bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of neutral oligosaccharides derived from mucus glycoproteins of human seminal plasma
Authors: HANISCH Franz‐G. , EGGE Heinz , PETER‐KATALINIĆ Jasna , UHLENBRUCK Gerhard
Published: March 2005

The primary structures of nine major saccharide alditols in the fraction of neutral carbohydrates derived from human seminal plasma mucin have been established on the basis of fast atom bombardment and electron impact mass spectrometry combined with methylation analysis, exoglycosidase digestion, and CrO3 oxidation, as follows.
Labeling of individual amino acid residues in the membrane‐embedded F0 part of the F1 F0 ATP synthase from Neurospora crassa
Authors: HOPPE Jürgen , GATTI Domenico , WEBER Hans , SEBALD Walter
Published: March 2005

Three F0 subunits and the F1 subunit β of the ATP synthase from Neurospora crassa were labeled with the lipophilic photoactivatable reagent 3‐(trifluoromethyl)‐3‐(m‐[125I]iodophenyl)diazirine ([125I]TID). In the proteolipid subunit which was the most heavily labeled polypeptide labeling was confined to five residues at the NH2‐terminus and five residues at the C‐terminus of the protein. Labeling occurred at similar positions compared with the homologous protein (subunit c) in the ATP synthase from Escherichia coli, indicating a similar structure of the proteolipid subunits in their respective organisms.

The inhibitors oligomycin and dicyclohexylcarbodiimide did not change the pattern of accessible surface residues in the proteolipid, suggesting that neither inhibitor induces gross conformational changes. However, in the presence of oligomycin, the extent of labeling in some residues was reduced. Apparently, these residues provide part of the binding site for the inhibitor. After reaction with dicyclohexylcarbodiimide an additional labeled amino acid was found at position 65 corresponding to the invariant carbodiimide‐binding glutamic acid. These results and previous observations indicate that the carboxyl side chain of Glu‐65 is located at the protein‐lipid interphase. The idea is discussed that proton translocation occurs at the interphase between different types if F0 subunits. Dicyclohexylcarbodiimide or oligomycin might disturb this essential interaction between the F0 subunits.
Differential protein synthesis in the induction of thyroid cell proliferation by thyrotropin, epidermal growth factor or serum
Authors: LAMY Françoise , ROGER Pierre P. , LECOCQ Raymond , DUMONT Jacques E.
Published: March 2005

Protein synthesis in the G1 period of the cell cycle has been investigated using two‐dimensional gel electrophoresis in primary cultures of dog quiescent thyroid cells, incubated in defined medium and induced to proliferate by the combined action of thyrotropin (TSH), epidermal growth factor (EGF) and serum or by each of these agents, acting alone.

1. The analysis of the proteins, pulse‐labeled for 3 h with [35S]methionine, in quiescent cells deprived of serum and in cells that had been stimulated for various periods of time by the addition of TSH, EGF and serum showed maximal modifications before entry into S phase: the labeling of at least ten proteins was enhanced while that of at least six proteins was decreased. The synthesis of one of these proteins (protein 1; Mr∼ 81000) was maximal 9–12 h after stimulation by the proliferative agents but began to decrease at 15–18 h and was still decreased at 29–32 h.

2. The study of the effect of each of the proliferation agents alone on the labeling of these sixteen proteins showed that TSH specifically stimulated the labeling of eight polypeptides (proteins 2–9) and that, in contrast, EGF and serum specifically increased the labeling of two other proteins (proteins 1 and 10). The labeling of one protein was decreased by each of the different agents (protein 6′) while TSH specifically decreased the labeling of four polypeptides (proteins 1′– 4′) and increased the labeling of one polypeptide (protein 5′) whose synthesis was decreased by EGF and serum.

3. The specific effect of TSH on one protein labeling (protein 7; Mr∼ 39000) was potentiated by EGF and serum while the specific effect of EGF and serum on another protein labeling (protein 1) was potentiated by TSH. There is thus a correlation between the level of synthesis of these two proteins and the proliferative state of the cells, which is much greater when the stimulating agents are acting together.

4. The induction of protein 1 synthesis by EGF was no longer observed when the cells were no longer proliferating. In the same way, TSH no longer stimulated the synthesis of protein 7 in thyroid cells at confluence.

In conclusion, the present study has identified some proteins (proteins 1 and 7) which, as judged by the peculiar stimulation and the kinetics of their synthesis, could be part of the final key events triggering DNA replication in thyroid cells. Our findings are discussed within the framework of the previously described coexistence of cyclic‐AMP‐dependent (stimulated by TSH) and cyclic‐AMP‐independent (stimulated by serum or EGF) regulatory pathways leading to the initiation of DNA synthesis.
Differential effects of defined chemical modifications on antigenic and pharmacological activities of scorpion α and β toxins
Authors: AYEB Mohamed EL , DARBON Hervé , BAHRAOUI El Mostafa , VARGAS Orietta , ROCHAT Hervé
Published: March 2005

Specific chemical modifications of scorpion α and β toxins have been used to study the involvement of particular residues in both the pharmacological and the antigenic sites of these toxins. Modification by 1,2‐cyclohexanedione of arginine‐27 of a β toxin, Centruroides suffusus suffusus toxin II, drastically decrease the antigenic activity without any influence on the pharmacological activity. Conversely, modification by the same reagent of arginine‐2 of an α toxin, Androctonus australis Hector toxin III, led to a 100‐times less pharmacologically potent derivative and did not induce a significant loss of antigenic activity. Excision of the N‐terminal pentapeptide of another α toxin, Buthus occitanus mardochei toxin III, by pepsin digestion led to a non‐toxic derivative retaining full antigenic activity. Thus, the N‐terminal part of the conserved hydrophobic surface of the toxin is highly implicated in the pharmacological activity, whereas the region of arginine‐27, located in the α helix situated on the back surface, opposite the conserved hydrophobic region, is fully implicated in the antigenic activity and is far from the pharmacological site.

These results are good arguments in favor of the idea that in scorpion toxins the surfaces implicated in the pharmacological and the antigenic activities do not overlap. Since the antigenic sites are present in highly variable sequence the development of an efficient polyvalent serotherapy is questionable.
Temperature dependence of dihydropteridine reductase activity
Authors: RANDLES David
Published: March 2005

The kinetic parameters Km and kcat of dihydropteridine reductase with a mixture of 6R and 6S quinonoid 7,8(6H)‐dihydrobiopterin were determined at several temperatures in the range 20–37°C. Both Km and kcat increased with temperature. Thermodynamic activation parameters were calculated and compared with those for the non‐enzymic reduction of quinonoid 7,8(6H)‐dihydrobiopterin by NADH. The temperature coefficients of the enzyme catalysed and uncatalysed reactions are 3.3 and 1.67 respectively.

The results are consistent with an ordered bi‐bi enzyme mechanism, in which the rate‐determining step is an isomerisation of the ternary complex. This isomerisation involves a positive entropy of activation, which overcomes an enthalpy of activation that is significantly higher for the enzymic than for the non‐enzymic reaction.
Optimization of protein synthesis in isolated higher plant chloroplasts
Authors: MULLET John E. , KLEIN Robert R. , GROSSMAN Arthur R.
Published: March 2005

Protein synthesis in isolated, intact pea chloroplasts was optimized and compared to translation within chloroplasts in vivo. Many polypeptides labeled with [35S]methionine in isolated intact chloroplasts did not comigrate with polypeptides which were labeled within chloroplasts in vivo. Antibodies to the large subunit of ribulose‐1,5‐bisphosphate carboxylase‐oxygenase (EC 4.1.1.39) immunoprecipitated [35S]‐labeled large subunit plus several lower‐molecular‐mass translation products of isolated chloroplasts. The lower‐molecular‐mass soluble translation products synthesized in pulse‐labeled chloroplasts were converted into full‐length large‐subunit polypeptides during a subsequent chase period. This result suggests that many of the polypeptides observed in pulse‐labeled chloroplasts are incomplete translation products which are the result of ribosome pausing at discrete points along chloroplast mRNAs. The pulse‐chase technique was used to follow synthesis of the 34.5‐kDa precursor of the psb A gene product and its processing to the mature 32‐kDa polypeptide in isolated chloroplasts. Chloroplast translation profiles obtained using the pulse‐chase assay were very similar to translation profiles obtained in vivo thus extending the utility of protein synthesis in isolated chloroplasts.
Stimulation of the adenylate cyclase activity of rabbit bone marrow immature erythroblasts by erythropoietin and haemin
Authors: BONANOU‐TZEDAKI Sophia A. , SETCHENSKA Milka S. , ARNSTEIN Henry R. V.
Published: March 2005

The effect of two agents of erythroid cell differentiation on the adenylate cyclase activity of fractionated rabbit bone marrow erythroblasts has been investigated.

1. Addition of 0.2 U/ml erythropoietin to cell cultures causes a transient increase in the activity of plasma membrane adenylate cyclase, which is maximal by 20 min and disappears within 4 h. The magnitude of the response to hormonal stimulation depends on the stage of erythroid cell development and is greater in the more immature cells.

2. Addition of 50 μM haemin to cultures of erythroblasts also causes an increase in the activity of adenylate cyclase, which differs from the effect of erythropoietin in kinetics and specificity of target cells. With immature cells the haemin‐induced stimulation starts after the first hour and continues to increase up to 20 h of culture.

3. Erythropoietin but not haemin can stimulate the basal activity of adenylate cyclase in an in vitro assay containing plasma membranes of immature erythroid cells. The degree of activation depends on the concentration of erythropoietin and is maximal with 0.2–0.5 U/ml hormone (5–12 nM). In the presence of guanine nucleotides the activation of adenylate cyclase by erythropoietin is increased further but the effect is not additive. With respect to the basal and the guanine‐nucleotide‐stimulated activities of adenylate cyclase erythropoietin acts differently from the β‐agonist l‐isoprenaline. The in vitro effect of erythropoietin is abolished by the β‐thio analogue of GDP, GDP[βS], and extensive washing of membranes makes hormone action GTP‐dependent.

4. The stimulation of adenylate cyclase by the addition of erythropoietin to the reaction mixture is inversely related to the extent of previous hormonal stimulation of the cells from which the membranes were prepared. This loss of hormonal responsiveness is due to desensitization or receptor down‐regulation and persists for up to 20 h.

5. We conclude that in immature erythroblasts erythropoietin acts via a receptor and a guanine nucleotide‐binding protein with high affinity for GTP (EC50 < 10 nM), whereas haemin appears to activate adenylate cyclase indirectly, as a consequence of progressive perturbations of the plasma membrane.
Hypermodified nucleoside carboxyl group as a target site for specific tRNA modification
Authors: GÓRNICKI Piotr , JUDEK Miroslaw , WOLAŃSKI Andrzej , KRZYŻOIAK Wlodzimierz J.
Published: March 2005

The free carboxyl group of hypermodified nucleosides N6‐methyl‐N6‐(threoninocarbonyl)adenosine (mt6A3‐) and 3‐(3‐amino‐3‐carboxypropyl)uridine (acp3U20:1) in tRNAMetm (yellow lupine), and N6‐(threoninocarbonyl)adenosine (t6A37) in tRNAMeti (yellow lupine) can be converted quantitatively and under very mild conditions into the respective anilides in a reaction with aniline and a water‐soluble carbodiimide. The tRNA reactions proceed with rates very similar to that reported previously for t6 A nucleoside. Detailed analysis of the products of tRNA modification with [3H]aniline on tRNA (chromatography on BD‐DEAE‐cellulose), oligonucleotide (polyacrylamide gel electrophoresis) and nucleoside (HPLC on Aminex A6) levels clearly indicates that only the hypermodified nucleoside residues undergo the reaction. The site of modification is confirmed for mono‐modified (at mt6 A37) and bis‐modified (at mt6 A37 and acp3U20:1) tRNAMetm, and for mono‐modified (at t6 A37) tRNAMeti by sequence analysis using 5'end 32P‐labeled tRNAs. The modification procedure seems to be universally applicable for all hypermodified nucleosides bearing a free carboxyl group and for different amine reagents designed for the studies on tRNA function.
Formation and metabolism of leukotriene C4 in macrophages exposed to bacterial lipopolysaccharide
Authors: LÜDERITZ Thomas , SCHADE Ulrich , RIETSCHEL Ernst Th.
Published: March 2005

Lipopolysaccharide (10 μg/ml) was found to stimulate resident mouse peritoneal macrophages to produce leukotriene C4 (36 ± 1.3 ng/106 cells, SEM, n= 20) within 16 h. Spontaneous synthesis in control cultures without lipopolysaccharide was < 1.6 ng/106 cells. Leukotriene C4 was characterized by reversed‐phase high‐performance liquid chromatography, ultraviolet spectrometry and radioimmunoassay. When the macrophages, prelabeled with [3H]arachidonic acid, were treated with lipopolysaccharide radioactivity was incorporated into leukotriene C4. The amount produced varied with the method of macrophage preparation and incubation conditions and was dependent on the amount of lipopolysaccharide added (0.5–60 μg/ml), on cell counts and on the incubation time (4–16 h). The released leukotriene C4 was converted to a compound identified as a C6‐cysteinylleukotriene, indicating metabolism of the leukotriene by the macrophages. Parallel determinations of prostaglandins E2 and F2α by radioimmunoassay demonstrated that leukotriene C4 and prostaglandin E2 are formed by mouse peritoneal macrophages to a similar degree.
Binding of ligands to horse liver alcohol dehydrogenase lacking zinc ions at the active sites
Authors: KOVÁŇ Jan , MATYSKA Luděk , ZEPPEZAUER Michael , MARET Wolfgang
Published: March 2005

1. The zinc‐deficient enzyme binds the fluorescence probes for the enzyme substrate pocket (auramine O, 13‐ethylberberine, chlorprothixene and acridine orange) more tightly than the native enzyme, whereas 1‐anilinonaphthalene 8‐sulphonic acid is bound with comparable affinity.

2. The use of fluorescence probes as reporter ligands revealed that the formation of binary complexes between the zinc‐deficient enzyme and aldehydes is possible (as with the native enzyme) and confirmed an increased affinity of coenzymes to the modified enzyme.

3. The absence of catalytic zinc ions brings about a loss of the essential stabilization effect in simultaneous NADH and aldehyde binding to liver alcohol dehydrogenase.

4. 2,2′‐Bipyridine, which chelates the active‐site zinc ion in the native enzyme, is bound rather loosely to the same site as aldehydes, auramine O and ethylberberine in the case of the zinc‐depleted enzyme.

5. The stopped‐flow measurements showed that the pH dependence of auramine O and ethylberberine binding to native and zinc‐depleted enzyme is essentially similar. These data are compatible with the presence of ionizable groups in the surroundings of the bound probes. This group might be either His‐67, bound to the zinc ion, or the zinc‐liganding water molecule in the case of the native enzyme (pK close to 9), or the free His‐67 residue in the case of the zinc‐deficient enzyme (pK about 8).
Activity of NMN+, nicotinamide ribose and analogs in alcohol oxidation promoted by horse‐liver alcohol dehydrogenase
Authors: SICSIC Sames , DURAND Philippe , LANGRENÉ Sylvie , GOFFIC François LE
Published: March 2005

Kinetic constants of horse‐liver alcohol‐dehydrogenase‐mediated oxidation of alcohol by nicotinamide mononucleotide and nicotinamide ribose were determined and the role of different adenine moieties complementing the reaction mixtures was investigated. Five nicotinamide ribose analogs were synthesized and their activities as NAD+ inhibitors and as cofactors in this dehydrogenase‐mediated oxidation of alcohol were assayed. In the light of these results, structural requirements of the pyridine ribofuranosyl part of the NAD+ are discussed.
Free energy coupling between H+‐generating and H+‐consuming pumps
Authors: PETRONILLI Valeria , PIETROBON Daniela , ZORATTI Mario , AZZONE Giovanni Felice
Published: March 2005

The ΔGp/ΔH ratio has been measured in mitochondria close to state 4 in the presence of various uncoupler or K+/valinomycin concentrations in media containing either 1 mM or 50 mM Pi. Care has been taken to control the factors affecting ΔGp and ΔH which could lead to an artefactual increase of the ΔGp/ΔH ratio above the highest accepted value for the H+/ATP stoichiometry (n= 4, synthesis + transport). In particular, to avoid overestimation of ΔGp due to inactivation of the ATPases at low ΔH or to the presence of adenylate kinase, the static head state was approached from the side of net ATP synthesis and ΔGp was measured in a state close to static head but still maintaining a residual rate of aerobic phosphorylation. For each concentration of uncoupler or K+, the Pi concentration and/or the adenylate energy charge (EC) as a function of time have been measured as indicators of net ATP synthesis. Only the values of ΔGp measured during a decrease in Pi concentration and/or an increase in EC have been considered to be meaningful for calculations of ΔGp/ΔH ratios. Both uncouplers and K+ transport cause a marked depression of ΔH and a parallel depression of the rate of ATP synthesis. However the low rate of ATP synthesis taking place under conditions of low ΔH eventually results, especially at high Pi concentrations, in a relatively large ΔGP. The ΔGP/ΔH ratios obtained at the lower ΔH values exceed 4 and approach 6. Although slightly higher ΔGP/ΔH ratios are obtained with valinomycin‐treated than with uncoupler‐treated mitochondria, the pattern of the rise of the force ratio as ΔH decreases is similar in both cases. An increase of the ΔGP/ΔH ratio above 4, the maximal accepted H+/ATP stoichiometry is thermodynamically incompatible with the delocalized protonic coupling model.
Studies on polypeptide composition, hydrolytic activity and proton conduction of mitochondrial F0F1H+ATPase in regenerating rat liver
Authors: BUCKLE Malcolm , GUERRIERI Ferruccio , PAZIENZA Angela , PAPA Sergio
Published: March 2005

A study of the F0F1ATPase complex of mitochondria isolated from regenerating rat liver following partial (70%) hepatectomy is presented. As we have previously reported, ATPase activity in submitochondrial particles prepared from regenerating rat liver 24 h following partial hepatectomy was depressed by 75% with respect to controls (submitochondrial particles from sham‐operated animals). Polyacrylamide gel electrophoresis and immunodecoration using an antibody raised against isolated bovine heart F1sector of the F0F1ATPase indicated a substantial decrease in F1content in the mitochondrial membrane from regenerating rat liver. Proton conduction by the F0F1ATPase complex was studied by following the anaerobic relaxation of the transmembrane proton gradient (ΔμH+) generated by succinate‐driven respiration. In control rat‐liver submitochondrial particles containing the F0F1moiety of the ATPase complex, anaerobic relaxation of ΔμH+showed biphasic kinetics, whilst the same process in particles derived from regenerating rat liver exhibited monophasic kinetics and was significantly more rapid. Oligomycin andN,N‐dicyclohexyl carbodiimide [(cHxN)2C] inhibited proton conductance by the F1‐F0ATPase complex in submitochondrial particles from both control and regenerating rat liver. Binding of [14C](cHxN)2C and immunodecoration using an antibody raised against bovine heart oligomycin‐sensitivity‐conferring protein (OSCP) indicated no difference in the content of either the (cHxN)2C binding protein or OSCP between control and regenerating rat‐liver mitochondrial membranes.

The results reported show that the structural and functional integrity of the F0‐F1ATPase of rat liver is severely perturbed during regeneration.
The binding of porphyrin cytochrome c to yeast cytochrome c peroxidase
Authors: KORNBLATT Jack A. , ENGLISH Ann M.
Published: March 2005

1. Porphyrin c, the iron‐free derivative of cytochrome c, is a reasonably good model for cytochrome c binding to cytochrome c peroxidase (CcP). It binds with the same stoichiometry but only one‐quarter as tightly as cytochrome c.

2. CcP (resting, FeIII) and CcP · CN can both bind up to two molecules of porphyrin c. The binding of the first porphyrin c is tight (Kd= 1 × 10−9 M, pH 6, ionic strength μ= 0, 4°C) and results in quenching of the porphyrin c fluorescence. The binding is sensitive to ionic strength. The binding of the second porphyrin c is looser (Kd unknown) and does not result in quenching of the porphyrin fluorescence. The binding of porphyrin c to the cyano form and the resting forms of CcP cannot be distinguished by our methods.

3. ES is the first acceptor of electrons from c(II) and can bind at least two molecules of porphyrin c. The binding of the first porphyrin c is extremely tight, results in substantial quenching and is insensitive to ionic strength. The binding of porphyrin c to the loose site (Kd= 2 × 10−9 M, pH 6, 4°C, μ= 0) results, unlike the resting and cyano forms, in quenching of fluorescence of the second porphyrin c. The binding of the second porphyrin c to ES is sensitive to ionic strength.

4. The calculated distances between porphyrin c and the hemes of CcP(FeIII) and ES are approximately 2.5 nm.
Biosynthesis of the wall acidic polysaccharide in Bacillus cereus AHU 1356
Authors: KOJIMA Naoya , ARAKI Yoshio , ITO Eiji
Published: March 2005

Biosynthetic studies on an acidic polysaccharide, comprising galactose, rhamnose, N‐acetylglucosamine and sn‐glycerol 1‐phosphate, were carried out with a membrane system obtained from Bacillus cereus AHU 1356. Incubation of the membranes with UDP‐[14C]Gal, TDP‐[14C]Rha and UDP‐[14C]GlcNAc resulted in the formation of four or more labeled‐sugar‐linked lipids and a labeled polysaccharide. Data on structural analysis of the sugar moieties released from the glycolipids, together with results of enzymatic conversion of [14C]galactose‐linked lipid and [14C]Rha‐Gal‐linked lipid to higher‐oligosaccharide‐linked lipids and polysaccharide, led to the conclusion that the acidic polysaccharide is probably synthesized through the following pathway: Gal‐PP‐lipid→Rha‐Gal‐PP‐lipid→GlcNAc‐Rha‐Gal‐PP‐lipid→Rha‐GlcNAc‐Rha‐Gal‐PP‐lipid→glycerol‐P‐Rha‐GlcNAc‐Rha‐Gal‐PP‐lipid→[‐(glycerol‐P‐)Rha‐GlcNAc‐Rha‐Gal‐]n‐PP‐lipid→polysaccharide. Theglycero‐phosphate residues seem to be derived from phosphatidylglycerol.
Structural studies on the minor teichoic acid of Bacillus coagulans AHU 1631
Authors: KOJIMA Naoya , UCHIKAWA Ken‐ichi , ARAKI Yoshio , ITO Eiji
Published: March 2005

The minor teichoic acid linked to glycopeptide was isolated from lysozyme digests of Bacillus coagulans AHU 1631 cell walls, and the structure of the teichoic acid moiety and its junction with the peptidoglycan were studied. Hydrolysis of the teichoic‐acid–glycopeptide complex with hydrogen fluoride gave a nonreducing oligosaccharide composed of glucose, galactose and glycerol in a molar ratio of 3:1:1 which was presumed to be dephosphorylated repeating units of the polymer chain. From the results of structural analysis involving NaIO4 oxidation, methylation and acetolysis, the above fragment was characterized as glucosyl(β1 → 3)glucosyl(β1 → 6)galactosyl‐(β1 → 6)glucosyl(α1→1/3)glycerol. In addition, the Smith degradation of the complex yielded a phosphorus‐containing fragment identified as glycerol‐P‐6‐glucosyl(β1→1/3)glycerol. These results led to the most likely structure for the repeating units of the teichoic acid, ‐6[glucosyl(β1 → 3)]glucosyl(β1 → 6)galactosyl(β1 → 6)glucosyl(α1→1/3)glycerol‐P‐.

The minor teichoic acid, just like the major teichoic acid bound to the linkage unit, was released by heating the cell walls at pH 2.5. The mild alkaline hydrolysis of the minor teichoic acid after reduction with NaB3H4 gave labeled saccharides characterized as glucosyl(β1 → 6)galactitol and glucosyl(β1 → 3)glucosyl(β1 → 6)galactitol, together with a large amount of the unlabeled repeating units of the teichoic acid chain. Thus, the minor teichoic acid chain is believed to be directly linked to peptidoglycan at the galactose residue of the terminal repeating unit without a special linkage sugar unit.
Properties of catalase purified from a methanol‐grown yeast, Kloeckera sp. 2201
Authors: MOZAFFAR Sabiha , UEDA Mitsuyoshi , KITATSUJI Katsura , SHIMIZU Sakayu , OSUMI Masako , TANAKA Atsuo
Published: March 2005

Catalase, a marker enzyme of peroxisomes, was purified to homogeneity from whole cells of Kloeckera sp.2201 (a strain of Candida boidinii) grown on methanol by means of ammonium sulfate fractionation followed by hydroxyapatite, Sephacryl S‐300 and DEAE‐Sepharose column chromatographies. Crystallized catalase was brown‐coloured and needle‐like. The molecular mass of the enzyme was about 240000 daltons consisting of four identical subunits of 62000 daltons. The minimum size of catalase molecule was estimated to be about 6x10 nm from an electron micrograph. Judging from the absorption spectrum, the enzyme seemed to belong to a group of T‐type catalase. The Km value of the enzyme for hydrogen peroxide (catalatic activity) was 25 mM, while that for methanol (peroxidatic activity) was 83 mM. Catalase from Kloeckera sp. cells showed a certain degree of ‐similarity to the enzyme purified from alkane‐grown Candida tropicalis [T. Yamada et al. (1982) Eur. J. Biochem. 125, 517–521 and 129, 251–255] in its immunochemical properties.
The malate/aspartate shuttle and pyruvate kinase as targets involved in the stimulation of gluconeogenesis by phenylephrine
Authors: LEVERVE Xavier M. , VERHOEVEN Arthur J. , GROEN Albert K. , MEIJER Alfred J. , TAGER Joseph M.
Published: March 2005

The mechanisms responsible for the stimulation by phenylephrine of gluconeogenesis from dihydroxyacetone and glycerol were studied in perifused rat hepatocytes.

1. The stimulation by phenylephrine of glucose formation from dihydroxyacetone was biphasic. Transient stimulation of about 25% after 3 min was followed by a stable stimulation of about 15% 7 min later. Concomitantly there was a transient inhibition by phenylephrine of pyruvate kinase flux during the first few minutes followed by a more stable inhibition after about 10 min. The stable inhibition could quantitatively account for the stable stimulation of gluconeogenesis by the hormone when dihydroxyacetone was the gluconeogenic substrate. The effects of phenylephrine were independent of cAMP.

2. With the reduced gluconeogenic substrate, glycerol, a transient aminooxyacetate‐sensitive stimulation of gluconeogenesis occurs, followed by a stable stimulation of smaller magnitude [Leverve, X. M., Groen, A. K., Verhoeven, A. J. and Tager, J. M. (1985) FEBS Lett. 181, 43–46]. This transient phase of stimulation was abolished by addition of low concentrations of pyruvate, in accordance with our previous proposal that phenylephrine transiently accelerates the mitochondrial oxidation of cytosolic NADH via the malate/aspartate shuttle. The transient stimulation of gluconeogenesis from glycerol was accompanied by a transient increase in the cytosolic/mitochondrial aspartate concentration gradient.

3. Phenylephrine caused a transient stimulation of mitochondrial aspartate efflux in two other systems dependent on the operation of the malate/aspartate shuttle, i. e. gluconeogenesis from lactate and the oxidation of ethanol.

4. It is proposed that addition of phenylephrine to hepatocytes brings about a transient increase in the mitochondrial membrane potential and hence an increased rate of transport of aspartate from the mitochondria to the cytosol.
Molecular and functional properties of protein SS1 from small ribosomal subunits of Streptomyces aureofaciens
Authors: MIKULÍK Karel , ŠMARDOVÁ Jana , JIRÁŇOVÁ Anna , BRANNY Pavel
Published: March 2005

Small ribosomal subunits of gram‐positive cells of Streptomyces aureofaciens contain an acidic protein designated SS1. Purified protein SS1 has the same mobility in sodium dodecyl sulfate/polyacrylamide gel as ribosomal protein S1 of Escherichia coli (apparent Mr 68000). Protein SS1 was dissected under mild conditions with trypsin and generated fragments were compared with well‐characterized fragments of protein S1. The protein SS1 contains a structure homologous with the C‐terminal fragment of protein S1.

The affinity of protein SS1 to poly(U) is virtually identical with that of E. coli protein S1. In contrast to protein S1, the addition of SS1 to partially S1‐depleted ribosomes of E. coli had no stimulatory effect on poly(U)‐directed synthesis of polyphenylalanine. At molar excess of SS1 over ribosomes, the protein had comparable inhibitory effect on polypeptide synthesis as had S1 of E. coli.

Ribosomes of S. aureofaciens required about one order of magnitude higher concentration of poly(U) for maximum synthetic activity than did ribosomes of E. coli. The addition of proteins SS1 or S1 to ribosomes of S. aureofaciens had no stimulatory effect on translation of poly(U). Our data indicate that the high‐molecular‐mass acidic protein SS1 of small ribosomal subunits of S. aureofaciens exhibits only a part of the functional properties of E. coli protein S1.
Soluble fibrin consists of fibrin oligomers of heterogeneous distribution
Authors: RÖTKER Jürgen , PREISSNER Klaus T. , MÜLLER‐BERGHAUS Gert
Published: March 2005

Soluble fibrin is observed in patients with intravascular coagulation and represents an intermediary product of conversion of fibrin monomers into a fibrin clot whereby the presence of fibrinogen may suppress fibrin clot formation. The interactions between fibrin and fibrinogen and the occurrence of fibrin oligomers in soluble fibrin were studied by sucrose density ultracentrifugation. Different concentrations of soluble fibrin, prepared by mixing 125I‐fibrin (24 nM – 1.5 μM) with a constant concentration of 131I‐fibrinogen (6 μM) were analyzed at 37°C in stable linear sucrose density gradients containing a uniform concentration of unlabelled fibrinogen (6 μM) and calcium ions in order to mimic the physiological situation. At any fibrin concentration, 125I‐fibrin sedimented faster than 131I‐fibrinogen through 5 – 30% (w/v) sucrose gradients. Sedimentation rates of fibrin increased from 9 S to 23 S depending on the initial fibrin concentration. The relative amount of residual fibrin monomer not incorporated into oligomers was calculated from the sedimentation profiles. At any fibrin concentration, the portion of free monomer was always more than twofold higher for batroxobin‐generated (desAA‐) fibrin than for thrombin‐generated (desAABB‐) fibrin. Apparent association constants for desAABB‐fibrin were 3–10 times higher than those for desAA‐fibrin indicating a stronger interaction between monomers of the former type of fibrin. In the presence of excess fibrinogen the predominant species in soluble desAA‐fibrin were monomers and dimers, whereas dimers, trimers and higher‐molecular‐mass oligomers were present in soluble desAABB‐fibrin. Strong interactions between both types of fibrin were demonstrated from their cosedimentation, whereby the size of these copolymers was shown to be governed by the oligomer size of the desAABB‐fibrin type. These results provide evidence for the occurrence of differently sized oligomers of fibrin in soluble fibrin and for the concept of a cooperative polymerization process between both types of fibrin devoid of any stable complexes between fibrin and fibrinogen.
Purified pyruvate kinases type M2 from unfertilized hen's egg are substrates of protein kinase C
Authors: NODA Shigeru , HORN Friedemann , LINDER Dietmar , SCHONER Wilhelm
Published: March 2005

To characterize pyruvate kinase isoenzymes from cells with the capability to proliferate, this enzyme was purified from yolk and vitelline membrane of unfertilized hen's egg.

Pyruvate kinase type M2 from vitelline membrane was obtained in a homogeneous form after a 1150‐fold purification to a specific enzymatic activity of 450 μmol · min−1· mg−1. It was saturated half‐maximally with phosphoenolpyruvate at KPPrv0.5= 0.36 mM phosphoenolpyruvate and was activated by fructose 1,6‐bisphosphate and L‐serine at suboptimal substrate concentrations. After 11000‐fold purification to a specific enzymatic activity of 60 μmol min−1· mg−1, the pyruvate kinase isoenzymes type M2 (KPPrv0.5= 0.32 mM) and M1 (KPPrv0.5= 0.04 mM) were obtained from the yolk substance. Kinetic differences were noted between the pyruvate kinase type‐M2 isoenzymes from vitelline membrane and yolk.

A comparison of the amino acid composition of the purified pyruvate kinase isoenzymes from hen's egg revealed that all isoenzymes were related to pyruvate kinase type M1 from chicken breast muscle. The M2‐type isoenzyme from vitelline membrane was related to the M2‐type isoenzyme from chicken tumors, but was not related to the M2‐type pyruvate kinase from chicken lung or liver.

Protein kinase C from chicken oviduct phosphorylated in vitro both pyruvate kinase M2 isoenzymes from the unfertilized hen's egg preferably at serine and less at threonine residues. Pyruvate kinase type M1 from egg yolk was a weak substrate of protein kinase C. An activation of pyruvate kinase type M2 from vitelline membrane was observed at suboptimal concentrations of phosphoenolpyruvate under the conditions of phosphorylation, in the presence of phosphatidylserine.
Somatic antigens of Pseudomonas aeruginosa
Authors: KNIREL Yuriy A. , VINOGRADOV Evgeny V. , SHASHKOV Alexander S. , WILKINSON Stephen G. , TAHARA Yasutaka , DMITRIEV Boris A. , KOCHETKOV Nikolay K. , STANISLAVSKY Evgeny S. , MASHILOVA Galina M.
Published: March 2005

Lipopolysaccharides from Pseudomonas aeruginosa O1 (Lányi classification), O3 (Habs classification), O13 and O14 (Wokatsch classification), and strain NCTC 8505, which is also related to serogroup O3 (Habs), have structurally similar O‐specific polysaccharide chains built up of tetrasaccharide repeating units involving L‐rhamnose (Rha), 2‐acetamido‐2‐deoxy‐D‐glucose (GlcNAc), 2‐acetamido‐2‐deoxy‐L‐galacturonic acid (GalNAcA), and a di‐N‐acyl derivative of bacillosamine (BacN): 2,4‐diacetamido‐2,4,6‐trideoxy‐D‐glucose or 2‐acetamido‐2,4,6‐trideoxy‐4‐[(S)‐3‐hydroxybutyramido]‐D‐glucose. The latter derivative was obtained free by solvolysis with hydrogen fluoride of carboxyl‐reduced Habs O3 polysaccharide, and was identified by 1H‐nuclear magnetic resonance spectroscopy and by mass spectrometry of the corresponding methylated alditol. Habs O3, Lányi O1, and Wokatsch O14 polysaccharides contained O‐acetyl groups.

Solvolysis with hydrogen fluoride of the native Habs O3 polysaccharide resulted in selective cleavage of the glycosidic linkages of 6‐deoxy sugars to give the trisaccharide fragment involving all three N‐acylated amino sugars. Similar solvolysis of NCTC 8505 polysaccharide afforded a mixture of disaccharide and trisaccharide with N,N′ ‐diacetylbacillosamine at the reducing end. Smith degradation of Habs O3 polysaccharide resulted in selective oxidation of rhamnose to give a glycoside of a trisaccharide with glyceraldehyde as the aglycone. Smith degradation of NCTC 8505 polysaccharide was complicated by the formation of the glycoside of a trisaccharide with an aglycone of unknown structure. A trisaccharide with rhamnose at the reducing end was also isolated after Smith degradation of the latter polysaccharide.

Analysis of the composition and structure of all oligosaccharides obtained, and detailed examination of the 13C‐nuclear magnetic resonance spectra of these oligosaccharides, and of both intact and modified polysaccharides, revealed the following structures of the repeating units. The structure for the NCTC 8505 polysaccharide differs from that proposed previously [Tahara, Y. and Wilkinson, S. G. (1983) Eur. J. Biochem. 134, 299–304] in the configurations assigned to the glycosidic linkages of rhamnose and bacillosamine. The results obtained show the P. aeruginosa strains studied to represent three different O‐serotypes in a single O‐serogroup.
Mucin synthesis
Authors: BROCKHAUSEN Inka , MATTA Khushi L. , ORR Jeanne , SCHACHTER Harry , KOENDERMAN Anky H. L. , van den EIJNDEN Dirk H.
Published: March 2005

A UDP‐GlcNAc:R1‐β1‐3Gal(NAc)‐R2 [GlcNAc to Gal(NAc)] β6‐N‐acetylglucosaminyltransferase activity from pig gastric mucosa microsomes catalyzes the formation of GlcNAcβ1‐3(GlcNAcβ1‐6)Gal‐R from GlcNAcβ1‐3Gal‐R where ‐R is ‐β1‐3GalNAc‐α‐benzyl or ‐β1‐3(GlcNAcβ1‐6)GalNAc‐α‐benzyl. This enzyme is therefore involved in the synthesis of the I antigenic determinant in mucin‐type oligosaccharides. The enzyme also converts Galβ1‐3Galβ1‐4Glc to Galβ1‐3(GlcNAcβ1‐6)Galβ1‐4Glc. The enzyme was stimulated by Triton X‐100 at concentrations between 0 and 0.2% and was inhibited by Triton X‐100 at 0.5%. There is no requirement for Mn2+ and the enzyme activity is reduced to 65% in the presence of 10 mM EDTA. Enzyme products were purified and identified by proton NMR, methylation analysis and β‐galactosidase digestion. Competition studies suggest that this pig gastric mucosal β6‐GlcNAc‐transferase activity is due to the same enzyme that converts Galβ1‐3GalNAc‐R to mucin core 2, Galβ1‐3(GlcNAcβ1‐6)GalNAc‐R, and GlcNAcβ1‐3GalNAc‐R to mucin core 4, GlcNAcβ1‐3(GlcNAcβ1‐6)GalNAc‐R. Substrate specificity studies indicate that the enzyme attaches GlcNAc to either Gal or GalNAc in β(1‐6) linkage, provided these residues are substituted in β(1‐3) linkage by either GlcNAc or Gal. The insertion of a GlcNAcβ1‐3 residue into Galβ1‐3GalNAc‐R to form GlcNAcβ1‐3Galβ1‐3GalNAc‐R prevents insertion of GlcNAc into GalNAc. These studies establish several novel pathways in mucin‐type oligosaccharide biosynthesis.
Site‐directed alkylation and site‐site interactions in bovine seminal ribonuclease
Authors: DONADIO Stefano , TAMBURRINI Maurizio , DI DONATO Alberto , PICCOLI Renata , D'ALESSIO Giuseppe
Published: March 2005

Active‐site histidine residues of bovine seminal RNase have been found to react with bromoacetic acid and with 2′(3′)‐O‐bromoacetyluridine (BrAcUrd) at a much faster rate than free histidine. The former reagent reacts preferentially at the pros‐N of His119 the latter is specific for the tele‐N of His12. Alkylation with bromoacetic acid is mutually exclusive for either His119 or His12 and takes place predominantly at His119, while with BrAcUrd alkylation was found to be selective for His12. These results are very similar to those obtained with the same reagents on RNase A, confirming that seminal and pancreatic ribonucleases have similar geometries at their active sites. On the other hand, the kinetics of reaction of bromoacetyluridine with seminal RNase reveal a ‘half‐of‐the sites’ reactivity of the enzyme for this reagent, which is found to discriminate between the two structurally identical active sites of the dimeric enzyme.
Prostaglandin‐E2 9‐ketoreductase from human uterine decidua vera
Authors: KRÙER Susanne , SCHLEGEL Werner
Published: March 2005

Prostaglandin‐E2 9‐ketoreductase, the enzyme which catalyzes the reaction from prostaglandin F2α(PGF2α to prostaglandin F2α (PGF2α), has been purified 232‐fold from human uterine decidua vera.

The molecular mass of the enzyme, as estimated by fast protein liquid chromatography, was 29 kDa. Sodium dodecyl sulfate disc gel electrophoresis of the denatured enzyme revealed a molecular mass of 31 kDa. These data suggest that the enzyme consists of a single polypeptide chain.

The rate equation of the enzyme reaction for two substrates was used for the determination of five kinetic constants. The equilibrium constant with respect to PGE2 was 83 μM, the Michaelis constant, Km, for PGE2 was 93 μM. For NADPH, the equilibrium constant was 1.0 μM and Km was 1.6 μM. The maximal velocity for the forward reaction was V1= 217 pmol/min. The inhibition constants for the analgesic agents indomethacin and fentiazac were Ki= 850 μM and Ki= 450 μM and for the steroid progesterone Ki= 1.5 mM, respectively.

Prostaglandin‐E2 9‐ketoreductase might be responsible for the control of the PGE2/PGF2α ratio in human decidua vera. The enzyme, therefore, might be an important factor in the cascade of events leading to uterine contractions and parturition.
Plasma membrane proton ATPase from human kidney
Authors: SALLMAN Alan L. , LUBANSKY Harry J. , TALOR Zvi , ARRUDA Jose A. L.
Published: March 2005

Distal urinary acidification is thought to be mediated by a proton ATPase (H+‐ATPase). We isolated a plasma membrane fraction from human kidney cortex and medulla which contained H+‐ATPase activity. In both the cortex and medulla the plasma membrane fraction was enriched in alkaline phosphatase, maltase, Na+,K+‐ATPase and devoid of mitochondrial and lysosomal contamination. In the presence of oligomycin (to inhibit mitochondrial ATPase) in the presence of ouabain (to inhibit Na+,K+‐ATPase) and in the absence of Ca (to inhibit Ca2+‐ATPase) this plasma membrane fraction showed ATPase activity which was sensitive to dicyclohexylcarbodiimide and N‐ethylmaleimide. This ATPase activity was also inhibited by vanadate, 4,4′‐diisothiocyano‐2,2′‐disulfonic stilbene and ZnSO4. In the presence of ATP, but not GTP or UTP, the plasma membrane fraction of both cortex and medulla was capable of quenching of acridine orange fluorescence, which could be dissipated by nigericin indicating acidification of the interior of the vesicles. The acidification was not affected by presence of oligomycin or ouabain indicating that it was not due to mitochondrial ATPase or Na+,K+‐ATPase, respectively. Dicyclohexylcarbodiimide and N‐ethylmaleimide completely abolished the acidification by this plasma membrane fraction. In the presence of valinomycin and an outward‐directed K gradient, there was increased quenching of acridine orange, indicating that the H+‐ATPase is electrogenic. Acidification was not altered by replacement of Na by K, but was critically dependent on the presence of chloride. In summary, the plasma membrane fraction of the human kidney cortex and medulla contains a H+‐ATPase, which is similar to the H+‐ATPase described in other species, and we postulate that this H+‐ATPase may be involved in urinary acidification.
Characterization of the phosphorylation of rat mammary ATP‐citrate lyase and acetyl‐CoA carboxylase by Ca2+ and calmodulin‐dependent multiprotein kinase and Ca2+ and phospholipid‐dependent protein kinase
Authors: HARDIE D. Grahame , CARLING David , FERRARI Stefano , GUY Paul S. , AITKEN Alistair
Published: March 2005

ATP‐citrate lyase and acetyl‐CoA carboxylase purified from lactating rat mammary gland are phosphorylated stoichiometrically by the calmodulin‐dependent multiprotein kinase from rabbit skeletal muscle. The reactions are completely dependent on the presence of both Ca2+ and calmodulin.

ATP‐citrate lyase and acetyl‐CoA carboxylase are also phosphorylated stoichiometrically by the Ca2+‐and phospholipid‐dependent protein kinase (protein kinase C) purified from bovine brain. Phosphorylation of these substrates is stimulated 6‐fold and 40‐fold respectively by Ca2+ and phosphatidylserine.

The calmodulin‐dependent and phospholipid‐dependent protein kinases phosphorylate the same serine residue on ATP‐citrate lyase that is phosphorylated by cyclic‐AMP‐dependent protein kinase. The sequence of the tryptic peptide containing this site on the mammary enzyme is identical with the sequence of the peptide containing the site on ATP‐citrate lyase that is phosphorylated in isolated hepatocytes in response to insulin and/or glucagon.

The calmodulin‐dependent, phospholipid‐dependent and cyclic‐AMP‐dependent protein kinases phosphorylate distinct sites on acetyl‐CoA carboxylase. However, one of the three phosphorylated tryptic peptides derived from enzyme treated with the phospholipid‐dependent kinase is identical with the major phosphopeptide (T1) derived from enzyme treated with cyclic‐AMP‐dependent protein kinase.

Phosphorylation of acetyl‐CoA carboxylase by the phospholipid‐dependent protein kinase inactivates acetyl‐CoA carboxylase in a similar manner to cyclic‐AMP‐dependent protein kinase. With either protein kinase slightly greater phosphorylation and inactivation is seen after pretreatment of acetyl‐CoA carboxylase with protein phosphatase‐2A, but the effects of the protein phosphatase treatment are not completely reversed. Inactivation by the phospholipid‐dependent protein kinase is Ca2+‐and phospholipid‐dependent, is reversed by protein phosphatase‐2A, and correlates with the degree of phosphorylation.

The relevance of these findings to insulin‐ and growth‐factor‐promoted phosphorylation of ATP‐citrate lyase and acetyl‐CoA carboxylase in intact cells is discussed.
Purification and characterization of Bacillus coagulans oligo‐1,6‐glucosidase
Authors: SUZUKI Yuzuru , TOMURA Yoshihiro
Published: March 2005

A p‐nitrophenyl‐α‐D‐glucopyranoside‐hydrolyzing oligo‐1,6‐glucosidase of Bacillus coagulans ATCC 7050 (facultative thermophile) was purified to homogeneity. The relative molecular mass, Stokes radius, sedimentation coefficient at 20°C in water, molecular absorption coefficient at 280 nm and pH 6.8, and isoelectric point were estimated as 60 000, 3.29 nm, 4.8 × 10−13 s, 1.34 × 105 M−1, and 4.3, respectively. The amino‐terminal amino acid was threonine. There was no common antigenic group between the enzyme and each of its homologous counterparts from Bacillus cereus ATCC 7064 (mesophile) and Bacillus thermoglucosidasius KP 1006 (obligate thermophile). These oligo‐1,6‐glucosidases strongly resembled one another in their amino acid composition, except that the proline content increased with the elevation of thermostability in the order, mesophile → facultative thermophile → obligate thermophile enzymes.