bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of the Mannan Structure from the Cell‐Wall Mutant Candida sp. M‐7002 and Its Wild Type
Authors: HAMADA Takashi , NAKAJIMA Tasuka , IZAKI Kazuo , MATSUDA Kazuo 
Published: March 2005

The cell‐wall mutant of a hydrocarbon‐assimilating yeast, Candida sp. M‐7002 and its wild type have shown a significant difference in mannose content. Each mannan was isolated from the mutant and the wild‐type cells by fractionation with Cetavlon and copper reagent. Both mannans contain d‐mannose, d‐glucose and phosphate. The mutant mannan has a relatively high content of protein (18% in weight bases) whereas the wild‐type mannan has a low protein content (5.1%) with a high amount of carbohydrate (<90%)Structural analyses by enzymatic and chemical methods showed that both mannans had an mannosidic(α1‐6)‐linked back bone substituted at O‐2 by side chains fo varying length. The side chains of the mutant mannan were shown to consist of single mannose units and disaccharide units whose linkages were predomonstrated that the mannans also contain mannosyl oligosaccharides linked to protein through O‐glycosidic linkage.The chemical properties of the mannan of the Candida mutant indicates that the mutation might occur not only in the side chain structure but also in the (α1‐6)‐linked mannan back bone.
tRNA Thiolated Pyrimidines as Targets for Near‐Ultraviolet‐Induced Synthesis of Guanosine Tetraphosphate in Escherichia coli
Authors: THOMAS Gilles , THIAM Khoudia , FAVRE Alain 
Published: March 2005

Illumination with near‐ultraviolet light triggers synthesis of ppGpp(guanosine 3;‐diphosphate 5′‐disphosphate) not only in growingEscherichia coli cells containing the putative chromophore 4‐thiouridine in their tRNAs[Ramabhadran, T.V. and Jagger, J.(1976) Proc. Natl Acad.Sci USA, 73, 59‐69], but also in nuv− cells which lack 4‐ thiouridine.The burst of ppGpp in nuv− cells is, however, induced exclusively by light of wavelengths shorter than 350 nm. Its maximum level is half that obtained in the parental strain. This ppGpp synthesis is also under the control of the relA gene, indicating that it is due to the accumulation of uncharged tRNAs.A candidate likely to trigger this effect is a 5‐methylaminomethyl‐2‐thiouracil residues present in the fiest position of the anticodon loop of tRNA Glu, tRNALys and one tRNAgln isoacceptor.in conditions in vitro, this base is highly photoreactive at wavelengths shorter than 350nm. Furthermore, near‐ ultraviolet‐photomodified tRNAGlu and tRNALys become poor substrates of their acylation enzyme.
The Specificity of the Sodium Channel for Monovalent Cations.
Authors: FRELIN Christian , VIGNE Paul , LAZDUNSKI Michel 
Published: March 2005

The specificity of bthe Na+ channel for monovalent cations has been analyzed in three different ways using neutrotoxins and excitable cells in culture.

Influx experiments using radioactive monovalent cations have shown that all neurotoxins or combination of neurotoxins which change the gating system of the Na+ channel (veratridine, batrachotoxin, dihydrograyanotoxin, scorpion and sea‐anemone toxins) induce a considerable alteration of the permeability of the channel for monovalent cations. Theer is a much decreased ionic selectivity of the channel. Toxin‐induced changes in ionic selectivity are the same or very similar in neuroblastoma cells and in skeletal myotubes. Thefact that toxins which change the kinetics of the gating system also change channel selectivity indicates that the selectivity filter and the gating system may be coupled entities.
Efflux experiments using neuroblastoma cells preloaded with 22N+, 42K+ or 86Rb+ indicate that the toxin stimulated release of these monovalent cations through the Na+ channel is not seen in the presence of cholinium in the external medium. It is seen only if the external medium contains permeant monovalent cations. The ‘chemical’ activation of the Na+ channel by neurotoxins necessitates the saturation at the external face of the channel of sites specific for permeant monovalent cations. Dose‐response curves for the effect of increasing external concentrations of Na+, K+, Rb+ or guanidinium on 22Na+ efflux indicate positive cooperativity in the stimulating effect of these cations. The best apparent affinity is observed for guanidinium, then for Na+, K+ and Rb+.
Competition experiments between monovalent cations and a radiolabelled tritiated derivative of tetrodotoxin for binding to the tetrodotoxin receptor of the Na+ channel in neuroblastoma cells indicatae that monovalent cations bind cooperatively to external sites for Na+. The Hill coefficients and the dissociation constants for Na+ and guanidinium binding to the Na+ channel measured with this approach compare well to values found from 22Na+efflux experiments.
Endoplasmic Reticulum Nuclease
Authors: KOUIDOU Sofia , TRIANTOS Althanasios , KAVOUKOPOULOS Evangelos , TRAKATELLIS Antonios 
Published: March 2005

An endonuclease, which was originally identified for its RNA polymerase inhibitory activity was isolated from rat liver endoplasmic reticulum.The enzyme yields on gel chromatography four active fractions of different molecular weights (Mr 5.3 × 104,9 × 104,1.55 × 105 and Sepharcyl S‐200 fraction at V0) Each fraction contains polypeptide chains which give a single band on sodium dodecylsulphare electrophoresis (Mr5.4 × 104). This indicates that the enzyme is an oligomeric protein and each of its subunits exhibits the same or very similar molecular weights.Deoxyribonucleoside and ribonucleoside triphophates can bind to the endoplasmic reticulum nuclease, Binding is enhanced in the presence of divalent cations particularly Mg2.The enzyme exhibits mainly Rnase activity but can also degrade denatured DNA and DNA RNA hybrids which contain breaks in one of the two starands. Poly (A) and mainly poly(U) are most susceptible to its nucleolytic activity whereas poly(C) is completely resistant.
Domain Structure of Isocitrate Dehydrogenase from Azotobacter vinelandii
Authors: SLOBBE Wilma , VOORDOUW Gerrit 
Published: March 2005

Isocitrate dehydrogenase from Azotobacter Vinelandii, which consists of a single polypeptide chain of Mr 80,000, is completely unfolded in 3 M guanidine hydrochloride at pH 7.5 and 22 C as judge by hydrodynamic criteria and a study of tryptophan fluorescence.
Transfer of denatured isocitrate dehydrogease to native solution conditions (less than 0.1 M guanidine hydrochloride) leads to rapid reactivation (t1/2 ∼ 1 min at 22 C,pH 7.5). Practically complete reactivation (85‐95%) can be achieved under properly chosen conditions.
At high protein concentration(c > 0.5 ‐ 1.0 mg/ml) intermolecular aggregation competes with proper folding and reactivation. Renatured isocitrate dehydrogenase is indistinguishable from the original native enzyme provided that aggregated material is removed by centrifugation and gel filtration.
The denaturation by guanidine hydrochloride proceeds in two separable steps: N⇄D1⇄D2.The midpoint of the N⇄ D1 transition is at 0.3 M, that of the D1⇄ D2 transition at 1.2‐1.5 M guanidine hydrochloride. The sedimentation coefficient (s20,w) drops from 4.5 S for the native conformation N to 4.5 S for D1 and 2.3 S for D2 The relative values for the tryptophan fluorescence of D1 and D2 are 50% and 25% of that of N, respectively (γex = 295nm, γem = 330nm).
Protease digestion studies using thermolysin or trypsin or trypsin in 0.4 M guanidine hydrochloride suggest that isocitrate dehydrogenase from A vinelandii is composed of three domains which, according to their molecular masses, are designated as the 45‐kDa and the 19‐kDa, the 16‐kDa domains. The 45‐kDa and 16‐kDa domains are resistant to protease digestion in 0.4 M guanidine hydrochloride, while the 19‐kDa domain is rapidly digested under these conditions.
The(un)folding intermediate D1 thus represents a conformation comprising folded 45‐kDa and 16‐kDa domains and a largely unfolded 19‐kDa domain. In D2 all three domains have unfolded. The observation that intermolecular aggregation is favored by low concentrations of guanidine hydrochloride (0.3‐0.5 M) indicates that this process may depend on intermolecular interactions between the 45‐kDa and 16‐kDa domains.
Quasi‐Lipoxygenase Activity of Haemoglobin
Authors: KÜHN Hartmut , GÖTZE Regina , SCHEWE Tankred , RAPOPORT Samuel M. 
Published: March 2005

Haemoglobin catalyses at low concentrations (0.01–1 μM) a quasi‐lipoxygenase reaction with remarkably high substrate specificity. Formation of lipohydroperoxides was demonstrated only with free 9‐cis,12‐cis‐octadecadienoic acid (Linoleic acid), its glycerol monoester, or 9‐cis,12‐cis‐octadecadienol (Linoleoyl alcohol). All‐cis,‐9,12, 15‐octadecatrienoic acid (α‐linolenic acid), all‐cis,‐5,8,11,14‐eicosatetraenoic acid (arachidonic acid) and all‐cis,‐9,12, 15‐ eicosatetraenoic acid (dihomo‐ν‐linolenic acid), phospholipids and biological membranes were not attacked. Saturated and unsaturated free fatty acids other than linoleic acid were strong inhibitors of the haemoglobin‐catalyzed oxygenation of linoleate. The reaction was also inhibited by cyanide, carbon monoxide, phenolic antioxidants and the lipoxygenase inhibitor salicylhydroxamic acid, as well as by some anti‐inflammatory drugs (prednisolone, dexamethasone, acetylsalicylic acid); indomethacin did not inhibit. Protein inhibitors of liproxygenase reactions were found in lung homogenates of various species. The molecular activity of the haemoglobin‐catalyzed oxygenation of linoleate was comparable with those of true lipoxygenases and was strongly suppressed by denaturation (heating, urea, pronase treatment, acid splitting). The apparentS0.5, the activation energy and the pH‐optimum did not differ from those of most of the true lipoxygenases.The reaction product hydroperoxylinoleic acid but not hydrogen peroxide was an activator of the haemoglobin‐catalyzed oxygenation of linoleate. Hydroxylinoleic acid was a competitive inhibitor of the activation. At haemoglobin concentrations higher than 1 μM the quasi‐lipoxygenase activity was completely suppressed presumably owing to the formation of inhibitory concentrations of hydroxylinoleate via the lipohydroperoxidase [21] activity of haemoglobin.The quasi‐lipoxygenase reaction of haemoglobin exhibits a suicidal behaviour, caused by destruction of haem groups. The properties of the free haemin‐catalyzed reaction were similar to those of haemoglobin. Among other haemoproteins tested myoglobin and cytochrome P‐450LMshow comparable activities. The mechanism of the haemoglobin‐catalyzed oxygenation of linoleate seems to involve a change of valency of the haem iron.
A New Principle of Regulation of Enzymic Activity
Authors: TSCHESCHE Harald , MACARTNEY Henry W. 
Published: March 2005

Latent collagenase form human polymorphonuclear leukocytes was isolated and shown to be activatable by disulfides, e.g. cystine, oxidized glutathione and insulin. Activation proceeds via a disulfide‐thiol exchange mechanisms by which the active proteinase (Mr 65500–67000) is released from the inactive latent enzyme (Mr 91000–94000) which is a mixed disulfide of an inhibitor (Mr20000–25000) and the collagenase [Macartney and Tschesche (1980) FEBS Lett. 119, 327‐332].In a system in vitro the activation can be coupled to the glutathione cycle. Reduced glutathione and hydrogen peroxide in the presence of any of the enxymes glutathione peroxidase, NADH peroxidase, lactoperoxidase, horse radish peroxidase or human leukocyte myeloperoxidase activate the latent collagenase. Pre‐incubation of the peroxidase with the inhibitor sodium azide prevents the activation of the latent enzyme via hydrogen peroxide and reduced flutathione. The oxidizing equivalents can also be generated by employing the hydrogen‐peroxide‐generating system of glucose and glucose oxidase. The peroxidase‐catalysed activation by the glucose/glucose oxidase system can be inhibited by including either catalase or glutathione reductase in the activating system. NADH also inhibits the glucose‐coupled activation system, but this effect can be counteracted by including NADH peroxidase.The result indicate that the activation of the latent collagenase can be regulated by the glutathione cycle. The activation could be coupled in vitro to the glucose metabolism via the system glucose/glucose oxidase. Regulation in vitro might perhaps be coupled to the phagocytosis‐associated respiratory burst known to be linked to the hexose monophosphate shunt activity of the cell.In an experiment in vitro, with the concentrations of reduced and oxidized glutathione normally found in vivo, the glutathione cycle could be used either to activate or to inactivate the latent collagenase, depending on the addition of either hydrogen peroxide as oxidizing equivalents or NADPH or NADH as reducing equivalents. This provides the first example in which regulation of proteolyticactivity activity could be linked to the glutathione redox potential of the cell.
The Charge Heterogeneity of the Mitochondrial Acetyl‐CoA Acetyltransferase from Rat Liver
Authors: HUTH Walter 
Published: March 2005

The charge heterogeneity of the mitochondrial acetyl‐Coa acetyltransferase (EC 2.3.1.9), referred to transferases A and B, seems to be performed in vivo and can be demonstrated in vivo by a spontaneous transformation of transferase A into transferase B. these two enzymes, although remarkably similar in amino acid composition, show structural dissimilarities. This becomes evident from their tryptic maps, which are substantially different.Isoelectric focusing in the presence of urea confirms the charge heterogeneity of the mitochondrial acetyl‐CoA acetyltransferase by indicating different patterns of protein bands for transferases A and B. transferase B lacks the protein band with an isoelectric point of 7.2 which is normally shown with transferase A. However, this 7.2‐pI protein band can be demonstrated after a [3H]CoASH treatment of transferase B in the presence of urea by protein sustaining and by autoradiography. This change in the isoelectric focusing band pattern after interaction of the subunits with CoASH is interpreted as being the result of an apparently covalent secondary modification of the protein side chains.The CoASH‐modification may be the only molecular basis of the acetyl‐CoA acetyltransferase change heterogeneity, a view, which is further substantiated by the CoASH‐mediated transformation of transferase B into transferase A. An additional heterogeneity of the enzyme as caused by a limited proteolysis seems unlikely but cannot be definitely excluded.
Binding of Magnesium and Chloride Ions to Human Hemoglobin A
Authors: ACHILLES Wolfgang , CUMME Gerhard A. , WINNEFELD Klaus , FRUNDER Horst 
Published: March 2005

Ultrafiltration and direct determination of [Mg2+]by ion exchange were used to study the binding of Mg2+ and Cl− to isoionic human hemoglobinA value of 58.8+1.71/mol was determined for the volume of hydrated hemoglobin from ultrafiltration of hemoglobin solutions containing 0.15–0.8 M glucose.In solutions with 5.7 mmol hemoglobin,150mmol KCl and 0.5–3.5 mmol MgCl2/l total water,0.6 mol and 2 mol cl− were bound/mol oxygenated and deoxygenated hemoglobin respectively. A value of about 111/mol was determined for the association constant of Mg2= to hemoglobin monomer.Free Mg2+ concentrations were measured in hemoglobin solutions containing KCl, MgCl2, ATP and d‐ glycerate‐2.3‐bisphosphate at concentrations close to those of red cells. The experiments yielded 0.65 mmol/l free Mg2+ after oxygenation and 0.82 mmol/l after deoxygenation.The data indicate that only small changes of free Mg2+ levels in red cells are caused by physiological changes of pO2.
NADH Dehydrogenase and NADh Oxidation in Membrane Vesicles from Bacillus subtilis
Authors: BERGSMA Jack , STRIJKER Rein , ALKEMA Jet Y. E. , SEIJEN Hendrik G. , KONINGS Wil N. 
Published: March 2005

Crossed immunoelectrophoresis of membrane vesicles of Bacillus subtilis W23, solubilized in 5% Triton X‐100, against anti‐vesicle imunoglobulins resulted in at least 43 immunoprecipitates.With the zymogram staining techique the precipitation lines of the enzymes succinic dehydrogenase, malate dehydrogenase, ATPase, NADPH dehydrogenase and NADH dehydrogenase were identified.NADH‐dehydrogenase activity was found in three precipitation lines. Preparative polyacrylamide gel electrophoresis of NADH dehydrogenase, solubilized with 0.1% Triton X‐100, showed three to five major protein bands with NADH dehydrogenase activity.The sodium dodecyl sulphate/polyacrylamide gel electrophoresis patterns of these bands all contained a band of the catalytic subunit with an apparent Mr of 64000. Antibodies against this catalytic subunit were prepared. The presence of only one NADH dehydrogenase in the cytoplasmic membrane was indicated by the facts that solubilized (NADH dehydrogenase) immunoglobulins and that membranes solubilized with 5% Triton X‐100 showed the same three precipitation lines with NADH dehydrogenase activity when either anti‐(NADH dehydrogenase) or anti‐(membrane vesicle) immunoglobulins were used.This NADH dehydrogenase is located at the inner surface of the cytoplasmic membrane(like succinic dehydrogenase and ATPase) as was shown with immunoabsorption experiments.Membrane vesicles oxidize NADH, presented at the outer surface, at a high rate. The mechanism of NADh oxidation was studied.NADH is oxidized via the respiratory chain. Uptake of NADH by the membrane vesicles could not be demonstrated and ermoval of 74% of NADH dehydrogenase resulted only in a 24% decrease of NADH oxidase activity. Membrane vesicles from the menaquinone‐deficient strain B subtilis aroD do not oxidize externally added NADH but BADH oxidation can be restord by addition of the menaquinone analogue, menadione. These resuts suggest that externally added NADH donates electrons directly to menaquinone‐7 at the outer surface of the vesicle membrane and that NADH dehydrogenase is not involved in this oxidation process.
Biosynthesis of Pyruvate Kinase Isozymes in Rat Liver
Authors: WU Stephen Wing Nin , WONG Susanna Siu Chun , YEUNG Desmond Chak Yew 
Published: March 2005

The L and M1 isozymes ofpyruvate kinase were purified to homogeneity from rat liver and muscle respectively and their specific antibodies were employed to quantify the isozyme concentration in rat liver during development.
Total enzyme activity decreases towards birth and reaches a minimum on the 3rd postnatal day, but the activity increases dramatically after weaning.
Immunoprecipitation revealed that the M2 type predominates in the prenatal period but decreases sharply just before birth.
The L isozyme contribution is augmented upon weaning and is sustained until the rat is adult and a L/M ratio of 9:1 is maintained.
By means of incorporation studies with [3H]leucine followed by immunoprecipitation, the increase in L‐type activity when approaching term and after weaning is explained by a twofold increase in its rate of synthesis coupled with a concomitant reduction of the M2‐type synthesis.
Modification of the Phosphatidylcholine‐Transfer Protein from Bovine Liver with Butanedione and Phenyiglyoxal
Authors: AKEROYD Robert , LANGE Louis G. , WESTERMAN Jan , WIRTZ Karel W. A. 
Published: March 2005

Modification of arginine residues with 2,3‐butanedione and phenylglyoxal completely inhibits the transfer activity of the phosphatidylcholine transfer protein from bovine liver. Removal of borate and butanedione leads to a slow reactivation of the protein.
Both α‐dicarbonyl reagents modify three of the ten arginine residues present per protein molecule. The extent of modification is linearly related to the loss of activity.
Inactivation with butanedione is greatly diminished when the protein is bound to strongly negatively charged vesicles. Under these conditions a rapid modification of two arginine residues is observed. This suggests that the transfer protein contains one arginine residue essential for activity, probably as a binding site for the negatively charged phosphate group of the phosphatidylcholine molecule.
This study provides convincing evidence that arginine residues may play an essential role in phospholipid‐ protein interactions.
Improved Assay and Mechanism of the Reaction Catalyzed by the Chitin Synthase from Saccharomyces cerevisiae
Authors: FÄHNRICH Marianne , AHLERS Jan 
Published: March 2005

An improved filtration method is introduced to perform kinetic investigations on the chitin synthase reaction. This method is especially suited for the assay of a large number of samples necessary in enzyme kinetic studies.
From initial rate data the possibility could be excluded that the two‐substrate reactions occurs by a random or a ping‐pong mechanism.
Investigations of product inhibition exclude a rapid random mechanism but favour an ordered mechanism with UDP‐N‐acetylglucosamine as the first substrate.
This result was confirmed by isotope exchange studies.
Oligosaccharides in Lysosomal Enzymes
Authors: HASILIK Andrej , VON FIGURA Kurt 
Published: March 2005

The distribution of the different types of oligosaccharides in cathepsin D and in β‐hexosaminidase syn‐ thesized in cultured human fibroblasts was studied by using endo‐β‐N‐acetylglucosaminidase H as a probe for high‐mannose oligosaccharides. The enzymes were specifically labelled in the protein or the carbohydrate moiety. In both enzymes, resistant and cleavable oligosaccharides were found. The resistant oligosaccharides prevailed the secreted enzymes. Precursor molecules of cathepsin D contained two oligosaccharide side chains. Multiple forms of the precursor are synthesized with both, one or none of two oligosaccharides sensitive to the action of the endo‐β‐N‐acetylglucosaminidase H.In fibroblasts unable to phosphorylate lysosomal enzymes (mucolipidosis 11) the excessively secreted lysosomal enzymes contained predominantly oligosaccharides resistant to endo‐β‐N‐acetylglucosaminidase H.
Evidence for a Mg2+‐Induced Conformational Change at the ATP‐Binding Site of (Na++ K+)‐ATPase Demonstrated with a Photoreactive ATP‐Analogue
Authors: REMPETERS Gerold , SCHONER Wilhelm 
Published: March 2005

The 3′‐ribosyl ester of ATP with 2‐nitro‐4‐azidophenyl propionic acid has been prepared and its ability to act, as a photoaffinity label of (Na++ K+)‐ATPase has been tested.
In the dark 3′‐O‐[3‐(2‐nitro‐4‐azidophenyl)‐propionyl]adenosine triphosphate (N3‐ATP) is a substrate of (Na++ K+)‐ATPase and a competitive inhibitor of ATP hydrolysis.
Upon irradiation by ultraviolet light, N3‐ATP photolabels the high‐affinity ATP‐binding site and is covalently attached to the α‐subunit and an approximately 12000‐Mr component.
Photolabeling of the α‐subunit by N3‐ATP irreversibly inactivates (Na++ K+)‐ATPase.
Photoinactivation is strictly Mg2+‐dependent. Na+ enhances the inactivation. ATP or ADP and K+ protect the enzyme against inactivation.
Mg2+;, in concentrations required for photoinactivation, protects (Na++ K+)‐ATPase against inactivation by tryptic digestion under controlled conditions.
It is assumed that a conformational change of the ATP‐binding site of (Na++K+)‐ATPase occurs upon binding of Mg2+ to a low‐affinity site.