demonstrated by electron paramagnetic resonance spectroscopy at 10 K. It is not clear whether the c‐type cytochrome in the same fractions is part of the sulfur‐reducing apparatus of Sp. 5175.
Effects of monocyte‐lymphocyte interaction on the synthesis of leukotriene B4
Authors: JAKOBSSON Per‐Johan , ODLANDER Björn , CLAESSON Hans‐Erik 
Published: March 2005

Human monocytes in monolayers were challenged with the calcium ionophore A23187. Methanol trapping of the products in the cell‐free supernatants, followed by analysis on HPLC and by ultraviolet spectroscopy, revealed the presence of two compounds, which exhibited a conjugated‐triene spectrum and chromatographed with the compounds formed when synthetic leukotriene (LT) A4 was added to warm acidified methanol. Furthermore, addition of purified LTA4 hydrolase to the cell‐free supernatant of monocytes, stimulated with the ionophore A23187, resulted in increased levels of LTB4. These results indicate that monocytes release LTA4 extracellularly after activation with the calcium ionophore.Incubation of monocytes together with monoclonal lymphocytic cells, of both B and T cell lineage, yielded increased levels of LTB4 whereas the non‐enzymatic isomers of this compound, i.e. Δ6‐trans‐LTB4 and 12‐epi‐Δ6‐trans‐LTB4, declined. In addition, the sum of LTB4 and its non‐enzymatically formed isomers increased in mixed cultures of monocytes and monoclonal lymphocytic cells as compared to monocytes alone.The present study indicates that activated monocytes release LTA4, which is converted into LTB4 by monoclonal lymphocytic cells. Furthermore, the increase of the total amounts of leukotrienes on incubation of monocytes with lymphocytic cells, suggests the presence of an additional mechanism leading to activation of the 5‐lipoxygenase pathway in monocytes.
Characterization of the yeast acidic ribosomal phosphoproteins using monoclonal antibodies
Authors: VILELLA Maria Dolores , REMACHA Miguel , ORTIZ Blanca L. , MENDEZ Enrique , BALLESTA Juan Pedro García 
Published: March 2005

In order to characterize the acidic ribosomal proteins immunologically and functionally, a battery of monoclonal antibodies specific for L44, L44′ and L45, the three acidic proteins detected in Saccharomyces cerevisiae, were obtained. Eight monoclonal antibodies were obtained specific for L45, three for L44′ and one for L44. In addition, two mAbs recognizing only the phosphorylated forms of the three proteins were obtained. The specific immunogenic determinants are located in the middle region of the protein structure and are differently exposed in the ribosomal surface. The common determinants are present in the carboxyl end of the three proteins. An estimation of the acidic proteins by ELISA indicated that, in contrast to L44 and L45, L44′ is practically absent from the cell supernatant; this suggests that protein L44′ does not intervene in the exchange that has been shown to take place between the acidic proteins in the ribosome and in the cytoplasmic pool. It has also been found that, while IgGs specific for L44 and L45 do not inhibit the ribosome activity, the anti‐L44′ effectively blocks the polymerizing activity of the particles.These results show for the first time that the different eukaryotic acidic ribosomal proteins play a different functional role.
Different isoforms of an apoprotein (apolipophorin III) associate with lipoproteins in Locusta migratoria
Authors: VAN DER HORST Dick J. , VAN DOORN Jan M. , VOSHOL Hans , KANOST Michael R. , ZIEGLER Rolf , BEENAKKERS Ad M. Th. 
Published: March 2005

Insects transport lipid for flight in the form of diacylglycerol‐rich low‐density lipoproteins (low‐density lipophorin, LDLp), which in the hemolymph are produced from high‐density lipophorin (HDLp) by reversible association with several molecules of an apolipoprotein, apolipophorin III (apoLp‐III, Mr∼ 18000–20000) during lipid loading.Two isoforms of apoLp‐III (a and b) were purified both from adult Locusta migratoria migratorioides hemolymph and LDLp, which have identical apparent Mr but differ in amino acid composition, NH2‐terminal amino acid sequence, and isoelectric points (5.35 ± 0.01 for apoLp‐IIIa, 5.10 ± 0.01 for apoLp‐IIIb). The NH2‐terminal sequence of apoLp‐IIIb is identical to the primary structure of apoLp‐III deduced from cloned cDNA [Kanost et al. (1988) J. Biol. Chem. 263, 10568–10573], whereas the NH2‐terminal sequence of apoLp‐IIIa is identical to that of apoLp‐IIIb but preceded by Arg‐Pro‐, which is the C‐terminal of the putative signal peptide coded by cDNA upstream from that coding for apoLp‐IIIb. The ratio apoLp‐IIIa apoLp‐IIIb free in hemolymph is identical to that in LDLp (5:9); since 14 molecules of apoLp‐III appear to be bound in one molecule of LDLp, an average of 5 molecules of apoLp‐IIIa and 9 of apoLp‐IIIb are involved in formation of each LDLp particle. In vivo studies using 35S‐labeled apoLp‐IIIa and b demonstrate that each of the isoforms can associate with HDLp to produce LDLp reversibly; in an in vitro system, production of LDLp containing exclusively apoLp‐IIIa or apoLp‐IIIb demonstrates independent participation of each isoform in LDLp formation.
Observation of a stable intermediate form in the reaction of human hemoglobin with carbon monoxide
Authors: COLETTA Massimo , GERACI Giuseppe 
Published: March 2005

The reaction of human hemoglobin with carbon monoxide has been investigated near the equilibrium isosbestic wavelength (i.e.426 nm). As previously reported by others [Gray, R. D. & Gibson, Q. H. (1971) J. Biol. Chem. 246, 5176–5178], in the presence of 0.1 M phosphate pH 7.0 a rise‐and‐fall kinetic pattern can be observed at this wavelength, which indicates the presence of at least one spectroscopically detectable intermediate species. In this paper we demonstrate that (a) the intermediate species is thermodynamically stable; (b) both phases refer to bimolecular processes; (c) only the initial fast phase is observed when deoxyhemoglobin is reacted with substoichiometric amounts of CO (i.e. final [CO]/[heme] ≤ 0.5); (d) only the second slow phase is observed when hemoglobin that is partially saturated with CO (Ȳ≤ 0.5) is reacted with saturating CO concentrations; (e) the CO dissociation rate constant measured on the intermediate formed after a partial CO saturation at a final Ȳ∼ 0.4 has a value similar to that observed starting from the fully liganded form.These results can be accounted for by a two‐state allosteric model [Monod, J., Wyman, J. & Changeux, J.‐P. (1965) J. Mol. Biol. 12, 88–118] under the assumption that either (a) 426 nm is an isosbestic wavelength for the To–R spectral changes but not for the To–T liganded reaction; or (b) a functional heterogeneity of the two types of subunits is present in the T state and at this wavelength this feature is spectroscopically detectable.