bluuebunny/crossref_metadata_2025 huggingface dataset:
Fully Active Fe‐Protein of the Nitrogenase from Azotobacter vinelandii Contains at least Eight Iron Atoms and Eight Sulphide Atoms per Molecule
Authors: BRAAKSMA Arnold , HAAKER Huub , VEEGER Cees
Published: March 2005

The Fe‐protein of the Azotobacter vinelandii nitrogenase enzyme complex contains a variable iron and sulphide content. The iron and sulphide content of the protein is dependent upon the specific activity. Up to a specific activity of 1000 nmol C2H4 produced · min−1· mg Av2−1, three iron and three sulphide atoms per molecule Av2 are found. At specific activities above 1000 nmol C2H4 produced · min−1· mg Av2−1, a linear relationship between specific activity and iron and sulphide content of Av2 is found. The maximum values found are 8.8 iron atoms and 8.6 sulphide atoms/molecule at a specific activity of 2250 nmol C2H4 produced · min−1· mg Av2−1. Also the experimental molar absorption coefficients at 430 nm of the oxidized and reduced forms depend on the specific activity. The highest values found are 15.9 mM−1 cm−1 and 9.1 mM−1 cm−1, respectively.

Since occasionally the preparations with specific activities around 3000 nmol · min−1· mg−1 are isolated which contain more than 10 iron atoms and 11 sulphide atoms per molecule, it cannot be excluded that under certain physiological conditions Av2 contains even more than two [4 Fe‐4S] clusters.

The addition of MgATP induces a conformational change in the Fe‐protein which results in a higher reactivity with iron chelators. But irrespective of the specific activity, the amount of iron extracted from the protein after addition of MgATP never exceeds four atoms/molecule.

The results are discussed with respect to the present molecular model of the Fe‐protein.
Chemically Synthesized Galactosyl Ficaprenyl Diphosphate as an Intermediate in the Biosynthesis of the Salmonella O‐Antigenic Polysaccharide
Authors: KANEGASAKI Shiro , WRIGHT Andrew , WARREN Christopher D.
Published: March 2005

The membrane fraction from a mutant of Salmonella anatum deficient in UDPgalactose‐4‐epimerase, utilized synthetic ficaprenyl α‐d‐galactosyl diphosphate as a substrate in the biosynthesis of the O‐polysaccharide portion of lipopolysaccharide which has a mannosylrhamnosylgalactose repeating sequence. The galactosyl lipid was prepared by chemical synthesis from d‐galactose and ficaprenol extracted from Ficus elastica. Membrane preparations catalyzed the transfer of rhamnose from TDP‐rhamnose onto membrane‐bound ficaprenyl galactosyl diphosphate forming rhamnosylgalactosyl ficaprenyl diphosphate; the reaction was dependent on the prior insertion of the synthetic glycosyl‐lipid into the membrane, and was proportional to incubation time up to 4 min at 29°C. When both TDP‐rhamnose and GDP‐mannose were added, the product formed was O‐polysaccharide. These results indicate that the chemically synthesized ficaprenyl galactosyl diphosphate can be an active substrate for the in vitro synthesis of the Salmonella O‐polysaccharide.
The Binding of Glucose to Native and Proteolytically Modified Yeast Hexokinase PI
Authors: MAYES Elaine L. , HOGGETT James G. , KELLETT George L.
Published: March 2005

The binding of glucose to native, dimeric yeast hexokinase PI and to its proteolytically modified product, the monomeric SI form, has been investigated by equilibrium dialysis and by titration of the quenching of intrinsic protein fluorescence. The effects of glucose concentration, pH and ionic strength on binding have been examined and the way in which these factors also affect the monomer‐dimer association‐dissociation reaction of the native‐enzyme has been investigated by analytical ultracentrifugation.

At pH 6.0 and pH 8.5 the dissociation constants of glucose from hexokinase SI monomer were 0.11 mmoll−1 and 0.23 mmol 1−1 respectively and each was independent of ionic strength over the range 0–1.0 mol KCl 1−1. The dependence of the dissociation constant of glucose from SI on pH showed a sharp transition between these two extremes over a narrow interval from pH 7 to pH 7.5 At pH 8 the affinity of glucose for proteolytically modified SI monomer was the same as that for native PI monomer formed in the presence of 1.0 mol KCll−1 in support of the idea that the former is a good analogue of the latter.

At pH 6.7 and low ionic strength, the binding of glucose to hexokinase PI dimer alone shows strong positive cooperativity. This behaviour is in marked contrast to that of the PII isoenzyme for which both binding sites on the dimer are equivalent in solution and binding is non‐cooperative. At pH 6.7, the quenching of intrinsic protein fluorescence as a function of glucose concentration (Hill coefficient, h= 1.3) markedly precedes binding (h= 1.6) to hexokinase PI, the concentration at half‐saturation for each being 0.21 mmol l−1 and 0.82 mmol l−1 respectively. Detailed analysis of the data according to the Adair equation is presented.

At pH 8.0, hexokinase PI exists in association‐dissociation equilibrium between monomer and dimer. At low ionic strength, despite the presence of some monomer which binds glucose more strongly than dimer, the affinity of hexokinase PI is markedly reduced at pH 8.0 compared with that at pH 6.7 where it existed solely as dimer. It is concluded from linkage calculations that the instrinsic affinity of the dimer for glucose decreases by the same factor of two as does the monomer over this pH range and that the binding of glucose to PI dimer is non‐cooperative at pH 8.0. There is thus a structural transition from a cooperative to a non‐cooperative dimer, possibly occurring over the same narrow interval from pH 7 to 7.5 as the change in affinity of the monomer. pH is a major factor in determining the relative properties of hexokinases PI and PII.

The ratio of the affinity of the monomer for glucose to that of the dimer is about six and is independent of pH above pH 6.7. Under conditions where hexokinase PI exists as a mixture of monomers and dimers, changes in the binding of glucose as a function of ionic strength can be accounted for entirely by changes in the monomer‐dimer equilibrium.
6‐Phosphogluconolactonase
Authors: BAUER Hans Peter , SRIHARI Thota , JOCHIMS Johannes C. , HOFER Hans Werner
Published: March 2005

6‐Phosphogluconolactonase was purified to apparent homogeneity in a four‐step procedure from bovine erythrocytes. The extent of purification and the kinetic properties of the enzyme were evaluated with an optical test that was based on the hydrolysis of synthetic 6‐phosphogluconolactone.

The active enzyme from bovine erythrocytes is a monomer with an approximate molecular weight of 30000. It exhibits Michaelis‐Menten kinetics and cofactors are not required for activity.

The enzyme was found in a number of tissues. Its activity, when compared to the activity of the glycolytic enzymes, illustrates the importance of glucose oxidation via the pentose phosphate pathway relative to glycolysis.
Time‐Dependent Fluorescence Intensity and Depolarization of Diphenlhexatriene in Micellar Complexes of Apolipoprotein C‐I and Dimyristoylglycerophosphocholine
Authors: JONAS Ana , PRIVAT Jean‐Paul , WAHL Philippe
Published: March 2005

The lipohilic fluorescent probe diphenylhexatriene was used to probe the lipid order and dynamics in apolipoprotein C‐I dimyristoylglycerophosphocholine (Myr2Gro‐P‐Cho) complexes. These complexes contain on the average 25 mol Myr2Gro‐P‐Cho/mol of apolipoprotein C‐I, have a molecular weight around 200000, and appear as discoidal, stacked particles by negative‐stain electron microscopy. Steady‐state fluorescence polarization of diphenylhexatriene as a function of temperature gives a broadened and shifted phase transition for Myr2Gro‐P‐Cho from the gel to liquid‐crystalline state, with a mid‐point around 27°C.

Time‐dependent fluorescence intensity and anisotropy measurements of the diphenylhexatriene probe at 15°C and 35°C give fluorescence decay curves which can best be fit by two exponential functions, in each case. The fluorescence lifetimes and their fractional amplitudes approach the corresponding parameters in Myr2Gro‐P‐Cho vesicles and suggest insignificant effects of the protein on the microenvironment and conformations of the probe. The rotational correlation times and their fractional anisotropies indicate similar local motions of the probe in complexes and in vesicles, but reveal a significant ordering effect of the protein at both temperatures. The overall complex rotation at 157deg;C has a correlation time of 136 ± 13 ns, consistent with the size (∼ 200 kDa) and shape (disc ∼ 5 × 15 nm) of the particle.
Binding of Ethidium Bromide to Self‐Complementary Deoxydinucleotides
Authors: DOGLIA Silvia , GRÄSLUND Astrid , EHRENBERG Anders
Published: March 2005

Optical spectra titrations were performed with ethidium bromide and the self‐complementary deoxydinuclectides pdCpdG, pdGodA, pdTpdA, and pdApdT. The titrations were performed in 7.5 mM phosphate buffer, pH 7.0, at 7°C, and with varying dinucleotide concentrations always in large excess of the dye concentration. Well‐defined isosbestic points were present in each titration after correction for dinucleotide light absorption.

The binding curves were evaluated in terms of simple bimolecular or termolecular reaction models. The bimolecular reaction model gave a significantly better fit to the experimental data, judging from a computerized non‐linear least‐squares fitting procedure. The following equilibrium constats were obtained: KC‐G= 2000 M−1; KG‐C= 950 M−1; KT‐A= 370 M−1; KA‐T= 350 M−1. From these data the absorption spectra of the completely bound dye were evaluated. These spectra showed bathchromic shifts of their maxima, increasing with the magnitude of K.

Fluorescence spectra of ethidium bromide/dinucleotide mixtures were recorded under conditions similar to those for absorption spectra. From the known equilibrium constants the contributions of the bound dye could be estimated. The following fluorescence enhancements Ib/If wer found: IbC‐G/If= 6.5: IbG‐C/If= 3.0; IbT‐A/If= 2.0; IfA‐T/If= 2.0.

From our results, in relation to other theoretical and experimental studies, we conclude that electrostatic phosphate‐dye interactions give rise to a major part of the binding energy, which varies with dinucleotide geometry. The more strongly bound complexes exhibit less exposure of the dye to the solvent.
Calcium‐Binding Proteins from Human Platelets
Authors: GOGSTAD Geir O. , KRUTNES May‐Britt , SOLUM Nils Olav
Published: March 2005

Calcium‐binding platelet proteins were examined by crossed immunoelectrophoresis of solubilized platelets against antibodies to whole platelets followed by incubation of the immunoplates with 45Ca2+ and autoradiography. When the immunoplates had been pretreated with EDTA at pH 9.0 in order to remove divalent cations, three immunoprecipitates were markedly labelled with 45Ca2+. These corresponded to the glycoprotein IIb‐IIIa complex, glycoprotein Ia and a presently unidentified antigen termed G18. These antigens were membrane‐bound and surface‐oriented. When an excess of EDTA was introduced in the incubation media the results revealed that the glycoprotein IIb‐IIIa complex and antigen G18, but not glycoprotein Ia, contained sites with a stronger affinity for calcium than has EDTA at pH 7.4. Immunoprecipitates of the separate glycoproteins IIb and IIIa both bound calcium in the same manner as the glycoprotein IIb‐IIIa complex. As another approach, platelet‐rich plasma was incubated with 45Ca2+ prior to crossed immunoelectrophoresis of the solubilized platelets. A single immunoprecipitate was weakly labelled. This did not correspond to any of the immunoprecipitates which were visible after staining with Coomassie blue. The labelling of this labelling of this antigen was markedly increased when the platelet‐rich plasma had been preincubated with EDTA and in this case a weak labelling of the glycoprotein IIb‐IIIa pricipitate also became apparent. No increased incorporation of calcium occurred in any of these immunoprecipitates when the platelets were aggregated with ADP in the presence of 45Ca2+.
Structural Investigations of the Mg · ATP Complex at the Active Site of Porcine Adenylate Kinase Using Phosphorothioate Analogs and Electron Paramagnetic Resonance of Mn(II) with Chiral 17)‐Labelled ATP Analogs
Authors: KALBITZER Hans Robert , MARQUETANT Rainer , CONNOLLY Bernard A. , GOODY Roger S.
Published: March 2005

The interaction between ATP, divalent metal ions and the active site of adenylate kinase from muscle has been studied by two different methods. No reversal of the relative reaction rates of the two diastereomers of adenosine 5′‐([α‐thio]triphosphate) was observed on changing the metal ion from Mg2+ to Cd2+, suggesting that the α‐phosphate group is not coordinated to the divalent metal ion in the enzyme · metal · ATP complex. This interpretation is confirmed by the lack of influence of 17O incorporated into the α‐phosphate group of ATP on the electron paramagnetic resonance (EPR) spectrum of Mn2+ at the active site of the enzyme. The specificity for the Sp diastereomer thus appears to be due to an interaction of the enzyme active site with the pro‐R oxygen of the α‐phosphate group. In contrast, the specificity of adenylate kinase for the diastereomers of adenosine 5′([β‐thio]triphosphate) is reversed from Sp to Rp on replacing Mg2+ by Cd2+, indicating an interaction of the pro‐R oxygen of the β‐phosphate group of ATP with the metal ion in the enzyme · metal · ATP complex. In agreement with this, ATP which is regio‐specifically labelled with 17O on the β‐phosphate group causes a line‐broadening of the EPR spectrum of Mn2+ at the active site, demonstrating coordination of at least one of the β‐phosphate oxygens with the metal. Using stereospecifically labelled [β‐17O]ATPs, it was found that (Rp)‐[β‐17O]ATP but not (Sp)‐[β‐17O]ATP caused a line broadening in the Mn2+ EPR spectrum similar to that seen with regio‐labelled [β‐17O]ATP, further indicating that the pro‐R oxygen of the β‐phosphate group of ATP is coordinated to the metal ion at the active site of adenylate kinase. These results lead to a value of 0.22–0.25 mT for the superhyperfine coupling constant of a single 17O‐Mn2+ interaction in a manganese‐ATP‐enzyme complex. ATP labelled with 17O on the γ‐phosphate group has no effect on the spectrum of Mn2+ at the active site, leading to the conclusion that Mg · ATP exists predominantly as a β‐monodentate complex at the active site of adenylate kinase. From the effect of H217O on the EPR spectrum of Mn2+ in the Mn · ATP complex at the active site it appears most likely that three, four or five water molecules are bound to the metal ion.
Control of Rat‐Liver Glutaminase by Ammonia and pH
Authors: VERHOEVEN Arthur J. , VAN IWAARDEN Johan F. , JOSEPH Suresh K. , MEIJER Alfred J.
Published: March 2005

Regulation by ammonia of phosphate‐dependent glutaminase in isolated rat‐liver mitochondria was studied at pH values near the cytosolic pH of 7.0.

Glutaminase activity, both in the absence and presence of bicarbonate, was completely dependent on the presence of ammonia.

Glutaminase activity, both in the absence and presence of bicarbonate, was strongly depressed by decreasing the pH of the incubation medium from 7.0 to 6.8 when the ammonia concentration was below 0.5 mM.

Bicarbonate stimulated glutaminase activity only in the presence of low concentrations of ammonia.

The data indicate that the reported inhibition of glutamine degradation in the perfused liver at low pH [e.g. Häussinger et al. (1980) Hoppe‐Seyler's Z. Physiol. Chem. 361, 995–1001] is due to a decreased affinity of glutaminase for ammonia.
Bicarbonate and the Pathway of Glutamate Oxidation in Isolated Rat‐Liver Mitochondria
Authors: WANDERS Ronald J. A. , MEIJER Alfred J. , GROEN Albert K. , TAGER Joseph M.
Published: March 2005

The factors affecting the pathway of glutamate oxidation were studied in isolated rat‐liver mitochondria in incubations of 2–3 min.

It was found that bicarbonate at a physiological concentration has a profound effect on the pathway of glutamate oxidation. Ammonia formation via glutamate dehydrogenase is stimulated by bicarbonate [from 5.48 ± 0.29 (n= 10) to 9.57 ± 0.73 (n= 8) nmol ± min−1± mg protein−1], whereas aspartate formation via the transamination pathway is inhibited [from 38.41 ± 2.24 (n= 9) to 24.56 ± 3.28 (n= 6) nmol ± min−1± mg protein−1].

Bicarbonate has no effect on the rate of transport of glutamate via the glutamate‐hydroxyl translocator.

The interaction of bicarbonate with the pathway of glutamate oxidation occurs primarily at the level of succinate dehydrogenase, due to competitive inhibition of the enzyme by bicarbonate.

Inhibition by bicarbonate of the transamination pathway leads to a decrease in intramitochondrial 2‐oxoglutarate, so that the deamination pathway is stimulated.

Using an equation which describes flux through glutamate dehydrogenase kinetically, it could be shown that the bicarbonate‐induced decrease in intramitochondrial 2‐oxoglutarate quantitatively accounts for the enhanced rate of deamination.

It is concluded that in the intact liver flux through glutamate dehydrogenase is sufficient to account for the ammonia formation required for urea synthesis from substrates such as alanine.
Characterization of a Salivary Agglutinin Reacting with a Serotype c Strain of Streptococcus mutans
Authors: ERICSON Thorild , RUNDEGREN Jan
Published: March 2005

A protein, which can agglutinate a Streptococcus mutans serotype c strain, was isolated from parotid saliva by affinity adsorption of the salivary agglutinin to the microorganism followed by a desorption with a 10 mM phosphate buffer. The agglutinin was subjected to preparative ultracentrifugation, gel filtration, and ultrafiltration. The native purified agglutinin is active only in the presence of Ca. Polyacrylamide gel electrophoresis, analytical centrifugation, and analyses of amino acids and carbohydrates showed that the native agglutinin was a fucose‐rich glycoprotein with a carbohydrate content of 45% and with a molecular weight of at least 5 × 106. After sodium dodecyl sulphate treatment the molecular weight was 4.4 × 105. There was a low content of proline and a high content of aspartic acid, serine and threonine. The concentration of agglutinin in parotid saliva is less than 0.5% of total protein. It has high biological activity: 0.1 μg agglutinin causes a rapid aggregation of approximately 108 bacteria.
Substrate Specificity of the Human Splenic Non‐specific Soluble β‐Glucosidase
Authors: MARET Arlette , SALVAYRE Robert , NEGRE Anne , DOUSTE‐BLAZY Louis
Published: March 2005

The soluble non‐specific 4‐methylumbelliferyl‐β‐d‐glucosidase, one of the three splenic groups previously reported [Maret, A., Salvayre, R., Nègre, A., and Douste‐Blazy, L. (1981) Eur. J. Biochem. 115, 455–461], was partially purified by gel filtration and heat‐inactivation (which inactivated the contaminating acid β‐galactosidase).

Its substrate specificity was accurately demonstrated by comparing the enzymic properties towards each substrate and two‐substrate mixed assays: 4‐methylumbelliferyl β‐d‐galactopyranoside, β‐d glucopyranoside, β‐d‐fucopyranoside, α‐l‐arabinopyranoside and β‐d‐xylopyranoside (relative rates 100, 95, 70, 15, 5, respectively in the standard conditions used) were competitively hydrolysed.

Information about the enzymic site was given by the effect of various substrate analogs: generally we observed a greater inhibition of β‐galactosidase activity than β‐glucosidase and β‐fucosidase activities. Moreover, p‐nitro‐phenyl‐β‐d‐mannopyranoside showed a strong inhibitory effect on the three main activities of this enzyme, similar to d‐glucono‐1,5‐lactone inhibition. A selective inhibition by Triton X‐100 on β‐d‐galactosidase activity and in a lesser degree on β‐d‐fucosidase activity was discussed.

The non‐specific soluble β‐glucosidase from a Gaucher patient and from normal subjects showed identical properties. Thus this enzyme is not affected by the mutation in the Gaucher type I spleen.
The Effects of Octylglucoside on the Interactions of Chloroplast Coupling Factor 1 (CF1) with Adenine Nucleotides
Authors: PICK Uri , BASSILIAN Sara
Published: March 2005

The effects of octylglucoside (OcGlc) micelles, which stimulate a Mg‐specific ATPase activity in chloroplast coupling factor 1 [Pick, U. and Bassilian, S. (1982)Biochemistry, 21, 6144–6152], on the interactions of the enzyme with adenine nucleotides have been studied.

OcGle specifically accelerates the binding and the release of ADP but not of ATP or adenosine 5′[β, γ‐imido]triphosphate (AdoPP[NH]P) from the tight‐sites. The binding affinity for ADP and for ATP is only slightly decreased (twofold) by the detergent. ATP competitively inhibits the binding of ADP and vice versa in the presence or absence of OcGlc.

OcGle‐induced inactivation of CF1‐ATPase is correlated with the release of bound nucleotides. In the absence of medium nucleotides ADP · CF1is rapidly inactivated while ATP · CF1and AdoPP[NH]P· CF1are slowly inactivated by OcGle in parallel with the release of bound nucleotide. In contrast, low concentrations of either ATP or ADP in the medium effectively protect against OcGlc inactivation while AdoPP[NH]P, whose binding to CF1is inhibited by OcGle, is ineffective even at millimolar concentrations. The results suggest that the occupancy of the tight‐sites protects the enzyme against OcGlc‐induced inactivation.

Mg ions specifically inhibit the release of bound ADP and the OcGlc‐induced inactivation of CF1. High concentrations of medium ATP and ADP (K50= 100 μM) also inhibit the OcGlc‐induced release of bound nucleotides in an EDTA medium. In contrast, in the absence of OcGlc, medium ADP and ATP accelerate the release of bound adenine nucleotides.

Mg‐ATP in the presence of OcGlc stimulates the release of bound ADP from CF1. Bound ATP is neither released nor hydrolyzed at the tight‐sites under these conditions where medium ATP is rapidly hydrolyzed. Mg‐ADP stimulates the release of bound ADP only in the presence of inorganic phosphate or of phosphate analogs, e.g. arsenate, pyrophosphate or selenate.

It is suggested that: (a) ATP and ADP bind to the same tight‐sites, but OcGlc activation specifically accelerates the exchange of bound ADP at the site. (b) CF1contains low affinity adenine nucleotide binding sites which may be the catalytical sites and which influence the tight‐sites by cooperative interactions. (c) Mg‐ATP in the presence of OcGlc induces a conformational change at the catalytical site which accelerates the release of ADP from the tight‐site.

The implications of these results to the role of adenine nucleotides in the regulation and mechanism of ATP hydrolysis by CF1are discussed.
Improved Purification and Further Characterization of Acetyl‐CoA Carboxylase from Cultured Cells of Parsley (Petroselinum hortense)
Authors: EGIN‐BÜHLER Barbara , EBEL Jürgen
Published: March 2005

Acetyl‐CoA carboxylase from irradiated cell‐suspension cultures of parsley (Petroselinum hortense) has been purified to apparent homogeneity. The procedure included affinity chromatography of the enzyme on avidin‐ monomer ‐ Sepharose 4B. Molecular weights of about 420000 for the native enzyme and about 220000 for the enzyme subunit were determined respectively by gel filtration or sucrose‐density‐gradient sedimentation and by electrophoresis in the presence of dodecyl sulfate. The purified enzyme showed an isoelectric point of 5.

The enzyme carboxylated the straight‐chain acyl‐CoA esters of acetate, propionate, and butyrate at decreasing rates in this order. The catalytic efficiency of the carboxylase was highest when ATP existed largely as MgATP2− complex. At the optimum pH of 8 the apparent Km values for the substrates were: acetyl‐CoA, 0.15mmol/1; bicarbonate, 1 mmol/1; MgATP2−, 0.07mmol/1. The carboxylase was inhibited by > 50mmol/1 NaC1, KCl, or Tris/HCl buffer. The putative allosteric activator, citrate, stimulated the enzyme only slightly at concentrations below 2 mmol/1, but strongly inhibited the carboxylase at higher concentrations.

The results of these studies demonstrate that several properties of the light‐inducible acetyl‐CoA carboxylase of parsley cells, an enzyme of the flavonoid pathway, are remarkably similar to those of acetyl‐CoA carboxylases from a variety of other organisms.
Properties and Reconstitution of a Cytochrome Oxidase Deficient in Subunit III
Authors: PENTTILÄ Timo
Published: March 2005

Three different preparations of beef heart cytochrome oxidase (EC 1.9.3.1) were reconstituted into the mem‐ branes of artificial liposomes, and the electrical charge/electron ratios were determined for charge translocation coupled to enzymic activity. Our previously characterised subunit‐Ill‐deficient preparation, which apparently lacks H+ translocation capacity [Saraste et al. (1981) Eur. J. Biochem. 115, 261–2681 has a decreased charge/ electron ratio (0.9–1.0) as determined from the uptake of potassium in the presence of valinomycin, in contrast to the intact reconstituted cytochrome oxidase (1.9–2.0). A third preparation that was depleted of three minor polypeptides by trypsin treatment (these polypeptides are also removed together with subunit III using the present method), but which retains subunit III, had a K−/e− ratio of 1.5 but also a relatively low respiratory control index.

The pH‐dependence of the Em of cytochromerr determined in the presence of cyanide is abolished i n the subunit‐Ill‐deficient enzyme.

Electron transfer activities are nearly identical for the original and subunit‐111‐depleted enzymes at an infinite concentration of cytochrome c in a polarographic assay with supplemented phospholipids. The optical spectral properties are vcry similar for both preparations, but with a small shift to the blue of the α‐peak in the modified enzyme.

These results support the hypothesis that the removal of subunit 111 abolishes the H+‐translocating function of cytochrome oxidase. This occurs by an intrinsic decoupling of Hf transport from electron transfer, and yields preparation with only half‐maximal efficiency of energy conservation.
Histone Acetylation in Chromatin Containing Mouse Satellite DNA
Authors: PASHEV Iliya G. , DIMITROV Stephan I. , IVANOV Ivan G. , MARKOV George G.
Published: March 2005

Histone acetylation in transcriptionally inactive chromatin has been studied with chromatin containing mouse satellite DNA. The latter was obtained by digestion of nuclei from Ehrlich ascites tumor cells with the restriction nuclease Bsp, which degrades main‐band DNA but leaves satellite DNA intact. The enzyme‐resistant material was separated by gel filtration. Satellite DNA amounted to 65% of the total DNA in this fraction. When the cells were grown in the presence of sodium n‐butyrate to inhibit histone deacetylation, a few, if any, hyperacetylated forms of core histones were found in satellite chromatin. Conversely, the highest quantity of tetraacetylated H4 molecules was found in the fractions containing the most extensively degraded chromatin.
The Core Protein of Alphaviruses
Authors: BOEGE Ulrike , WENGLER Gisela , WITTMANN‐LIEBOLD Brigitte
Published: March 2005

The primary structure of the core protein of Semliki Forest virus has been established by protein chemical characterization of 102 peptides, generated by digestion with trypsin, pepsin, thermolysin, and by partial acid cleavage of the protein. Besides a difference in one position, the sequence as established by these experiment is in agreement with the sequence predicted from the nucleotide sequence of the mRNA [Garoff et al. (1980) Proc. Natl Acad. Sci. USA, 77, 6376–6380]. The core protein has a blocked N terminus, consists of 267 amino acid residues, and has the following amino acid composition: Asp12, Asn9, Thr16, Ser10, Glu11, Gln15, Pro23, Gly20, Ala23, Val19, Met8, Ile11, Leu9, Tyr7, Phe6, His7, Lys37, Arg15, Trp5, Cys4, and an Mr of 29919. It contains 22.1% basic amino acids, mainly lysines, compared with a total of 8.6% acidic residues. The resulting surplus of positive charge is located in the N‐terminal half of the protein (predominantly arginines at positions 12–21 and lysines at positions 66–114). Other amino acids are also unevenly distributed; proline and glutamine are accumulated in the N‐terminal half of the sequence whereas histidine, glycine and the acidic residues are mainly present in the C‐terminal part. This distribution suggests that the virus core protein consists of two or more structural domains.
The Core Protein of Alphaviruses
Authors: BOEGE Ulrike , WENGLER Gisela , WITTMANN‐LIEBOLD Brigitte
Published: March 2005

The primary structure of the core protein of Sindbis virus has been established by protein chemical characterization of peptides derived by enzymatic digestion with trypsin, pepsin and thermolysin and by chemical cleavage with cyanogen bromide. The peptide chain consists of 264 amino acids and has the composition Asp8, Asn8, Thr17, Ser12, Glu12, Gln14, Pro28, Gly24, Ala22, Val16, Met10, Ile8, Leu14, Tyr4, Phe9, His6, Lys25, Arg23, and Trp4, and an Mr, of 29 382. Comparison of this structure with the primary structure of the SF virus core protein revealed several important common characteristics of alphavirus core proteins.

The N‐terminal halves (1–110) of the proteins are rich in basic amino acids and proline.

The C‐terminal part (∼ 110–264/267) is highly conserved: 70% of the amino acid residues are in identical positions.

The conserved part contains a possible catalytic centre for the presumed protease activity of the core protein.

The similarities between the primary structures of both core proteins are reflected in their predicted secondary structures.
Interaction of the Dissociable Glycerol‐3‐phosphate Dehydrogenase and Fructose‐1,6‐bisphosphate Aldolase
Authors: OVÁDI Judit , MOHAMED OSMAN Ibrahim Refaie , BATKE József
Published: March 2005

Cytoplasmic sn‐glycerol‐3‐phosphate dehydrogenase, labelled covalently with fluorescein isothiocyanate, shows an enzyme‐concentration‐dependent fluorescence anisotropy. The anisotropy versus enzyme concentration curves is shifted towards higher concentrations when substrates are present. The comparison of the dissociation constants estimated from anisotropy measurements and derived from kinetic experiments suggests that the substrate‐induced dissociation of the dimeric dehydrogenase is slow with respect to the enzymatic reaction catalyzed by either its monomeric or dimeric form.

The fluorescence anisotropy of the fluorescent dye‐labelled dehydrogenase increases with time upon addition of unlabelled fructose‐1,6‐bisphosphate aldolase approaching a limiting value. This fact indicates the binding of fructose‐1,6‐bisphosphate aldolase to glycerolphosphate dehydrogenase. A model is proposed assuming simuitaneous binding of tetrameric fructose‐1,6‐bisphosphate aldolase to monomeric and dimeric glycerolphosphate dehydrogenase with 1:1 stoichiometry. The dissociation constants, as parameters fitted to the experimental curves, were estimated as 0.2 μM and 1 μM for aldolase–dimeric‐glycerolphosphate‐dehydrogenase and aldolase–monomeric‐glycerolphosphate‐dehydrogenase complexes respectively.
Quaternary Structure and Spin Equilibria in Ferric Hemoglobins
Authors: NOBLE Robert W. , DE YOUNG Alice , DI IORIO Ernesto , WINTERHALTER Kasper H. , CERDONIO Massimo , MORANTE Silvia , VITALE Stefano
Published: March 2005

The effective magnetic moments for a number of human and carp methemoglobin derivatives were determined in solution at room temperature. The data permit us to confirm the dependence of the spin‐state equilibrium of azide methemoglobin on the quaternary state of the hemoglobin and to demonstrate a similar dependence for both human and carp aquomethemoglobin.

In addition, the pH dependence of the effective magnetic moment and the Soret spectrum of carp azide‐methemoglobin are compared.
Salt‐Induced Conformational Transitions in Chromatin
Authors: MAKAROV Vladimir L. , DIMITROV Stephan I. , PETROV Peter T.
Published: March 2005

The chromatin structure in solution has been studied by the flow linear dichroism method (LD) in a wide range of ionic strengths. It is found that increasing the ionic strength from 0.25 mM Na2EDTA, pH 7.0 to 100 mM NaCl leads to a strong reduction of the LD amplitude of chromatin and inversion of the LD sign from negative to positive at 2 mM NaCl. Chromatin exhibits a positive LD maximum value at 10–20 mM NaCl. These data enable us to conclude that in very low ionic strength (0.25 mM Na2EDTA) the nucleosome discs are oriented with their flat faces more or less parallel to the chromatin filament axis. Increasing ionic strength up to 20 mM NaCl leads to reorientation of the nucleosome discs and to formation of chromatin structures with nucleosome flat faces inclined to the fibril axis. A conformational transition of that kind is not revealed in H1‐depleted chromatin. The condensation of the chromatin filaments with increasing concentration of NaCl from 20 mM to 100 mM slightly influences the orientation of the nucleosomes.
Interaction between the Different Domains of Aminoacyl‐tRNA and the Elongation‐Factor‐Tu · Kirromycin Complex
Authors: GUESNET Joëlle , PARLATO Giuseppe , PARMEGGIANI Andrea
Published: March 2005

In this work, we have studied the effect of aa‐tRNA and derived 3′ aminoacylated fragments on the EF‐Tu GTPase in the presence of kirromycin, using two systems: without and with ribosomes. The aa‐tRNA fragments were obtained by enzymatic digestion. Procedures for the enzymatic preparation of C‐A‐Val and Val‐tRNA1Val 3′ half molecule, as well as a purification method for short 3′ aminoacylated fragments based on the amino group charge, were newly developed for this work. Aminoacyl‐adenosine was found to be able to stimulate the EF‐Tu · kirromycin GTPase, but only to a very small extent. Increasing the length of the aminoacylated fragments increased the stimulatory effect as follow: A‐Val ≪ C‐A‐Val < 3′ valyladenosine dodecanucleotide ≪ Val‐tRNA1Val 3′ half molecule < Val‐tRNA1Val. The presence of ribosomes did not affect the order of effectiveness, but increased the basic GTPase activity of EF‐Tu · kirromycin and the stimulation by aa‐tRNA, its 3′ half molecule and even more by its 3′ short fragments. The effect of aa‐tRNA and derived 3′ fragments in the absence of ribosomes was not influenced by MgCl2 concentrations of 5–30 mM whereas, in the presence of ribosomes, low concentrations of MgCl2 (5 mM) greatly reduced the stimulation of aa‐tRNA and, to a lesser extent, also the effect of the C‐C‐A‐aa as well as the basic activity of the EF‐Tu · kirromycin GTPase.

The extent of the stimulation by aa‐tRNA, and even more by C‐C‐A‐aa, depends on the nature of the amino acid. Among the aminoacyl side chains tested (Arg‐, Phe‐, Val‐, Met‐, Leu‐, Lys‐) arginine was found to be the most active and leucine the least.

Our results show that (a) the 3′ aminoacylated extremity is of prime importance for the stimulation of the EF‐Tu GTPase, (b) in the 3′ extremity there are critical sequences for the interaction with EF‐Tu and (c) other domains of the aa‐tRNA molecule are capable of influencing this reaction: one of the most important is the region including the TΨC loop and stem.
The Accessibility of the Active Site and Conformation States of the β2 Subunit of Tryptophan Synthase Studied by Fluorescence Quenching
Authors: LANE Andrew N.
Published: March 2005

The rate of quenching of the fluorescence of pyridoxal 5′‐phosphate in the active site of the β2 subunit of tryptophan synthase from Escherichia coli was measured to estimate the accessibility of the coenzyme to the small molecules iodide and acrylamide. The α subunit and the substrate l‐serine substantially reduced the quenching rate. For iodide, the order of decreasing quenching was: Schiff's base of Nα‐acetyl‐lysine with pyridoxal 5′‐phosphate > holoβ2 subunit > holoα2β2 complex ∼ holoβ2 subunit +l‐serine > holoα2β2 complex +l‐serine. The coenzyme in the β2 subunit is apparently freely accessible to both iodide and acrylamide (k∼× 109 M−1 s−1), but the α subunit and l‐serine decrease the rate by factors of 2–5.

Quenching of the fluorescence of the single tryptophan residue of the β2 subunit revealed that the apo and holo forms exist in different states, whereas the α subunit stabilises a third conformation. As the α subunit binds to the β2 subunit, the tryptophan residue, which is within 2.2 nm of the active site of the β2 subunit, probably rotates with respect to the plane of the ring of the coenzyme, such that fluorescence energy transfer from tryptophan to pyridoxal phosphate is greatly reduced.

The α subunit strongly protects the active‐site ligand indole propanol phosphate from quenching with acrylamide, consistent with the active site being deep in a cleft in the protein.

Iodide induces dissociation of the holoα2β2 complex [E. W. Miles & M. Moriguchi (1977) J. Biol. Chem. 252, 6594–6599]. The effect of iodide on the fluorescence properties of holoα2β2 complex allows us to estimate an upper limit for the dissociation constant for the α2β2 complex of 10‐8M, in the absence of iodide.
9,10‐Dihydrophenanthrenes as Phytoalexins of Orchidaceae
Authors: FRITZEMEIER Karl‐Heinrich , KINDL Helmut
Published: March 2005

Hydroxy derivatives of 9,10‐dihydrophenanthrenes, orchinol and hircinol, were isolated from bulbs of Orchidaceae which had been induced to accumulate phytoalexins. Incorporation of radioactive precursors, l‐phenylalanine and various hydroxycinnamic acids, has been investigated by feeding experiments in vivo. m‐Coumaric acid and dihydro‐m‐coumaric acid were found to be efficiently incorporated into the dihydrophenanthrene derivatives. Dihydro‐m‐coumaric acid was not only converted into the dihydrophenanthrenes but was also formed from l‐phenylalanine in the same tissue; it was thus proved to be an intermediate.

The role of dihydro‐m‐coumaric acid was substantiated by studies in vitro. An active stilbene synthase was detected in enzyme preparations from induced orchid bulbs and assayed with different CoA esters. The enzyme, characterized on the basis of its substrate specificity, selectively converted dihydro‐m‐coumaroyl‐CoA plus malonyl‐CoA into 3,3′,5‐trihydroxybibenzyl.

The role of 3,3′,5‐trihydroxybibenzyl as physiological intermediate was further corroborated by investigations with intact plants. Both its formation from phenylpropanoids and its conversion into orchinol was demonstrated.

The data provided evidence for a biosynthetic sequence originating from l‐phenylalanine and leading to 9,10‐dihydrophenanthrenes via m‐coumaric acid, dihydro‐m‐coumaric acid, and 3,3′,5‐trihydroxybibenzyl.
The Sub‐Cellular Localisation of Pyruvate Carboxylase and of Some Other Enzymes in Aspergillus nidulans
Authors: OSMANI Stephen A. , SCRUTTON Michael C.
Published: March 2005

The sub‐cellular localisation of enzymes has been defined by latency analysis, and fractionation by differential centrifugation, in cell‐free extracts prepared from the mycelium of Aspergillus nidulans by growth in the presence of 2‐deoxyglucose followed by treatment with a mixture of β‐glucuronidase, sulphatase and β‐glucanase and exposure to N2 cavitation at 5.2 PMa. In such extracts pyruvate carboxylase and NAD‐dependent and NADP‐dependent glutamate dehydrogenases are exclusively localised in the cytosol whereas all the other enzymes studied have sub‐cellular localisation patterns similar to those described for mammalian liver. Electrophoretic analysis has established the presence of unique mitochondrial and cytosolic isoenzymes for many of the enzymes, e.g. NAD–malate dehydrogenase, NADP–isocitrate dehydrogenase, glutamate/oxaloacetate transaminase, fumarase, which show a marked extent of incomplete latency and the presence of significant activity in the mitochondrial and cytosolic fractions prepared by differential centrifugation. A novel method is described for detection of citrate synthase activity following electrophoresis of the cell‐free extract. Application of this method confirms the absence of a unique cytosolic isoenzyme of citrate synthase and hence shows that citrate synthase activity detected in the soluble fraction results from damage to the mitochondria during isolation.

A scheme is proposed on the basis of these data to describe the organisation of lipid and amino acid synthesis from glucose in an organism which posessesses a cytosolic pyruvate carboxylase.
DNA Fragment Conformations
Authors: TRAN‐DINH Son , NEUMANN Jean‐Michel , TABOURY Jean , HUYNH‐DINH Tam , RENOUS Sylvie , GENISSEL Bernadette , IGOLEN Jean
Published: March 2005

The conformation and the helix‐coil transition of two complementary hexamers, d(C‐A‐C‐G‐T‐G) and d(G‐T‐G‐C‐A‐C), were investigated as a function of temperature by 1H‐NMR spectroscopy at 500 MHz and 90 MHz. At high temperature (t > 70–80°C), in the coil form, the proton chemical shifts depend strongly on the nature and position in the sequence of the residue. Purine and pyrimidine residues can be distinguished not only by the difference in chemical shift of the H 2′ and H 2” resonances but also by that of the H 3′ or H 4′ protons. For equivalent positions, the dA proton resonances are always located at lower fields than those of other residues. For residues of the same nature the chemical shift of the H 3′ or H 4′ protons decreases in the following order: δ(pdNp) > δ(dNp) > δ(pdN), where dN = dA, dG, dC or dT; moreover the terminal residue can easily be identified by either the H 1′ resonance [δ(pdN) > δ(pdNp, dNp)] or the H 2” resonance for the purine family [δ(pdN) < δ(pdNp, dNp) ≤δ(pdNp, dNp, pdN), where dN = dA, dG] and the H 2′ resonance for the pyrimidine family [δ(pdN) > δ(pdNp, dNp), where dN = dC or dT].

The chemical shift difference between the coil and helix forms is generally greater for the pyrimidine than for the purine residues and the proton resonances of the external residues often exhibit different temperature‐dependent profiles. The results suggest that, as in the helical form the bases of adjacent residues are close to each other, the pyrimidine base proton resonances are shifted not only by the change in environment but also by the ring‐current effects from neighbouring purine bases. In the intercalated purine‐pyrimidine sequences, the protons of an internal pyrimidine residue are statistically subjected to opposite ring‐current effects from the base of the preceding and following residues. The difference in temperature‐dependent behaviour of the H 2” resonances between the initial or internal purine residues (where the phosphate group is located at the 3′ position) on the one hand and the terminal residue on the other can be explained by the change in conformation of the phosphate groups from the coil to the helix form.

From measurements of proton coupling constants and observation of the NH resonances, it was concluded that the duplexes of both hexamers, d(C‐A‐C‐G‐T‐G) and d(G‐T‐G‐C‐A‐C), adopt the B‐helical conformation and open sequentially from the exterior with increasing temperature. The midpoint temperatures, t1/2, coil proportions and helix‐coil dissociation constants Kd, were determined from the melting curves. The dissociation rate constant, kd, was deduced versus temperature from the line width of the H 1′ and H 5 resonances. The midpoint temperature of each residue and the difference between the t1/2 external and central residues depend strongly on the number of the dG · dC base pairs and their positions in the sequence, whereas the dissociation enthalpy appears to be independent for sequences of the same size.
Protein Disulphide‐Isomerase Activity in Various Cells Synthesizing Collagen
Authors: MYLLYLÄ Raili , KOIVU Juha , PIHLAJANIEMI Taina , KIVIRIKKO Kari I.
Published: March 2005

A high correlation was found between the activities of protein disulphide isomerase and prolyl 4‐hydroxylase when assayed in cells synthesizing various collagen types or the same type at markedly different rates. The highest activities of both enzymes were found in freshly isolated chick‐embryo tendon and cartilage cells, intermediate activities in confluent cultures of human skin and lung fibroblasts and mouse 3T6 fibroblasts, and the lowest values in three human sarcoma cell lines, the difference in protein disulphide isomerase activity between the freshly isolated tendon cells and confluent simian‐virus‐40‐transformed human lung fibroblasts being about 25‐fold. All these differences are in good agreement with differences reported between the various cells in their rates of collagen synthesis. A great similarity was also found between the changes in the two enzyme activities measured per cell during the growth of 3T6 fibroblast cultures from the early logarithmic phase to the stationary phase. No correlation was found between protein disulphide isomerase activity and the type of collagen synthesized. The data suggest that protein disulphide isomerase may be involved in the formation of intra‐chain and inter‐chain disulphide bonds in procollagens, but there is no collagen type‐related variation in this enzyme activity of a magnitude that would explain the marked differences in the rates of formation of inter‐chain disulphide bonds between the various collagen types.
Structure of the Mouse Cytochrome P1‐450 Genomic Gene
Authors: NAKAMURA Michitoshi , NEGISHI Masahiko , ALTIERI Mario , CHEN Yuan‐Tsong , IKEDA Toshihiko , TUKEY Robert H. , NEBERT Daniel W.
Published: March 2005

Clone 46 was previously shown to represent mouse cytochrome P1‐450 cDNA by both translation arrest experiments and segregation of induced P1‐450 mRNA with induced aryl hydrocarbon hydroxylase activity among individual 3‐methylcholanthrene‐treated offspring of the (C57BL/6N)(DBA/2N)F1× DBA/2N backcross. With clone 46 as a probe, a MOPC 41 mouse genomic‐DNA library was screened. λ3NT12, a 16 × 103‐base‐pair insert of genomic DNA grown in a recombinant Charon 4A λ vector phage, was isolated and characterized. It was determined that clone 46 hybridizes to the extreme 5′ end of λ3NT12. pMJE12, a 3.0 × 103‐base‐pair fragment in the 5′ region of λ3NT12, was subcloned in plasmid pBR322 and used as a probe to screen again the same mouse‐DNA library; recombinant phages λ3NT13, and λ3NT14, and λAhP‐1 were isolated and characterized. The relative orientation of each of the four genomic clones on the mouse chromosome was determined. Only λAhP‐1 contains the entire P1‐450 genomic gene, which by R‐loop analysis spans about 46 × 102 base pairs and contains at least five exons. Clone 46 is shown to be a 3′ unique sequence of the genomic P1‐450 gene. The λAhP‐1 genomic‐DNA clone from the MOPC 41 plasmocytoma is shown by a series of restriction enzymes to be the same as genomic DNA from normal mouse liver. With a subclone in the 5′ portion of the P1‐450 gene, two and three hybridizable fragments are found with mouse genomic DNA that has been digested with EcoRI and BamHI, respectively.
Dicyclohexylcarbodiimide Does not Inhibit Proton Pumping by Cytochrome c Oxidase of Paracoccus denitrificans
Authors: PÜTTNER Irene , SOLIOZ Marc , CARAFOLI Ernesto , LUDWIG Bernd
Published: March 2005

The effect of dicyclohexylcarbodiimide (DCCD) on the proton pumping two‐subunit cytochrome c oxidase from Paracoccus denitrificans was investigated. Purified Paracoccus oxidase was reconstituted into phospholipid vesicles by cholate dialysis. Following incubation with increasing amounts of DCCD, proton ejection was recorded in response to reductant pulses with reduced cytochrome c. Concentrations of DCCD which greatly reduced proton pumping by bovine cytochrome c oxidase used as a control were found to exert only a minor effect on proton translocation by Paracoccus oxidase. Similarly, incubation of the bacterial enzyme with [14C]DCCD failed to reveal the specific covalent interaction previously demonstrated to occur with bovine cytochrome c oxidase, and here also shown for the oxidase of yeast. Thus, Paracoccus oxidase differs in its interaction with DCCD from the functionally analogous eukaryotic enzymes.