bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigations of the Structure of 3‐Methylcrotonyl‐CoA Carboxylase from Achromobacter
Authors: SCHIELE Ulrich , NIEDERMEIER Rosa , STÜRZER Magdalena , LYNEN Feodor 
Published: March 2005

It was shown by gel electrophoresis in sodium dodecylsulphate solution that 3‐methylcrotonylCoA carboxylase from Achromobacter IVS is composed of two different subunits with molecular weights of about 78000 and 96000, respectively. The biotin is bound to the heavier subunit. It was previously found that 3‐methylcrotonyl‐CoA carboxylase contains four biotin molecules per complex. A complex composed of four of each subunit would thus have a molecular weight of about 700000. This is compatible with the molecular weight of 760000 determined earlier by analytical ultracentrifugation.Both subunits were isolated preparatively. As the subunits, unlike the complex, are very sensitive to oxygen, special precautions had to be taken during isolation. The biotin‐containing subunit was isolated by chromatography on DEAE‐cellulose in 5 M urea. It no longer catalyzed the overall reaction, yet could still carboxylate free biotin. The biotin‐free subunit was separated after dissociation of the enzyme by three‐days' dialysis at pH 9.8 under nitrogen. On chromatography over a Sepharose‐bound avidin column, the biotin‐subunit was fixed and the biotin‐free subunit was eluted unretarded. The latter subunit showed no enzymic activity. After the addition of the biotin‐containing subunit, overall activity was regenerated.The speed of reassociation is very much enhanced by 3‐methylcrotonyl‐CoA. It was shown by reassociation experiments under different conditions that probably an initial complex AxBy is formed, possessing a binding site for 3‐methylcrotonyl‐CoA. Upon, the binding of this substrate the conformation may be changed to a form favourable for reconstitution.Finally, the structures of biotin enzymes from different sources are compared. In the course of evolution there is a tendency toward integration of the different constituent proteins into only one polypeptide chain.
Biochemical Research on Oogenesis
Authors: WEGNEZ Maurice , MAZABRAUD André , DENIS Herman , PÉTRISSANT Guy , BOISNARD Monique 
Published: March 2005

The nucleotide sequence of initiator tRNA, tRNAfMet, from vitellogenic oocytes of Xenopus laevis was determined. The sequence was deduced from analysis of all T1 and pancreatic oligonucleotides and comparison with the sequence of initiator tRNA from other animal species. At least 80% of all initiator tRNA molecules from oocytes have the same nucleotide sequence. This means that most and probably all initiator tRNA genes which are active in oocytes are identical to one another. No structural difference was observed between liver and oocyte initiator tRNAs.Initiator tRNA from X. laevis has the same nucleotide sequence as initiator tRNA from several species of mammals. The genes coding for this RNA have therefore remained unchanged in the mammalian and amphibian lines for at least 300000000 years.
Subcellular Distribution of Histone‐Degrading Enzyme Activities from Rat Liver
Authors: HEINRICH Peter C. , RAYDT Gerhard , PUSCHENDORF Bernd , JUSIĆ Mirna 
Published: March 2005

Chromatin prepared from liver tissue contains a histone‐degrading enzyme activity with a pH optimum of 7.5–8.0, whereas chromatin isolated from purified nuclei is devoid of it. The histone‐degrading enzyme activity was assayed with radioactively labelled total histories from Ehrlich ascites tumor cells. Among the different subcellular fractions assayed, only lysosomes and mitochondria exhibited histone‐degrading enzymes. A pH optimum around 4.0–5.0 was found for the lysosomal fraction, whereas 7.5–8.0 has been found for mitochondria. Binding studies of frozen and thawed lysosomes or mitochondria to proteinase‐free chromatin demonstrate that the proteinase associated with chromatin isolated from frozen tissue originates from damaged mitochondria.The protein degradation patterns obtained after acrylamide gel electrophoresis are similar for the chromatin‐associated and the mitochondrial proteinase and different from that obtained after incubation with lysosomes.The chromatin‐associated proteinase as well as the mitochondrial proteinase are strongly inhibited by 1.0 mM phenylmethanesulfonyl fluoride. Weak inhibition is found for lysosomal proteinases at pH 5. Kallikreín‐trypsin inhibitor, however, inhibits lysosomal proteinase activity and has no effect on either chromatin‐associated or mitochondrial proteinases. The higher template activity of chromatin isolated from a total homogenate compared to chromatin prepared from nuclei may be due to the presence of this histone‐degrading enzyme activity.
Pyruvate Carboxylase Affinity Labelling of the Magnesium Adenosine Triphosphate Binding Site
Authors: EASTERBROOK‐SMITH Simon B. , WALLACE John C. , KEECH D. Bruce 
Published: March 2005

The 2′,3′‐dialdehyde derivative of ATP (oATP) was prepared by periodate oxidation and on the following criteria was considered to be an effective affinity label. The magnesium complex of this derivative (Mg‐oATP2−) was shown to be a linear competitive inhibitor with respect to MgATP2− in both the acetyl‐CoA‐dependent and ‐independent activities of the enzyme but was a non‐competitive inhibitor with respect to bicarbonate, and an uncompetitive inhibitor with respect to pyruvate. Mg‐oATP was covalently bound to pyruvate carboxylase by reduction using sodium borohydride with concurrent irreversible inactivation of the enzyme. Although bicarbonate, pyruvate and oxaloacetate were ineffective, both MgATP2− and acetyl‐CoA protected the enzyme against this chemical modification. At 100% inactivation, 1.1 ± 0.1 mol of Mg‐oATP2− were bound to the enzyme per mole of biotin. Acetyl‐CoA had no effect on this stoichiometry.Chromatography of samples of an enzymic digest of Mg‐o[14C]ATP2− ‐labelled enzyme revealed one major band of radioactivity which co‐chromatographed with authentic Lys‐oATP.
Regulation of Phenylalanine Ammonia‐Lyase Activity in Cell‐Suspension Cultures of Petroselinum hortense
Authors: HAHLBROCK Klaus 
Published: March 2005

The time courses for induced changes in the phenylalanine ammonia‐lyase activity at five different stages during the growth cycle of cell‐suspension cultures from parsley (Petroselinum: hortense Hoffm.) were investigated. Large increases in the enzyme activity, induced either by irradiation or by dilution of a cell culture into fresh medium, were followed by an exponential decline to the initial low level. The maximum inducible level of specific enzyme activity varied within a range of about six‐fold, depending on the mode of induction and the growth stage of the cell culture.The general shapes of the curves for the changes in enzyme activity were similar under the various experimental conditions. However, the precise positions of the peaks in the activity varied from about 12 – 27 h after the onset of induction. Proportionally large variations were found for the peak widths at half‐maximum and, in the case of induction by continuous irradiation, for the periods of time required for half‐maximal induction of the system with light. The apparent half‐life of the enzyme, as calculated from the rate of decline of the activity subsequent to the peak, remained approximately constant under all conditions investigated.A general model is proposed which would explain the regulation of phenylalanine ammonialyase activity in Petroselinum hortense cell cultures by an interplay of potentially large variations in the rate of synthesis and an approximately constant rate of degradation of the enzyme. This conclusion is supported both by the various experimental results and by theoretical derivations of curves for the apparent changes in the enzyme‐synthesizing activity and in the rates of provision of this activity at the site of enzyme synthesis.
Lipid Ordering and Enzymic Activities in Chromaffin Granule Membranes
Authors: BASHFORD C. Lindsay , JOHNSON Louise N. , RADDA George K. , RITCHIE Gillian A. 
Published: March 2005

The interaction of a variety of fluorescent probes with the membranes of adrenal medullary chromaftin granules is described.
Changes in the motional properties of the bound probes with temperature were investigated and evidence is presented which indicates that ordering of the membrane lipids occurs below 33 °C.
The ordering is characteristics of the membrane lipids and is retained by sonicated aqueous dispersions of the total lipid extracted from chromaffin granule membranes.
The ATPase and NADH : acceptor oxidoreductase activities of the chromaffin granule membrane have discontinuous Arrhenius temperature versus activity relationships with ‘transitions’ at 33 °C.
The ATPase has a second transition at 36.5 °C.
The 33 °C ‘transition’ for the NADH : acceptor oxidoreductase is removed by treatment with the detergent Triton X‐100.
The correlation between the onset of lipid ordering and the change in activation energy of the membrane‐bound enzyme activities is discussed in terms of the co‐operative interactions of the different membrane components. The possible role of lipid ordering in exocytosis is discussed.
Affinity Labelling of tRNA Nucleotidyltransferase from Baker's Yeast with tRNAPhe Modified on the 3′‐Terminus
Authors: STERNBACH Hans , SPRINZL Mathias , HOBBS John B. , CRAMER Friedrich 
Published: March 2005

2′‐Deoxy‐2′‐amino‐cytidylic acid can be incorporated into position 75 of tRNAPhe from yeast by tRNA nucleotidyltransferase yielding tRNAPhe‐C‐C(2′NH2). tRNAPhe‐C‐C(2′NH2) can be reacted with the N‐hydroxysuccinimide esters of bromoacetic acid and of mercuriacetic acid to yield the derivatives tRNAPhe‐C‐C(2′NHCOCH2Br) and tRNAPhe‐C‐C(2′NHCOCH2Hg+OH‐).Each of these reactive tRNAs inactivates tRNA nucleotidyltransferase from yeast with similar kinetics. The enzyme can be protected against inhibition by its substrates tRNAPhe‐C and tRNAPhe ‐C‐C as well as ATP and CTP.A covalent. isolatable 1:1 complex between tRNAPhe ‐C‐C(2′NHCOCH2Br) and the enzyme was formed, but could not be found when the enzyme had previously been inactivated with p‐hydroxymerecuribenzoate.
Non‐equivalence of Human: Hemoglobin Chains in the Oxidation‐Reduction and Heme‐Transfer Reactions
Authors: BANERJEE Ramaprasad , LHOSTE Jean‐Marc 
Published: March 2005

13C nuclear magnetic resonance (NMR) spectroscopy has been applied to the investigation of chain non‐equivalence for two reactions of human hemoglobin: oxidation‐reduction and heme‐transfer. The method is based on previous observations that in the carbonyl region, Hb13CO gives two well‐resolved resonances which arise from 13C of carbonyls bound respectively to the α and β chains; moreover, integration of spectra allows one to estimate their relative abundance.A mixture of ferrous and ferric hemoglobins in dye‐mediated oxidation‐reduction equilibrium can be formally considered to be equivalent to two redox couples in equilibrium, namely α111/α11 and β111/β11: from a knowledge of these ratios, one can conclude whether the chains are equivalent or not in their oxidation‐reduction properties. In this work, these ratios were evaluated by reacting the redox systems with 13CO and integrating the 13C NMR spectra. The results show differences in the intrinsic oxidation‐reduction potentials of the chains in hemoglobin tetramer, E1/2(β) being higher than El/2(α) in neutral solution but not at pH 9 and above. The binding of inositol hexakisphosphate does not modify the difference between β and α though substantially increasing the overall potential. The results are discussed in the light of current hypotheses to account for the change of Hill coefficient with pH for the reaction studied.The non‐equivalence of chains is shown also for heme transfer from methemoglobin. For the phosphate‐free protein, the β chains lose heme more rapidly than the α chains; the addition of inositol hexakisphosphate results in the decrease of overall heme ‐transfer as well as of chain heterogeneity.
Error Structure of Enzyme Kinetic Experiments
Authors: ASKELÖF Per , KORSFELDT Margareta , MANNERVIK Bengt 
Published: March 2005

Knowledge of the error structure of a given set of experimental data is a necessary prerequisite for incisive analysis and for discrimination between alternative mathematical models of the data set. A reaction system consisting of glutathione S‐transferase A (glutathione S‐aryltransferase), glutathione, and 3,4‐dichloro‐l‐nitrobenzene was investigated under steady‐state conditions. It was found that the experimental error increased with initial velocity, ν, and that the variance (estimated by replicates) could be described by a polynomial in νVar(ν)=K0+K1.ν+K2.ν2or by a power functionVar (ν)=K0+K1oνK2.These equations were good approximations irrespective of whether different ν values were generated by changing substrate or enzyme concentrations. The selection of these models was based mainly on experiments involving varying enzyme concentration, which, unlike ν, is not considered a stochastic variable. Different models of the variance, expressed as functions of enzyme concentration, were examined by regression analysis, and the models could then be transformed to functions in which velocity is substituted for enzyme concentration owing to the proportionality between these variables.Thus, neither the absolute nor. the relative error was independent of velocity, a result previously obtained for glutathione reductase in this laboratory [BioSystems 7, 101–119 (1975)]. If the experimental errors or velocities were standardized by division with their corresponding mean velocity value they showed a normal (Gaussian) distribution provided that the coefficient of variation was approximately constant for the data considered. Furthermore, it was established that the errors in the independent variables (enzyme and substrate concentrations) were small in comparison with the error in the velocity determinations.For weighting in regression analysis the inverted value of the local variance in each experimental point should be used. It was found that the assumption of proportionality between variance and ν (where a is an empirically determined exponent) was a good approximation for the weighting. The value of α was 1.6 in the present case. The weight function was tested in the fitting of a rate equation to a kinetic‐data set involving variable substrate concentrations.Recommendations are given regarding the establishment of the error structure in a general case and its application in regression analysis.
4a‐Alkyldihydroflavin Coenzyme Synthesis and Modification of Flavodoxin
Authors: SCOLA‐NAGELSCHNEIDER Gisela , BRÜSTLEIN Martin , HEMMERICH Peter 
Published: March 2005

A stable (i.e. non‐oxidisable), reduced flavocoenzyme, 4a‐(n‐propyl)‐4a,5‐dihydro‐riboflavin 5′‐monophosphate, has been synthesized. The method of choice was selective photo‐allylation in position 4a. Stabilization of this substituent was achieved by hydrogenation after N(5) protection, while hydrogenation of the unprotected compound would have led to removal of the allyl substituent. The mode of this substituent mobility has been checked with deuterated photosubstrate. Upon hydrogenation, the allyl substituent becomes firmly and irreversibly fixed to the flavin surface. The diastereomers thus formed have been separated and characterized by their ultraviolet, circular dichroic and nuclear magnetic resonance spectra.After subsequent side chain phosphorylation it has been shown that only one of the diastereomeric dihydro‐flavocoenzymes is firmly bound by Peptostreptococcus elsdenii apoflavodoxin. This leads to the. hypothesis that (a) the non‐planar 4a,5‐dihydro‐flavin isomer and, therefore, C(4a) as active flavin center, may have biological relevance, white (b) the flavin site in fiavodoxin is accessible to some extent for a stacking interaction with a redox‐partner group
Localization of the GTP‐Binding Site in the Ribosome · Elongation‐Factor‐G · GTP Complex
Authors: GIRSHOVICH Alexander S. , POZDNYAKOV Valery A. , OVCHINNIKOV Yuri A. 
Published: March 2005

For localization of the GTP‐binding center in the Escherichia coli MRE‐600 ribosome elongation‐factor‐G GTP system a method of photo‐affinity labelling is used with two types of radioactive analogs oI GTP with a photo‐activated group attached to ribose or to γ‐phosphate residues of a nucleotide molecule: (2‐nitro,4‐azidobenzoyl)hydrazone of periodate‐oxidized GTP or guanosine 5′‐(α,β‐methylene)triphosphate and the γ‐(4‐azidobenzyl)amide of GTP.These photo‐analogs form a specific ternary complex with the ribosome and elongation factor G (EF‐G). Separately neither the ribosome tor the EF‐G bind analogs. The formation of the ternary complex with analogs is inhibited by an excess amount of the native nucleotide.The ribose analog of GTP is hydrolyzed by the EF‐G‐dependent GTPase, but the γ‐amide of GTP is not.Irradiation of complexes containing photo‐analogs leads to a highly specific labelling of EF‐G, while ribosome labelling is insignificant and non‐specific. The presence of an excess amount of the native nucleotide inhibits photo‐labelling of EF‐G without significant changes in the labelling of ribosomes. The labelling of free EF‐G is low. Thus, within the ternary complex the GTP‐binding center is localized on the EF‐G.Fusidic acid does not influence the binding of GTP γ‐amide in a ternary complex, but blocks the specific labelling of the EF‐G with no effect on the non‐specific labelling of ribosomes. A hypothesis is proposed on the direct contact of this antibiotic with a GTP molecule in the GTP‐binding center of EF‐G.
Purification of DNA‐Dependent RNA Polymerase C from Zajdela Hepatoma Cells
Authors: CAIZERGUES‐FERRER Michèle , ROUGH Claude , ZALTA Jean‐Pierre 
Published: March 2005

A C‐type DNA‐dependent RNA polymerase has been extracted and purified from the cytoplasmic fraction of Zajdela hepatoma cells. The purification method and the separation from other RNA polymerase are described.RNA‐polymerizing properties of this RNA polymerase C are studied and compared with those of other RNA polymerases.We reach the conclusion that the cytoplasmic C enzyme is clearly different from A and B nuclear enzymes and that the nuclear RNA polymerase III and the cytoplasmic RNA polymerase C behave like closely related enzymes.
Heterogeneity of Erythrocyte Catalase
Authors: SCHERZ Bernhard , KUCHINSKAS Edward J. , WYSS Sonja R. , AEBI Hugo 
Published: March 2005

Dissociation of the erythrocyte catalase tetramer into its dimer form proceeds readily in urea: the rate depends on urea concentration, time of inactivation and temperature, but is not influenced by enzyme concentration. This process is accompanied by loss of catalase activity, generation of peroxidase activity and an alteration of antigenic properties.
Recombination of dimer subunits to the tetramer molecule can be accomplished by removing urea by dialysis. Under optimal conditions up to 80% of the original catalase activity can be recovered. The reconstituted material is identical with the native catalase regarding enzymatic and antigenic properties, but differs with respect to electrophoretic mobility and heat stability.
Normal human erythrocyte catalase and the enzyme species present in blood of individuals heterozygous for Swiss‐type acatalasemia differ with regard to electrophoretic mobility and heat stability. Moreover, dissociation of the heterozygote catalase tetramer into dimer subunits occurs at lower urea concentrations when compared to the normal enzyme. Since heterozygote erythrocyte catalase exerts properties intermediate to those of the normal enzyme and of the variant found in homozygotes for Acatalasemia, this enzyme species is considered to represent a molecular hybrid.
Hybridization of catalases can be accomplished in vitro by dissociating a mixture of normal and heterozygote catalase preparations in 8 M urea and subsequent dialysis. The resulting enzyme species reveals intermediate electrophoretic mobility and heat stability when compared with its native constituents.
Antibodies to Poly(I) · poly(C)
Authors: GUIGUES Michèle , LENG Marc 
Published: March 2005

Sera of rabbits immunized with poly(I) · poly(C) adsorbed to methylated serum albumin react with poly(I) · poly(C), poly(I) and poly(C). Three families of antibodies have been isolated by affinity chromatography.The antibodies to poly(I) · poly(C) precipitate poly(I) · poly(C) but neither poly(I) nor poly(C). In 0.15 M NaCl, pH 7.5 at 4°C the antibodies to poly(I) precipitate only poly(I) and the antibodies to poly(C) precipitate only poly(C), but in 10 mM NaCl pH 7.5 and at room temperature they react with poly(I) · poly(C). These reactions occur because the antibodies to poly(I) and to poly(C) destabilize poly(I) · poly(C). These antibodies behave as melting proteins. The thermal stability of poly(I) · poly(C) is decreased in presence of antibodies to poly(I) but is increased in presence of antibodies to poly(I) · poly(C). It has also been found that the antibodies to poly(I) · poly(C) lower the tm of the transition poly(A) · 2 poly(U) to poly(A) poly(U) and increase the tm of the transition poly(A) · poly(U) to poly(A) + poly(U).The purified antibodies to poly(I) · poly(C) react with several double‐stranded polyribonucleotides such as poly(A) · poly(U), poly(I) · poly(brC), and poly(I) · poly(ioC), but do not react with poly(G) · poly(C), poly(X) · poly(U) or DNA.Association constants for the binding of Fab fragments to poly(l) · poly(C) and poly(I) · poly(brC) have been deduced from fluorescence experiments. In presence of poly(I) · poly(brC) the fluorescence off ab fragments is quenched. The analysis of the binding isotherms shows that about 3–4 base pairs are covered by the binding site of the Fab fragments, a value in agreement with that deduced from the precipitin curve (five base pairs). From the variation of the association constants as a function of salt concentration it is concluded that only two phosphate groups are involved in electrostatic interactions.
Mapping of Promoter Sites on the Genome of Bacteriophage M13
Authors: EDENS Luppo , VAN WEZENBEEK Peter , KONINGS Ruud N. H. , SCHOENMAKERS John G. G. 
Published: March 2005

With the aid of transcription studies on restriction fragments of bacteriophage M13 DNA it has been demonstrated that at least eight promoter sites are located on the M13 genome. Five of these promoters initiate the synthesis of RNA chains which contain at their 5′‐terminal end pppG (G promoters), while the other three promoters initiate RNA chains which start exclusively with pppA (A promoters). The positions of these promoter sites on the physical map are: 0.82 (G0.82), 0.88 ((G0.88). 0.94 (G0.94), 0.01 (G0.01), 0.08 (G0 08), 0.36 (A0 36), 0.51 (A0.51) and 0.56 (A0.56). The G promoters were found to be clustered within a distance of one‐third of the genome length from the central termination site for transcription (map position 0.77). The A promoters, however, were found at greater distances from this termination signal. Based upon the incorporation of [γ‐32P]ATP or [γ‐32P]GTP, the capacity of these promoters to initiate the synthesis of RNA chains varies. The strongest G promoters are G0.82, G0.94 and G0.08 and the strongest A promoter is A0.36.As judged from their position on the genetic map, it is postulated that two promoters. namely G0.94 and G0.01, are located within gene II. The other promoters are most probably located inunediately in front of the gene VIII/VII boundary (G0.82), and immediately in front of gene V (G0 88), gene II (G0.08), gene IV (A0.36), gene I (A0.51) and gene VI (A0.56). No evidence has been obtained so fou‐ for the existence of a promoter immediately in front of gene III.