bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of Cardiac Sarcoplasmic Reticulum ATP‐ADP Phosphate Exchange and Phosphorylation of the Calcium Transport Adenosine Triphosphatase
Authors: SUKO Josef , HASSELBACH Wilhelm 
Published: March 2005

The terminal phosphate of (γ‐32P)ATP is rapidly incorporated into cardiac sarcoplasmic reticulum membranes (0.7–1.3 μmol/g protein) in the presence of calcium and magnesium. Cardiac sarcoplasmic reticulum membranes catalize an ATP‐ADP phosphate exchange in the presence of calcium and magnesium.
Half‐maximum activation of the phosphoprotein formation and ATP‐ADP phosphate exchange is reached at an ionized calcium concentration of about 0.3 μM. The Hill coefficients are 1.3.
Transphosphorylation and ATP‐ADP phosphate exchange require magnesium and are maximally activated at magnesium concentrations close to or equal to the ATP concentration.
The phosphoprotein level is reduced to about 45% at an ADP/ATP ratio of 0.1. The rate of calcium‐dependent ATP splitting declines, whilst the rate of the calcium‐dependent ATP‐ADP phosphate exchange increases when the ADP/ATP ratio is varied from 0.1 to 1. The sum of both, the rate of ATP splitting and the rate of ADP‐ATP phosphate exchange remains constant.
Phosphoprotein formation and ATP‐ADP phosphate exchange are not affected by azide, dinitrophenol, dicyclohexyl carbodiimide and oubain, whilst both activities are reduced by blockade of –SH groups localized on the outside of the sarcoplasmic reticulum membrane.
The isolated phosphoprotein is acid stable. The trichloroacetic acid denatured 32P‐labelled membrane complex is dephosphorylated by hydroxylamine, which might indicate that the phosphorylated protein is an acyl‐phosphate.
Polyacrylamide gel elctrophoresis (performed with phenol/acetic acid/water) of phosphorylated sarcoplasmic reticulum fractions demonstrates that the 32P‐incorporation occurs into a protein of about 100000 molecular weight.
It is suggested that the phosphoprotein represents a phosphorylated intermediate of the calcium‐dependent ATPase which formation occurs as an early step in the reaction sequence of calcium translocation by cardiac sarcoplasmic reticulum similar as in skeletal muscle.
The Structure of Kinetoplast DNA
Authors: KLEISEN Coen M. , WEISLOGEL Paul O. , FONCK Kees , BORST Piet 
Published: March 2005

Degradation of highly purified kinetoplast DNA (kDNA) networks with restriction endonucleases yields ‘extra’ bands in agarose gels that are absent from digests of mini‐circles. Each of the five endonucleases tested, i.e. AluI, HapII, EcoRI, Hsu and HindII + III, yields a unique set of ‘extra’ bands. The ‘extra’ bands consist of linear DNA; they are not mini‐circle oligomers and their added molecular weights, calculated from mobility in gels, are around 2 × 107. Double digests with two restriction endonucleases yield a new set of ‘extra’ bands, showing that the ‘extra’ bands obtained with different enzymes are all derived from the same complex component of kDNA. In digests of 32P‐labelled kDNA an average of 2.3% of the radioactivity is recovered in the ‘extra’ bands.
Treatment of kDNA networks with the single‐strand‐specific S1 nuclease of Aspergillus oryzue preferentially releases a linear DNA with a molecular weight of 26 × 106, calculated from mobility in gels. We present evidence that the ‘extra’ bands obtained with restriction endonucleases are derived from this component.
DNA‐DNA renaturation analysis of fragmented kDNA shows the presence of a minor complex component with a complexity of about 3 × 107, making up less than 10% of the total kDNA.
From these results we conclude that 3–5% of the kDNA consists of a homogeneous class of maxi‐circles catenated in the mini‐circle network. The molecular weight of these maxi‐circles is about 26 × 106 and they contain a unique, non‐repetitive, non‐mini‐circle nucleotide sequence. This component is a prime candidate for the true mitochondrial DNA of trypanosomes.
Purification of Protease II from Escherichia coli by Affinity Chromatography and Separation of Two Enzyme Species from Cells Harvested at Late Log Phase
Authors: PACAUD Michèle. 
Published: March 2005

N‐Acetyl‐d‐arginine linked to an agarose matrix has been used to purify protease II from Escherichia coli by affinity chromatography. The specific adsorption of protease II to this absorbent was achieved in 220 mM potassium phosphate buffer pH 7.6, and the enzyme was‐eluted with l‐arginine.Enzyme preparations from cells harvested at late log phase have been resolved into two molecular species, which differ in specific activity, kinetic constants and carbohydrate content. Both species appeared homogeneous by electrophoresis in conventional buffers and also in the presence of sodium dodecyl sulfate. Only one enzyme species was obtained by the same procedure using bacteria harvested at the middle of exponential growth.
Lactose Synthetase
Authors: IVTT Raymond J. , ROSEMEYER Michael A. 
Published: March 2005

Galactosyl transferase and α‐lactalbumin were concurrently, isolated from bovine milk.The galactosyl transferase had an S20,W of 3.0 S and D20,W of 60 μm2/s. It exists as a monomer of 46000 molecular weight, as determined by sedimentation equilibrium and dodecylsulphatepolyacrylamide electrophoresis. Aggregation of the enzyme was promoted by N‐acetylglucosamine.Sedimentation velocity experiments show an association between galactosyl transferase and α‐lactalbumin in the presence of N‐acetylglucosamine. In contrast, UDP‐galactose, UDP or lactose do not promote protein‐protein association.A complex between galactosyl transferase and α‐lactalbumin was isolated by gel filtration in the presence of excess α‐lactalbumin and either N‐acetylglucosamine or glucose. The complex was stable over arrange of concentrations of these components.The complex is a discrete homogeneous entity with a molecular weight of 60000, corresponding to one molecule of galactosyl transferase and one molecule of α‐lactalbumin.The estimated association constants for the ternary complexes of the two proteins and either of the sugars, suggest that α‐lactalbumin enhances equally the binding of N‐acetylglucosamine or glucose to the galactosyl transferase.
The Enzyme Complex Citramalate Lyase from Clostridium tetanomorphum
Authors: BUCKEL Wolfgang , BOBI Angeliki 
Published: March 2005

The enzyme citramalate lyase from Clostridium tetanomorphum is not stable in crude extracts. However, the inactive enzyme can be reactivated by incubation with dithioerythritol followed by acetylation with acetic anhydride. Reactivation was also obtained with acetate, ATP. MgCl2 and acetate :SH‐enzyme ligases (AMP) from C. tetanomorphum or Klebsiella aerogenes.
Incubation of the inactive enzyme with iodoacetate resulted in rapid loss of enzymic activity as determined by reactivation with acetic anhydride whereas the active enzyme was stable in the presence of iodoacetate. Using iodo[2‐l4C]acetate the sites of carboxymethylation and acetylation where identified as cysteamine residues of the enzyme. The results demonstrate that the active enzyme contains acetyl thiolester residues which play the central role in the catalytic mechanism.
Citramalate lyase was purified by a procedure almost identical to that already described for citrate lyase from K. aerogenes. The molecular weight of citramalate lyase is equal to that of citrate lyase (Mr= 5.2–5.8 × 105) as estimated by gel chromatography and sucrose gradient centrifugation. Polyacrylamide gel electrophoresis of citramalate lyase in sodium dodecylsulfate yielded three polypeptide chains (Mr: α 5.3–5.6 × 104; β 3.3–3.6 × 104; γ 1.0–1.2 × 104) in probably equal molar amounts. These data lead to a hexameric structure (α,β,γ)6 of the complete enzyme.
Pantothenate (5 mol/mol of enzyme) and the essential cysteamine residues were exclusively present in the γ‐chain, the acyl carrier protein of citramalate lyase. The acyl exchange and cleavage functions, probably catalysed by the α and β‐subunits, were measured with acyl‐CoA derivatives which were able to substitute for the natural acyl carrier.
The results demonstrate that citramalate lyase is an enzyme complex with structure and functions closely resembling those of citrate lyase. Although the similarity between citramalate lyase and citrate lyases from various organisms suggests a close evolutionary relationship, these occur in very different, unrelated bacteria. A parallel situation found in the distribution of the nitrogenase system among procaryotes is discussed
The Prosthetic Group of Citrate‐Lyase Acyl‐Carrier Protein
Authors: DIMROTH Peter 
Published: March 2005

The acyl carrier protein of citrate lyase contains adenine, phosphate, sugar, cysteamine, β‐alanine and pantoic acid in a molar ratio of 1:2:2:1:1:1. Peptides containing these components in the same stoichiometric relationship were isolated after proteolytic digestion of acyl carrier protein. All components wefe.linked together in a single prosthetic group. This was released from the peptide by mild alkaline hydrolysis. Under these conditions a phosphodiester bond is cleaved which links the prosthetic group to a serine residue of the peptide.Incubation of the prosthetic‐group‐containing peptide with phosphodiesterase I yielded 4′phosphopantetheine and adenylic acid. The 5′‐AMP was not free but was substituted by presumably an acidic sugar residue, which was released by mild acid hydrolysis yielding free 5′‐AMP.It was concluded from these results that the prosthetic group of citrate Iyase acyl carrier protein consists of a substituted isomeric dephospho‐CoA. This is bound to the protein by the 5′‐phosphate group of adenylic acid. The 4′‐phosphopantetheine residue is bound by a phosphodiester linkage to the 2′ or 3′ position of ribose and the remaining hydroxyl group of ribose is substituted with presumably an acidic sugar residue. The structural similarities of this prothetic group and coenzyme A are discussed and related to the catalytic properties of citrate Iyase.
The Binding of Indole to the α‐Subunit and β2‐Subunit and to the α2β2‐Complex of Tryptophan Synthase from Escherichia coli
Authors: WEISCHET Wolfgang O. , KIRSCHNER Kasper 
Published: March 2005

The binding of indole and indolepropanol phosphate, an analogue of the substrate indoleglycerol phosphate, to the individual α and β2‐subunits and to the α2β2‐complex of tryptophan synthase was studied by equilibrium dialysis. The use of [14C]indole and indolepropanol [32P]phosphate permitted simultaneous binding studies to be carried out. Competition between indole and indolepropanol phosphate in binding to a particular site was taken as evidence for that site being part of the active site of the α‐subunit.The binding of indole to the active site of the α‐subunit is weak (Kd= 18 mM). A second distinct site binds indole more strongly (Kd= 1.5 mM) and interacts with the active site indirectly. It is therefore designated an effector site. Furthermore, the binding of indole and/or indolepropanol phosphate appears to stabilize different conformations of the α‐subunit. The β2‐subunit binds indole only weakly (Kd= 12 mM) to many (n = 10) sites per polypeptide chain. The α2β2‐complex retains one or two sites per αβ‐equivalent of relatively high affinity (Kd= 1.2 mM). The active sites of the component α and β‐subunits probably belong to the second class of many (n = 40) sites of low (Kd= 30 mM) affinity for indole. These findings support conclusions from the literature that both bi‐substrate reactions involving indole catalyzed by tryptophan synthase and its subunits must follow strictly ordered addition mechanisms with the respective other substrate adding first.
Peroxidase from Human Cervical Mucus
Authors: SHINDLER Julian S. , CHILDS Robert E. , BARDSLEY William G. 
Published: March 2005

A peroxidase has been isolated from pooled specimens of human cervical mucus. Investigation of substrate and inhibitor specificity, pH optimum, chromatography on Sephadex G‐200, disc electrophoresis, ultraviolet absorption spectrum and the effects of temperature and ionie strength variation are described.A study of the steady‐state kinetics is reported using hydrogen peroxide and 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) as substrates and a catalytic mechanism is proposed
The Mechanism of the Synthesis of Indoleglycerol Phosphate Catalyzed by Tryptophan Synthase from Escherichia coli
Authors: WEISCHET Wolf gang O. , KIRSCHNER Kasper 
Published: March 2005

The mechanism of indoleglycerol phosphate synthesis from indole and d‐glyceraldehyde 3‐phosphate catalyzed by tryptophan synthase has been investigated by steady‐state kinetic techniques. The equilibrium constant and the progress curves were measured by use of the difference in absorbance between indole and indoleglycerol phosphate. Stopped‐flow measurements show that only the nonhydrated form of d‐glyceraldehyde 3‐phosphate serves as substrate. The product analogue indolepropanol phosphate was used as an inhibitor to discriminate between possible mechanisms.The data agree well with an ordered addition mechanism with d‐glyceraldehyde 3‐phosphate adding first. Mechanisms involving random addition of substrates or ordered addition with indole adding first can he excluded because indolepropanol phosphate is a competitive inhibitor only towards glyceraldehyde 3‐phosphate. The high affinity of tryptophan synthase for indoleglycerol phosphate leads to product inhibition even at small extents of reaction. Glyceraldehyde 3‐phosphate combines with the enzyme with an apparent second‐order rate constant, which is not diffusion controlled and generates a site with. high affinity for indole
Steady‐State Kinetic Studies of the Synthesis of Indoleglycerol Phosphate Catalyzed by the α Subunit of Tryptophan Synthase from Escherichia coli
Authors: WEISCHET Wolfgang O. , KIRSCHNER Kasper 
Published: March 2005

For the α subunit of tryptophan synthase and at constant concentration of d‐glyceraldehyde 3‐phosphate the saturation curves with respect to indole concentration are weakly sigmoidal. This phenomenon can be explained by interaction between indole bound to the effector site established previously and the active center of the monomeric α subunit. Kinetic studies of the inhibition of indoleglycerol phosphate synthesis by the analogue indolepropanol phosphate show that the inhibition is competitive with respect to d‐glyceraldehyde 3‐phosphate and non‐competitive with respect to indole. Mechanisms with random addition of substrates or ordered addition with indole binding first can therefore be excluded. A quantitative fit of the data has been obtained to an ordered addition mechanism with d‐glyceraldehyde 3‐phosphate binding first and with a distribution of the enzyme between two states differing in V, governed by the binding of indole to the efector site.The kinetic constants obtained for the α subunit have been compared with those of the α2β2 complex of tryptophan synthase. Protein‐protein interaction of the α subunit with the β2 subunit (a)‐does not alter the catalytic mechanism of indoleglycerol phosphate synthesis, (b) suppresses the substrate activation by indole, and (c) changes the various equilibrium, rate and steady‐state constants in the sense of conveying higher substrate specificity and catalytic efficiency to the α‐subunit. The ocurrence of local and gross conformational changes in the tryptophan synthase system is discussed
A Comparison between the Common Type and a Rare Genetic Variant of Human Cupro‐Zinc Superoxide Dismutase
Authors: MARKLUND Stefan , BECKMAN Gunhild , STIGBRAND Torgny 
Published: March 2005

Human cupro‐zinc superoxide dismutase is polymorphic in northern Sweden. The genetic variant type has a lower mobility at electrophoresis in alkaline buffer. The enzyme was isolated from erythrocytes from one of the rare homozygotes and its properties compared to those of the common type.The isoelectric point of the variant was higher (4.85) than that of the common type (4.7). Small differences in amino acid composition were found but no definite amino acid substitutions could be pointed out. The molecular weights were equal as judged from electrophoreses in polyacrylamide gels in the presence of dodecylsulphate. The ultraviolet spectra were similar.Parameters related to the active site of the enzyme were very similar; i.e. specific activity and sensitivity to inhibition by cyanide and by H2O2. These parameters, especially the latter two, differ widely between species. Both enzymes were stable for weeks at neutral pH at 37 °C, whereas the common type was significantly more stable at pH 4 and pH 11 and also at incubation in neutral buffer at 70 °C.It appears that the active site of the variant is conserved whereas the stability of the enzyme is affected.
Comparison of Some Properties of Chromatin Non‐Histone Proteins and Nuclear Sap Proteins
Authors: UMANSKY Samuil R. , ZOTOVA Rimma N. , KOVALEV Yurij I. 
Published: March 2005

The properties of rat liver and thymus non‐histone and nuclear sap proteins were compared. The distribution of total, labile‐bound and 0.35 M NaCl extractable non‐histone proteins from one organ on polvacrylamide‐gel electrophoresis in the presence of sodium dodecylsulphate is quite similar. On electrophoresis non‐labelled and 32P‐labelled non‐histone and nuclear sap proteins from one organ differ from one another both qualitatively and quantitatively. We did not find an appreciable difference between non‐labelled non‐histone proteins isolated from liver and thymus. The distribution of 32Plabelled non‐histone proteins from various organs differs quantitatively rather than qualitatively. Non‐labelled and 32P‐labelled nuclear sap proteins from liver and thymus differ significantly. ‘Free’ nuclear sap proteins and the proteins of ribonucleoprotein particles from thymus nuclei contain a great quantity of identical polypeptides, whereas other polypeptides are specific to each of these protein fractions. Upon incubation of nuclei with [γ‐32P]ATP the label is incorporated into all the fractions of nuclear protein. The nuclear proteins are phosphorylated at decreasing rates in the order: labile‐hound non‐histone proteins > firmly bound non‐histone proteins > ‘free’ nuclear sap proteins = proteins of ribonucleoprotein particles > histones.Nuclear sap and non‐histone proteins contain protein kinases capable of phosphorylating both these proteins and histones. Histone phosphorylation is sharply inhibited after addition of DNA, the protein kinases of nuclear sap phosphorylating less effectively the histones complexed with DNA than the non‐histone proteins. Both non‐histone and nuclear sap proteins contain fractions interacting in vitro with DNA. Denatured DNA hinds twice as much 32P‐labelled nuclear sap proteins and a little more 32P‐labelled non‐histone proteins than native DNA. Denatured DNA binds nonhistoric and nuclear sap proteins much more effectively than native DNA. It was shown by the membrane filter technique that the major part of the nuclear sap and non‐histone proteins interacting with native DNA binds to it non‐specifically. A certain portion of non‐histone and nuclear sap proteins interacts specifically with homologous denatured DNA. The possible role of non‐histone and nuclear sap proteins in the regulation of transcription is discussed.
Enzymic Synthesis of Lignin Precursors
Authors: WENGENMAYER Herta , EBEL Jürgen , GRISEBACH Hans 
Published: March 2005

A cinnamoyl‐coenzyme A reductase catalyzing the NADPH‐dependent reduction of substituted cinnamoyl‐CoA thiol esters to the corresponding cinnamaldehydes was isolated from cell suspension cultures of soybean (Glycine max L. var. Mandarin). A 1660‐fold purification of the enzyme was achieved by (NH4)2SO4 fractionation, chromatography on DEAE‐cellulose, hydroxyapatite and Sephadex G‐100 and affinity chromatography on 5′‐AMP‐Sepharose. The apparent molecular weight of the reductase was found to be about 38000 on the basis of the elution volume from a Sephadex G‐100 column. Maximum rate of reaction was observed between pH 6.0 and 6.2 in 0.1—0.2 M citrate buffer at 30 °C. The enzyme was markedly inhibited by thiol reagents.The reductase showed a high degree of specificity for cinnamoyl‐CoA esters. Feruloyl‐CoA was the substrate with the lowest Km value (73 μM) and highest V (230 nkat/mg) followed by 5‐hydroxyferuloyl‐CoA, sinapoyl‐CoA, p‐coumaroyl‐CoA, caffeoyl‐CoA and cinnamoyl‐CoA. No reaction took place with acetyl‐CoA. The Km value for NADPH varied with the type of substrate. Km values of 28, 120, and 290 pM were found with feruloyl‐CoA, sinapoyl‐CoA, and p‐coumaroyl‐CoA, respectively. The rate of reaction observed with NADH was only about 5 % of that found with NADPH.The reaction products CoASH and NADP+ inhibited the reaction. The Ki values were in the range of 0.5—1 mM and the inhibition was of a noncompetitive (mixed) type. The role of the reductase in the biosynthesis of lignin precursors is discussed.
Two Forms of RNA Polymerase B in Yeast
Authors: DEZÉLÉE Sybille , WYERS Françoise , SENTENAC André , FROMAGEOT Pierre 
Published: March 2005

Special care to prevent proteolysis during yeast RNA polymerase B purification leads to the appearance of two forms of enzymes, BI and BII, with different molecular weight (465000 and 435000, respectively). The two forms of enzyme can be separated by ion‐exchange chromatography or polyacrylamide gel electrophoresis. Their subunit structures were compared by sodium dodecylsulfate gel electrophoresis, the only observed difference between the two enzymes is in the molecular weight of the heaviest subunit which is 220000 for enzyme BI and 180000 for enzyme BII. Otherwise, the two enzymes have seven common subunits of molecular weights 150000, 45000, 26000, 22500, 14500, 12500 and 9000. Two additional polypeptide chains of 32000 and 16500 Mr are dissociated from the enzyme upon polyacrylamide gel electrophoresis or DEAF Sephadex chromatography.The largest subunit of enzyme BI (Mr 220000) can be specifically cleaved in vitro by a yeast protease extract, generating a polypeptide chain indistinguishable from the largest subunit of enzyme BII. This proteolytic cleavage of enzyme BI in vitro is inhibited by phenylmethylsulfonyl fluoride and does not significantly change the activity of the enzyme with single‐stranded or double‐stranded DNA as template. The precursor‐product relationship of the different forms of class B RNA polymerises in eukaryotic cells is discussed.
Influence of DNA Acidification on DNA Premelting and Template Properties
Authors: SAROCCHI Marie‐Thérèse , DAUSSE Jean‐Pierre , GUSCHLBAUER Wilhelm 
Published: March 2005

Acidification of a T7 DNA sample was found to be partly irreversible as ultraviolet difference spectra measured at various sub‐melting temperatures were different from those observed for a ‘normal’ DNA sample. This implies some subtle conformational change which is not reversed by return to neutral pH. In the same conditions, only poly(purine)· poly(pyrimidine) polymers behaved in a different manner, during premelting. according to whether they were previously acidified or not.The properties of acidified and reneutralized T7 DNA were also investigated for Escherichia coli RNA polymerase binding and transcription. An inhibition of RNA synthesis and chain initiation was observed. The results suggest that the binding of the enzyme is affected. RNA synthesized is specific but there is a decrease in the number and in the stability of the RNA‐polymerase · DNA complexes.