bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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.
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.
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.
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.
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.
Orthophosphate – pyrophosphate exchange catalyzed by soluble and membrane‐bound inorganic pyrophosphatases
Authors: DE MEIS Leopoldo , BEHRENS Maria Isabel , CELIS Heliodoro , ROMERO Irma , GÓMEZ PUYOU Marietta Tuena , PUYOU Armando GÓMEZ 
Published: March 2005

A comparative study of the orthophosphate—pyrophosphate exchange reaction catalyzed by the soluble pyrophosphatase from baker's yeast and by the membrane‐bound pyrophosphatase of Rhodospirillum rubrum chromatophores was performed.In both systems the rate of exchange increased when the pH of the medium was raised from 6.0 to 7.8 and when the MgCl2 concentration was raised from 0.1 mM to 20 mM. For the yeast pyrophosphatase the exchange rates measured at different pH values and in the presence of 6.7 to 8.8 mM free Mg2+ superimposed as a single curve when plotted as a function of the concentrations of either HPO42− or MgHPO4. This was not observed with the use of R. rubrum chromatophores. With yeast pyrophosphatase, the Km for Pi was higher than 10 mM and could not be measured when the free Mg2+ concentration in the medium was lower than 0.5 mM. There was a decrease in the Km for Pi when the free Mg2+ concentration was raised to 6.7–8.8 mM or when, in the presence of low free Mg2+, the organic solvents dimethylsulfoxide (20% v/v) or ethyleneglycol (40% v/v) were inclded in the assay medium. In the presence of 6.7–8.8 mM free Mg2+ the Km for total Pi was 7 mM at pH 7.0 and 12 mM at pH 7.8. For the ionic species HPO42− and MgHPO4, the Km values were 5.8 mM and 4.2 mM respectively. In the presence of 0.24–0.42 mM free Mg2+ and either 20% (v/v) dimethylsulfoxide or 40% (v/v) ethyleneglycol the Km values for total, Pi, HPO42− and MgHPO4 were 7.6, 3.5 and 0.5 mM respectively. With R. rubrum chromatophores, the Km for Pi in the presence of 5.5–7.5 mM free Mg2+ was very high and could not be measured.In the presence of 0.24–0.45 mM free Mg2+ the ratio between the velocities of hydrolysis and synthesis of pyrophosphate mesured at pH 7.8 with yeast pyrophosphatase and chromatophores of R. rubrum were practically the same. When the free Mg2+ concentration was raised to 5.5–8.8 mM this ratio decreased from 1028 to 540 when the yeast pyrophosphatase was used and from 754 to 46 when chromatophores were used.
Monoclonal antibodies inhibitory to human plasmin
Authors: SIM Pheng‐Siew , FAYLE David R. H. , DOE William F. , STEPHENS Ross W. 
Published: March 2005

Four monoclonal antibodies raised against purified human plasminogen were characterized for their effects on the activation of plasminogen and on three enzymic properties of plasmin: (a) thioesterolysis, (b) fibrinolysis, (c) conversion of high‐Mr urokinase to its low‐Mr form.None of the monoclonal antibodies inhibited plasminogen (plg) activation by urokinase. The monoclonal antibodies characterized in this study fell into three groups. Anti‐plg 1 inhibited (a), (b) and (c), while anti‐plg 2 inhibited activities (a), (b) and (c) to varying degrees but also formed complexes with plasmin that were stable to sodium dodecyl sulphate. Anti‐plg 3 and anti‐plg 4 inhibited only activity (c).Selective use of these monoclonal antibodies demonstrated unequivocally that plasmin mediates the activation of the proenzyme form of urokinase‐type plasminogen activator. Besides their use in affinity chromatography, therefore, these antibodies are valuable for defining the role of plasmin in the mechanisms of extracellular matrix degradation.
Proteolytically induced variations in the enzymatic properties of tissue plasminogen activator
Authors: NORRMAN Bo , POHL Gunnar , JÖRNVALL Hans , WALLÉN Per 
Published: March 2005

Tissue plasminogen activator was treated with Sepharose‐bound trypsin or chymotrypsin. Trypsin rapidly converted the one‐chain activator to the two‐chain form. This caused a marked increase in the amidolytic activity, while plasminogen activation initially increased but then decreased again. SDS/polyacrylamide gel electrophoresis in combination with [3H]diisopropylfluorophosphate active‐site labeling revealed that after the conversion to the two‐chain activator a minor cleavage occurred in the B chain, while the A chain was substantially degraded.Chymotrypsin caused a marked decrease in both amidolytic activity and plasminogen activation. SDS/polyacrylamide gel electrophoresis under reducing conditions revealed that two pairs of new bands had appeared, with Mr or about 50000/52000 and 17000/20000 respectively. N‐terminal sequence analysis identified cleavage sites at peptide bonds 420–421 and 423–424. These bonds are located in a region of the activator which is homologues to the segments of trypsin and chymotrypsin, where autocatalytic cleavages occur during their activations.However, treatment of two‐chain activator with chymotrypsin had markedly less effect on plasminogen activation and amidolytic activity. By treatment of samples of chymotrypsin‐digested one‐chain activator with plasmin, amidolytic activity could be largely restored. Thus, chymotrypsin may, by cleaving bonds 420–421 and 423–424, convert the active one‐chain activator into an ‘inactive’ zymogen, which is again ‘activated’ by plasmin cleavage.
The phosphoenolpyruvate‐dependent fructose‐specific phosphotransferase system in Rhodopseudomonas sphaeroides
Authors: LOLKEMA Juke S. , ROBILLARD George T. 
Published: March 2005

Redox titrations of the fructose‐specific carrier protein, EFruII, in Rhodopseudomonas sphaeroides show that only the reduced form of the enzyme is active. The oxidized form of the enzyme can still be phosphorylated but is unable to transfer the phosphoryl group to fructose. The redox properties of the enzyme change upon phosphorylation. The reduction rate of EFruII is slower than that of EFruII‐P, whereas the opposite is true for the oxidation rate. Consequently the midpoint potential of the redox centre is more negative on EFruII than EFruII‐P, most likely due to an upwards pK shift of the thiols upon phosphorylation. The measurements indicate that the phosphotransferase system is regulated by the redox potential in a way that is dependent on the substrate concentrations. We propose that the change in the midpoint potential during turnover could be a mechanism for an electron transport function of the carrier.The binding of Zn2+ protects the carrier dithiol against oxidation but the presence of Zn2+ does not stimulate the reduction of the oxidized carrier.
Membrane‐bound hydroxylases in elicitor‐treated bean cells
Authors: BOLWELL G. Paul , DIXON Richard A. 
Published: March 2005

1. Treatment of cell‐suspension cultures of bean (Phaseolus vulgaris cv. Canadian Wonder) with an elicitor preparation heat‐released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum resulted in rapid changes in the activities of two microsomal oxygenases, cinnamic acid 4‐hydroxylase, involved in accumulation of wall‐bound phenolics and phytoalexins, and proline 2‐oxoglutarate dioxygenase (prolylhydroxylase) involved in the post‐translational modification of hydroxyproline‐rich glycoproteins.2. An anti‐(cytochrome P‐450) monoclonal antibody, originally raised against rat cytochrome P‐450 isoform c, has been shown to bind to bean microsomes and recognise in Western blots an Mr‐48 000 polypeptide, which comigrates with a haeme‐containing protein on SDS/polyacrylamide gel analysis and which has been tentatively identified as a cytochrome P‐450 capable of the hydroxylation of cinnamic acid.3. A preparation of proline 2‐oxoglutarate dioxygenase purified to homogeneity was used to immunise rabbits for the production of antiserum. An elicitor‐induced polypeptide of Mr 65000 was identified as prolyl hydroxylase while an antigenically related polypeptide of Mr 60000 was also immunoprecipitated but not induced by elicitor treatment.4. Use of the two antibodies has demonstrated rapid transient increases in the synthesis of the Mr 48000 and Mr 65000 oxygenases in vivo and for mRNAs as measured in in vitro translations, particularly for the putative cytochrome P‐450. These increases slightly precede corresponding changes in the synthesis of the soluble enzyme phenylalanine ammonia‐lyase, in common with which these oxygenases probably share a mechanism of gene activation underlying the increased activities seen in response to elicitor treatment.