bluuebunny/crossref_metadata_2025_split 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.
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.
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.
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.
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.