bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fluorescence of Proteins in 6‐M Guanidine Hydrochloride
Authors: PAJOT Patrick 
Published: March 2005

To determine the tryptophan content in proteins, an analytical ultraviolet fluorescence method is proposed based on making uniform the environment of aromatic chromophores in 6–7 M guanidine hydrochloride.The fluorescence intensity scale is calibrated using standard solutions of free tryptophan. A correlation coefficient between the fluorescence of protein tryptophanyl residues and of free tryptophan was estimated in testing 17 well characterized proteins.This method is particularly suited to proteins carrying groups absorbing in the 290–370‐nm region, such as flavin, heme and pyridoxal phosphate and in the presence of substances such as 2‐mercaptoethanol which prohibit the use of the spectroscopic or magnetic circular dichroism methods. It is less time‐consuming than techniques requiring hydrolysis or chemical reactions.
Studies on the Mechanism of Site I Energy Conservation
Authors: OHNISHI Tomoko 
Published: March 2005

Of the several iron‐sulfur centers detected in the site I segment of the respiratory chain, centers N‐2 and N‐1a alone exhibit apparent phosphate‐potential dependent half‐reduction potentials, indicating their possible involvement in energy conservation.
At high phosphate potential, the apparent half‐reduction potential of center N‐2 shifts positively by 125 ± 20 mV, while center N‐1a shifts negatively by approximately 60 mV.
The redox state of individual iron‐sulfur centers in various metabolic states was analyzed. Center N‐2 is highly reduced (> 90%) in “state 4” mitochondria, while center N‐1a stays mostly oxidized.
In a submitochondrial system, ATP addition induced reduction of center N‐2 if the Eh of the suspension was poised from the high potential side of site I using the succinate/fumarate couple. In contrast, center N‐2 was oxidized upon energization, if the Eh of the system was poised from the low potential side using the NADH/NAD couple.
Based on these redox behaviors of center N‐2, a tentative hypothesis of site I energy transduction was proposed.
Plastocyanin : Possible Significance of Quaternary Structure
Authors: SIEGELMAN Mark H. , RASCHED Ihab R. , KUNERT Karl‐Josef. , KRONECK Peter , BÖGER Peter 
Published: March 2005

Some properties of the blue copper protein plastocyanin from the green alga Scenedesmus have been investigated and compared with that from spinach, including amino acid composition, isoelectric point and copper content. The protein from Scenedesmus contains two, that from spinach four copper atoms per molecular weight of 40000.A combination of sodium dodecylsulfate/polyacrylamide gel electrophoresis and quantification of sulfhydryl groups indicates a strong preference for a species composed of 4 polypeptide chains of identical amino acid composition representing the enzymically active entity. Due to various treatments the subunits of both plastocyanins are detected as either monomer species alone or as monomer and dimer in a molar ratio of 2 : 1 on sodium dodecylsulfate gels.The four ‐SH groups per molecule are of different reactivity: two ‐SH groups can be detected after destruction of the chromophore; two more (forming an S‐S bridge in the dimer) become evident after appropriate reduction.A KCN treatment for production of apoprotein is reported and the use of electrodialysis to improve incomplete apoprotein formation. These studies lend support to the proposal of a quaternary structure.Apoproteins were subjected to dodecylsulfate gel analysis, which proved to be an effective means of estimating both the extent of apoprotein formation and its reconstitution to the holoprotein.
The Incorporation of Newly Synthesized RNA into Nuclear Ribonucleoprotein Particles after Oestrogen Administration to Immature Rats
Authors: KNOWLER John T. 
Published: March 2005

Ribonucleoprotein particles, known as informofers or heterogeneous nuclear ribonucleoprotein particles (hnRNA · protein), have been extracted from rat uterine nuclei and found to have properties similar to those characterized from other tissues.Incorporation of RNA precursors into the RNA of these particles is stimulated up to 8‐fold by oestrogen administration to rats.When uteri are dissected from oestrogen‐treated rats and incubated in vitro with radioactive RNA precursor, only the RNA in the ribonucleoprotein particles is synthesized at a rate faster than can be accounted for by increase in the uptake of precursor. This contrasts with previous studies where both hormone treatment and incorporation of radioactive precursor were performed in vivo and the synthesis of all RNA species was stimulated [Knowler, J. T. and Smellie, R. M. S. (1971) Biochem. J. 125, 605‐614; Knowler, J. T. and Smellie, R. M. S. (1973) Biochem. J. 131, 689–697].
Structural Properties of Escherichia coli RNA Polymerase Subunits
Authors: LOWE Peter A. , MALCOLM Alan D. B. 
Published: March 2005

The surface of the RNA‐polymerase ‐ DNA complex possesses an exposed polypeptide loop.
Proteinases with differing specificities (trypsin, chymotrypsin, subtilisin and clostripain) preferentially cleave the exposed region.
The cleaved polypeptide is reassembled into RNA polymerase by renaturation from a solvent which promotes a random coil conformation.
Isolated β subunit has a proteolytically resistant nucleus of approximately 70000 molecular weight. This resistant polypeptide may be generated by trypsin, chymotrypsin, subtilisin or clostripain.
Isolated α subunits are comparatively resistant to proteolysis.
Although of similar molecular weights β and β′ appear to have unrelated primary sequences and markedly different conformations in free solution.
Digestion of the β subunit may be blocked by formation of the α2β subassembly.
Evidence is presented suggesting that β in the intact enzyme (α2ββ′) possesses the exposed polypeptide loop.
Role of Calcium Ions in the Thermostability of Thermolysin and Bacillus subtilis var. amylosacchariticus Neutral Protease
Authors: TAJIMA Masahiro , URABE Itaru , YUTANI Katsuhide , OKADA Hirosuke 
Published: March 2005

The stabilizing effect of calcium ions on thermolysin and Bacillus subtilis var. amylosacchariticus neutral protease has been investigated. Calcium and zinc ions were removed from the proteases by gel filtration over Sephadex G‐25 equilibrated with metal chelating agents. Using these enzymes with different metal content, heat inactivation kinetics were studied at various temperatures. Removal of calcium ions caused a sharp decrease in thermostability and diminished the values of the activation enthalpy (ΔH*) and entropy (ΔS*) for heat inactivation. There was little difference in stability between thermolysin containing 0.3 g‐atom fmol and B. subtilis neutral protease containing 1.4 g‐atoms/ mol.Calcium binding isotherms of the proteases were obtained by equilibrium gel chromatography with various concentrations of free calcium ions. Thermolysin had four independent calcium binding sites with an identical intrinsic binding constant (K) of 2.0 104 M−1. B. subtilis neutral protease had four independent sites. The K value for three sites was 1.1 105 M−1 and the binding constant for the other site was 1.5 103 M−1. There was little difference in total free energy change for calcium binding between these proteases.From these results it is concluded that the stabilizing effect of calcium on these enzymes is almost equal, and the extra thermal stability of thermolysin is likely to come from its polypeptide chain structure.
Haptoglobin‐Haemoglobin Interaction
Authors: LAVIALLE Francoise , ROGARD Monique , ALFSEN Annette 
Published: March 2005

The formation of two different complexes when haptoglobin (Hp) and haemoglobin (Hb) are mixed in a 1:1 molar ratio is demonstrated by isoelectrofocusing. In these two complexes, the affinity of Hp for Hb is shown to be different, since Hb can be displaced only from one of the complexes, by a further addition of Hp. This is confirmed by a quantitative study of the reaction stoichiometry, when [Hp]/[Hb] = 1 and [Hp]/[Hb] > 1, which allows an evaluation of the amount of each complex formed.All these data cannot be explained other than by the existence of two forms of Hp molecule and a reaction scheme which fits these experiments, is proposed.
Phosphatidic Acid Metabolism in Rat Liver Microsomes
Authors: TZUR Ruth , SHAPIRO Benyamin 
Published: March 2005

Rat liver microsomes contain phosphatidate phosphatases which split phosphatidic acid into inorganic phosphate and diacylglycerol and a system of phospholipases and lipases, which split phosphatidic acid into free fatty acids, glycerol and inorganic phosphate.In the presence of ATP, CoA and [1‐14C]palmitate, part of the monoacyl‐sn‐glycerol 3‐phosphate formed by phospholipase action is reesterified, yielding radioactive phosphatidic acid. The sum of di‐ and triacylglycerols formed from phosphatidic acid in the presence of ATP and CoA exceeded the amount of diacylglycerol fromed in their absence.The yield of neutral lipids from sn‐glycerol 3‐phosphate and monoacyl‐sn‐glycerol 3‐phosphate markedly exceeded that from phosphatidic acid.Comparison of the yields of di‐ and triacylglycerols from glycerol‐labelled and fatty‐acid‐labelled phosphatidic acid was used to establish the extent of deacylation and reacylation. About 60% of the diacylglycerol was formed by direct dephosphorylation. The triacylglycerols, on the other hand, were formed almost exclusively from recycled phosphatidic acid.
Enzymic Unwinding of DNA
Authors: ABDEL‐MONEM Mahmoud , HOFFMANN‐BERLING Hartmut 
Published: March 2005

Evidence from various sources in the literature suggests that, in connection with DNA, ATP dephosphorylation can be used to provide energy for mechanical effects. Starting from this concept we have studied a novel DNA‐dependent ATPase purified to 90% homogeneity from Escherichia coli. The enzyme has a peptide weight near 180000 and, in high salt, is a monomeric, probably highly anisometric molecule. In salt‐free buffer, where the ATPase activity is highest, the enzyme forms aggregates. ATP is the preferred substrate (Km 0.27 mM) and dephosphorylated at the γ‐position at a maximal rate near 104 molecules per enzyme monomer per min at 35 °C. A requirement for divalent cation is best satisfied by Mg2+ or Ca2+ and the requirement for DNA best by the single‐stranded, circular DNA of phages øX174 (Km 62 nM nucleotide) and fd indicating that the enzyme recognizes internal DNA regions. When saturated with E. coli DNA unwinding protein, øX DNA is not accepted but, once in contact with the DNA, the enzyme is little inhibited by unwinding protein. Apparently the unwinding protein interferes preferentially with the recognition of DNA.The enzyme does not detectably cleave DNA, and for this and genetic reasons is not identical with the recBC ATPase or the K12 restriction ATPase of the extracted cells. The enzyme is probably not identical either with the dnaB‐product‐associated ATPase or the ATPase activity found in DNA polymerise III holoenzyme under appropriate conditions, and it is certainly not identical with a DNA‐dependent ATPase of molecular weight 69000 from E. coli which has recently been purified. Attempts to ascribe the enzyme to other genes, including recA, lex and rep, have failed.
Identification of the 30‐S Ribosomal Proteins at the Decoding Site by Affinity Labelling with a Reactive Oligonucleotide
Authors: LÜHRMANN Reinhard , GASSEN Hans Günter , STÖFFLER Georg 
Published: March 2005

Proteins S18 and S1 were identified by immunological and electrophoretic techniques as constituents of the 30‐S ribosomal decoding site by the use of G‐U‐U‐i[3H]acn5U (where i[3H]acn5U =5‐iodo[3H]acetylaminouridine) as a chemical affinity label. S12 was modified to a minor extent and mainly wheel assays were carried out in the absence of tRNA

Evidence for the specificity of the covalent binding of the oligonucleotide probe is given by the following experiments: G‐U‐U‐iacn5U bound twice as much Val‐tRNA as compared to G‐U‐U‐U. The covalent binding was strongly inhibited by prebound poly(U)‐Phe‐tRNA. ‘Programmed’ ribosomes, i.e. ribosomes which contained only covalent bound oligonucleotide, stimulated the binding of Val‐tRNA.
Val‐tRNA bound to G‐U‐U‐iacn5U labelled ribosomes could not be transferred to puromycin. This might indicate that binding of the oligonucleotide occurred at the aminoacyl site.
80–90% of the radioactivity was found in the 30‐S subunit and only the protein moiety of the ribosome was modified.
Unambiguous identification of the labelled proteins was achieved by immunological techniques and revealed proteins S1 and S18 as constituents of the 30‐S ribosomal decoding site.
Activation Parameters of the Adenosine Triphosphatase of Micrococcus lysodeikticus A Comparison of the Soluble and Membrane‐Bound Forms of the Enzyme
Authors: AYALA Juan , NIETO Manuel , CARREIRA Josè , MUÑOZ Emilio 
Published: March 2005

The Arrhenius plots for the membrane‐bound ATPase and its soluble form purified from Micrococcus Iysodeikticus, presented discontinuities near 30°C at pH 7.5. Glycerol‐containing lipids were not responsible for these discontinuities. The values of the enthalpies of activation were 12 (soluble) and 22 (membrane‐bound) kcal/mol (50.2 and 92.0 kJ/mol) above 30°C and 42 (soluble) and 29 (membrane‐bound) kcal/mot (175.7 and 121.3 kJ/mol) below that temperature.The results suggested that both molecular forms of the ATPase were able to adopt at least two different structures, above and below the critical temperature. Of the two, only the hightemperature structure seemed to be enzymically active. In the case of lipid‐dependent ATPases, such as the Escherichia coli enzyme, the transition between both enzyme structures probably occurred with simultaneous “melting” of their lipid microenvironment.
A Study of the Interaction of 4‐Bromomercuriocinnamic Acid with α‐Chymotrypsin by 79Br and 81Br Pulsed Nuclear‐Magnetic Resonance
Authors: GARNETT Michael W. , HALSTEAD Thomas K. , HOARE David G. 
Published: March 2005

Br− has been used as a nuclear magnetic resonance (NMR) probe to study the reversible association of α‐chymotrypsin and an Hg‐labelled substrate (4‐bromomercuriocinnamic acid, BrHgCin) which rapidly exchanges Br‐. T1 was measured for 79Br and 81Br, using a pulse spectrometer.Values of the parameters that determineTl, Obs in aqueous solutions of KBr (pH = 5.5) containing α‐chymotrypsin and BrHgCin are reported. It is found that the rate of Br exchange is diffusion‐limited and faster than the rate of reorientation of the BrHgCin‐α‐chymotrypsin complex.The rate constant for the formation of the covalent BrHgCin‐α‐chymotrypsin complex determined by this technique agrees well with previously published data. The rapid rate of Br exchange with the complex, however, is incompatible with the side chain of BrHgCin being entirely buried in a nonpolar pocket on the enzyme but compatible with the side chain being exposed to the solution. The contribution to the NMR signal from the non‐covalent complex is negligible.
Binding of a Metyrapone Spin Label to Microsomal Cytochrome P‐450
Authors: RUF Hans Heinrich , NASTAINCZYK Wolfgang 
Published: March 2005

Probing of the active site of microsomal cytochrome P‐450 was carried out with a spin label derived from 2‐methyl‐1,2‐bis(3‐pyridyl)‐1‐propanone (metyrapone). Its optical binding spectra to cytochrome P‐450 resemble the spectra with metyrapone. The electron paramagnetic resonance spectrum of the Spin label in microsomes indicates binding with strong immobilization. Since the apparent optical and EPR binding constants agree very well (Ks ∼ 2 10−5 M), and metyrapone is found to displace the spin label, we conclude, that the spin label binds to the active site of cytochrome P‐450. Addition of detergents or an increase in temperature mobilizes the bound spin label slightly.The EPR signal accounts for only 60% of the bound spin label due to its dipolar interaction with the low‐spin ferric heme of cytochrome P‐450. From this finding, the distance between nitroxide and iron is evaluated to be 11 Å. This supports the model that one of the pyridine nitrogens of metyrapone is coordinated to the iron of cytochrome P‐450. The bound spin label shows virtually no interaction with ferricya'nide as if the active site of cytochrome P‐450 is a hydrophobic pocket not accessible to ions.
Glycopeptides from Bovine Liver Basement Membrane and Plasma Membrane
Authors: GIBBONS Richard A. , DIXON Stephen N. , SELLWOOD Richard 
Published: March 2005

Proteolytic digests of liver plasma‐cell membranes from the cow were fractionated to yield two homogeneous glycopeptides and a third preparation about 92% pure. The composition of the two homogeneous glycopeptides made it clear that they were derived from basement membrane material rather than the plasma membrane. Ruminants are unusual in having large amounts of basement membrane in the liver while other animals generally have little or none.Both basement‐membrane‐derived glycopeptides contained a glucosyl galactosyl disaccharide linked to hydroxylysine, the smaller one contained no other sugar structure but the larger one contained in addition an acidic heterosaccharide, the two chains probably being linked separately to the same molecule. Smith degradation and β‐elimination operations show that this heterosaccharide has an inner structure containing mannose and hexosamine, with the sugars galactose, N‐glycollyl‐neuraminic acid and fucose situated more peripherally. The amino‐acid‐hetero‐saccharide linkage is alkali stable.The third glycopeptide, which may be plasma‐membrane‐derived, differs from the heterosaccharide saccharide described above in that it contains no glucose and contains some O‐seryl and O‐threonyl amino‐acid‐sugar linkage. It, too, has a periodate‐resistant structure of hexosamine and mannose.
DNA‐Dependent RNA Polymerase C from Xenopus laevis Ovaries Ability to Transcribe Intact Double‐Stranded DNA
Authors: LONG Eric , DINA Dino , CRIPPA Marco 
Published: March 2005

DNA‐dependent RNA polymerase C, partially purified from Xenopus laevis ovaries, has been resolved by DEAE‐Sephadex chromatography in two forms, eluting at 0.2 M and 0.3 M ammonium sulfate, respectively. Both are sensitive to high concentrations of α‐amanitin (200 μg/ml). Their ionic strength dependence and divalent cation requirements are indistinguishable. Quantitatively, RNA polymerase C represents the major form of RNA polymerase activity solubilized from the ovaries.Both RNA polymerases C are able to transcribe efficiently either high‐molecular‐weight Xenopus DNA or intact adenovirus DNA, as compared to nicked DNA. In contrast, RNA polymerase A has little activity on an intact DNA template. The salt dependence of the RNA polymerases C activity is different on the two kinds of template. Nicked DNA is efficiently transcribed up to a salt concentration of 100 mM ammonium sulfate. On intact DNA, optimal transcription is obtained at 40 mM ammonium sulfate and is inhibited by higher salt concentrations.
The Purification and Characterisation of the Human‐Serum Binding Protein for the 25‐Hydroxycholecalciferol (Transcalciferin) Identity with Group‐Specific Component
Authors: BOUILLON Roger , VAN BAELEN Hugo , ROMBAUTS Wilfried , DE MOOR Pieter 
Published: March 2005

The binding protein for 25‐hydroxyvitamin D3 has been isolated from human serum by monitoring the recovery of 3H‐labeled 25‐hydroxyvitamin D3. After a 500‐fold purification a pure protein was obtained as judged from the constant specific activity (ratio of absorbance versus radioactivity) on agarose and DEAE‐Sephadex chromatography and on the presence of a single band on both cellulose acetate and polyacrylamide gel electrophoresis.The molecular weight of the purified protein was measured by gel filtration on agarose (56000), Sephadex G‐75 (58000) and dodecylsulfate‐polyacrylamide gel electrophoresis (56000). On sucrose gradient ultracentrifugation a sedimentation coefficient of 4.1 S was found. The isoelectric point was 4.89 S on isoelectric focusing. The stability of the protein at 60°C was enhanced by the presence of excess 25‐hydroxyvitamin D3. On tandem crossed immunoelectrophoresis the purified binding protein was found to be identical to the one present in whole serum.The activity of the isolated protein was demonstrated by a Ka at 4 C of 1.2 1010 I mol−1. A binding capacity of 0.8 binding site/molecule was measured on a Sephadex G‐25 column.During immunological studies with this protein it became evident that the binding protein is identical with another serum protein known as group‐specific component (Gc).In analogy to other serum binding proteins we propose to call this group‐specific component/25‐hydroxyvitamin‐D3‐binding protein transcalciferin.
Carboxymethylated Liver Alcohol Dehydrogenase
Authors: HAREMAN Michael J. 
Published: March 2005

The rate constants for NADH binding and dissociation for carboxymethylated alcohol dehydrogenase have been determined and compared to those for the native enzyme.
Steady‐state and transient kinetic experiments Lave shown that the hydrogen transfer step is rate‐determining for oxidation of ethanol by carboxymethylated alcohol dehydrogenase. The rate constant of 0.19 s−1 is considerably slower than that for the native enzyme.
The steady‐state parameter, V/[E], was obtained for each of a series of alcohols and correlated with the Taft σ* parameter. The linear relationship obtained indicates that the same step, hydrogen transfer, is rate‐determining for all the alcohols. The Q* value obtained is the same as for the native enzyme; the implications of this for the mechanism of hydrogen transfer are discussed.
Studies on the Structure and Function of Chick‐Oviduct Chromatin
Authors: STRÄTLING Wolf H. , O'MALLEY Bert W. 
Published: March 2005

Chromatin prepared at various stages of hormone‐mediated development of the chick oviduct was investigated for the relative proportions of transcriptionally active (fraction I) and repressed (fraction II) fractions by ECTHAM‐cellulose chromatography. During primary stimulation with estrogen, the amount of chromatin DNA in fraction I plotted as a function of time of stimulation showed a bell‐shaped profile, similar to the profile obtained earlier for the number of chromatin sites available to RNA polymerase for initiation of RNA synthesis. Chromatin from a transcriptionally inactive system, hen erythrocytes, eluted mainly (98 %) as fraction II.The, transcriptionally active fraction I of estrogen‐stimulated oviduct contained a 4‐fold greater RNA polymerase II activity than was found in fraction II. This could be explained by a differential inhibition of RNA polymerase activity in fraction II since enzyme preparations extracted and purified from both chromatin fractions showed equal activities. In support of this finding, fraction I eluted from ECTHAM‐cellulose showed a 4‐fold greater concentration of rifampicin‐resistant RNA chain initiation sites as compared to fraction II.When chromatin from oviduct mince was incubated with labeled progesterone and 17β‐estradiol and was chromafographed on ECTHAM‐cellulose, the transcriptionally active fraction also contained a 4‐fold greater concentration of bound hormone (per weight DNA) as compared to the repressed fraction