bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
Nuclear Poly(A) Polymerase from Rat Liver and a Hepatoma
Authors: ROSE Kathleen M. , JACOB Samson T.
Published: March 2005

Poly(A) polymerase was extracted from isolated nuclei of rat liver and a rapidly growing solid tumor (Morris hepatoma 3924A). The enzyme from each tissue was purified by successive chromatography on DEAE‐Sephadex, phosphocellulose, hydroxyapatite and QAE‐Sephadex. Purified enzyme from both liver and tumor was essentially homogeneous as judged by polyacrylamide gel electrophoresis. Under nondenaturing conditions, enzyme activity corresponded to visible protein and, upon denaturation, a single polypeptide was detected. The enzymes had absolute requirements for Mn2+ as the divalent ion, ATP as the substrate and an oligonucleotide or polynucleotide as the primer. Both enzymes were inhibited by sodium pyrophosphate, N‐ethylmaleimide, Rose Bengal, cordycepin 5′‐triphosphate and several rifamycin derivatives. The reactions were unaffected by potassium phosphate, α‐amanitin and pancreatic ribonuclease. However, the liver and hepatoma enzymes differed from each other with respect to apparent Km, primer saturation levels and, sensitivity to pH changes. The most striking differences between the enzymes were in their calculated molecular weights (liver, 48000; hepatoma, 60000) and amino acid compositions. Finally, the level of the hepatoma enzyme relative to that of the liver enzyme was at least 1.5‐fold higher when expressed per nag DNA.
Phosphate Binding to Chromatophores of Rhodospirillum rubrum
Authors: SUTER Willi , LUTZ Hans U. , BACHOFEN Reinhard
Published: March 2005

Equilibrium dialysis has been used to determine the binding of phosphate to chromatophores of Rhodospirilhum rubrum. Assuming a complete exchange of the added 32Pi with endogenous phosphate, the saturation with phosphate retained in any form by chromatophores was reached at about 20 nmoles Pi per mg of bacteriochlorophyll. The retention of phosphate had a pH optimum at pH 6.5 to 6.8. At pH 8.0 only chromatophores which have not been liberated from DNA and RNA show a considerable retention of phosphate. However, illumination of chromatophores prior to dialysis in the presence of ADP leads to a retention of phosphate at pH 8.0 which persists during dark dialysis in the absence of added magnesium.
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.
Effect of Inhibitors on the Substrate‐Dependent Quenching of 9‐Aminoacridine Fluorescence in Inside‐Out Membrane Vesicles of Escherichia coli
Authors: SINGH Akhand P. , BRAGG Philip D.
Published: March 2005

The effect of various inhibitors on the substrate‐dependent quenching of the fluorescence of 9‐aminoacridine was measured in inside‐out membrane vesicles of Escherichia coli. The rate of fluorescence quenching in the presence of inhibitors was dependent on the rate of electron transfer through the respiratory chain with NADH, succinate d‐lactate or dl‐glycerol 3‐phosphate as substrates. Several patterns of response were given by the inhibitors. Inhibitors competitive with substrate, or those acting only on the dehydrogenases, gave a direct relationship between the extent of inhibition of oxidase activity and the rate of quenching. A biphasic relationship was given by 2‐heptyl‐4‐hydroxyquinoline N‐oxide and piericidin A which was due to these compounds acting both as inhibitors of the respiratory chain and, at higher concentrations, as uncoupling agents. Uncouplers inhibited fluorescence quenching with minimal inhibition of oxidase activity. The transmembrane pH difference was calculated from the extent of fluorescence quenching and the intravesicular volume. The maximum pH difference of 3.3–3.7 units was generated by each of the substrates tested.
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.
Structure and Function of Rat‐Liver Ribosomes
Authors: REYES Ramon , VAZQUEZ David , BALLESTA Juan P. G.
Published: March 2005

Rat liver ribosomes and 60‐S ribosome subunits were treated with the primary‐amino‐group‐specific reagent 2‐methoxy‐5‐nitrotropone. Important differences in the sensitivity of several ribo‐ somal activities to inactivation by the reagent were observed. While elongation‐factor‐dependent activities are totally abolished in the treated particles, peptidyl transferase activity is either unaffected in 60‐S ribosomal subunits or even strongly stimulated in 80‐S ribosomes. Analysis of the ribosomal proteins modified by nitrotropone made it possible to draw some conclusions on their accesibility in the ribosomal structure and to relate some proteins with their involvement in the ribosome active centers. Thus, proteins L3, L13, L15 and L23 seem to be in a rather well protected position while proteins L10, L35, L37, X1 and X2 are totally exposed to the reagent. The protein accessibility also depends on the ribosome conformation, proteins L14 and L17, for instance, being sensitive in 80‐S ribosomes and protected in 60‐S subunits. In relation to the implication of proteins in functional centers, the data presented here together with other data obtained from protein‐deficient core particles seem to indicate a possible role of proteins L21 and/or L26 in the peptidyl transferase center.
Role of Transfer Ribonucleic Acids in the Regulation of Several Biosyntheses in Saccharomyces cerevisiae
Authors: MESSENGUY Francine , DELFORGE Jacqueline
Published: March 2005

In a thermosensitive isoleucyl‐tRNA synthetase mutant (ilsl‐1) of Saccharomyces cerevisiae where only the charge of isoleucyl‐tRNA measured in vivo is reduced, enzymes from the isoleucine but also arginine and histidine biosyntheses are derepressed. The ilsl‐1 mutant shows the same behaviour as a leaky auxotrophic mutant, although no amino acid pool is lower than in the wild‐type.
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.
Evidence for a Function of Core Protein in Complex III from Beef‐Heart Mitochondria
Authors: GELLERFORS Pär , LUNDÉN Mats , NELSON B. Dean
Published: March 2005

Purified complex III (ubiquinol‐cytochrome c reductase) from beef heart mitochondria was alkylated with iodo[1‐14C]acetamide. After 6–8 h of incubation with iodo[1‐14C]acetamide, duroquinol and ubiquinol‐2‐cytochrome c reductase activities were inhibited approximately 50%. During this time 4.5 ± 1.6 nmol of iodo[1‐14C]acetamide reacted per mg of complex III protein. Experiments carried out over 24 h indicated that enzyme activity could be inhibited to 70% and that alkylation of complex III was proportional to inhibition. The rates of cytochrome b and c1 reduction by uroquinol are also decreased upon treatment of complex III with iodoacetamide. Separation of the peptides of complex III by electrophoresis in sodium dodecylsulfate shows that all of the radioactivity is located in a single peptide of 50 000 molecular weight, which has been identified as one of the two core proteins. The possible functions of core protein are discussed.
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 ν

Var(ν)=K0+K1.ν+K2.ν2

or by a power function

Var (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.
The Dimer of the Escherichia coli Galactose‐Binding Protein
Authors: RASCHED Ihab , SHUMAN Howard , BOOS Winfried
Published: March 2005

Purified galactose‐binding protein when stored at 4°C as precipitate in 100% saturated ammonium sulfate solutions was found to contain about 10%, dimers after dissolving the precipitate in buffer. The dimer has the same amino acid composition and specific binding affinity as the monomer. Both forms are stable in 10 mM Tris‐HCI, pH 7.0, buffer and can easily be separated from each other by Sephadex G‐75 column chromatography. The dimer is stable in 0.1% sodium dodecylsulfate at room temperature. Boiling in 0.1% dodecylsulfate for‐ 10 min results in the formation of 60–70% monomers.
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.
A Functional Arginine Residue in Rabbit‐Muscle Aldolase
Authors: LOBB Roy R. , STOKES Alistair M. , HILL H. Allen O. , RIORDAN James F.
Published: March 2005

Rabbit muscle aldolase is irreversibly modified by the arginine‐selective α‐dicarhonvi, phenylglyoxal loss of activity correlating with the unique modifications of one arginine residue per subunit, as determined by amino acid analysis, and [7‐14C]phenylglyoxal incorporation. The affinity of the modified enzyme for dihydroxyacetone phosphate is significantly reduced while substantial protection against inactivation is afforded by fructose 1,6‐disphosphate, dihydroxyacetonc phosphate or phosphate ion. The nature of the substrate C‐1 phosphate binding site in this enzyme is discussed in the light of these and other results.
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.
A New Concept of the Function of Elongation Factor 1 in Peptide Chain Elongation
Authors: GRASMUK Hans , NOLAN Robert D. , DREWS Jürgen
Published: March 2005

An entirely new model for the mechanism of elongation factor 1 (EF‐1) function is presented. Experiments, in which mixtures of [3H]EF‐1, ribosomes from Krebs II ascites cells and various additional co‐factors were analyzed by chromatography on Sepharose 6B, show that EF‐1 binds to the ribosome early in the translation process and remains bound on the ribosome during translation. Optimal EF‐1 binding occurs on polynucleotide‐programmed ribosomes carrying a tRNA in their P‐site.

On the other hand it was clearly shown that EF‐2 attached at each translocation event and was then released before a new Phe‐tRNA could be bound.
5′‐Capping Structures of Artemia salina mRNA and the Translational Inhibition by Cap Analogs
Authors: GRONER Yoram , GROSFELD Haim , LITTAUER Uriel Z.
Published: March 2005

The mRNA of the brain shrimp Artemia salina has two types of blocked methylated 5′‐terminal structures (caps). About 75% of the mRNA molecules have the 5′‐end structure of m7G5′ppp5′‐AmpGp and about 25% have the structure of m7G5′ppp5′GmpGp. The only other type of methylated residue found in Artemia mRNA is N6‐methyladenosine and which is located at internal positions along the mRNA chain. Translation of Artemia cyst or nauplius poly(A)‐rich mRNA in wheat‐germ extracts was found to be inhibited by 7‐methylguanosine 5′‐monophosphate, a chemical analog of the cap, as well as by synthetic caps such as m7G5′ppp5′Gm. On the other hand, the elongation activity on endonegous mRNA in an Artemia cell‐free system was not sensitive to 7‐methylguanosine 5′‐monophosphate.
Acetyl‐Coenzyme‐A Carboxylase of Candida lipolytica
Authors: MISHINA Masayoshi , KAMIRYO Tatsuyuki , TANAKA Atsuo , FUKUI Saburo , NUMA Shosaku
Published: March 2005

The level of acetyl‐coenzyme‐A carboxylase activity in Candida lipolytica undergoes large variations depending upon the carbon source on which the yeast is grown. Cells grown on n‐alkanes or fatty acids exhibit a lower activity level than do cells grown on glucose. Among the n‐alkanes and fatty acids tested, n‐heptadecane, n‐octadecane, oleic acid and linoleic acid reduce the enzyme activity to the lowest levels, which are 16–18% of the activity level in glucose‐grown cells.

Immunochemical titrations and Ouchterlony double‐diffusion analysis with specific antibody as well as kinetic studies have indicated that the observed decrease in the level of acetyl‐CoA carboxylase activity is due to a reduction in the cellular content of the enzyme. Furthermore, isotopic leucine incorporation studies with the use of the immunoprecipitation technique have demonstrated that the relative rate of synthesis of the enzyme in oleic‐acid‐grown cells is diminished to 12% of that in glucose‐grown cells. Evidence has also been obtained to support the view that the enzyme in this yeast is not degraded at a rate high enough to contribute to the marked decrease in the cellular content of the enzyme. Thus, it is concluded that the reduction in acetyl‐CoA carboxylase content in fatty‐acid‐grown cells is due to diminished synthesis of the enzyme.
Glucose Effect in tgl Mutant of Escherichia coli K12 Defective in Methyl‐α‐D‐glucoside Transport
Authors: ERLAGAEVA Raisa S. , BOLSHAKOVA Tatyana N. , SHULGINA Marina V. , BOURD Genrich I. , GERSHANOVITCH Vladimir N.
Published: March 2005

The dependence of the rate of accumulation of methyl‐α‐d‐glucoside on its extracellular concentration was studied in the tgl mutant of Escherichia coli K12, isolated earlier. It has been shown that the kinetics of methyl‐α‐d‐glucoside transport differ sharply from those in wild‐type bacteria.

The β‐galactosidase synthesis in tgl strain is much less sensitive both to permanent and transient glucose catabolite repression. The level of cyclic AMP in mutant cells under the conditions of glucose catabolite repression is several times higher than in the parent strain.

The tgl mutation does not affect the manifestation of catabolite inhibition and inducer exclusion with glucose.

The data obtained are discussed in the light of a hypothesis concerning the existence of two sites, binding and phosphorylating, in the molecule of the glucose (and methyl‐α‐d‐glucoside) specific enzyme II of the phosphoenolpyruvate‐dependent phosphotransferase system. The tgl mutation alters the first site, and the second one is damaged by the gpt mutation.

It is suggested that the products of the tgl and gpt genes are necessary for the manifestation of the phenomena of glucose permanent and transient repression. The effects of catabolite inhibition and inducer exclusion are realized irrespective of the existence or absence of the tgl product.
Anabolic Ornithine Carbamoyltransferase of Pseudomonas
Authors: STALON Victor , LEGRAIN Christiane , WIAME Jean‐Marie
Published: March 2005

The anabolic ornithine carbamoyltransferase of Pseudomonas appears to be extremely specialized. Unlike the other carbamoyltransferases studied, this enzyme catalyzes the phosphorolytic cleavage of citrulline with a very poor efficiency.

The main goal of this paper is to understand what, in the catalytic process, causes this directed functional specialization. On the basis of kinetic data and thermodynamic properties of the reaction, it appears that the reaction mechanism is the same as for ornithine carbamoyltransferases from other sources, that is, of the sequential ordered type, where carbamoylphosphate is the first substrate to be bound and phosphate the last product to be released. In addition to this, and here lies the difference with other ornithine carbamoyltransferases, the anabolic transferase of Pseudomonas forms a binary dead‐end complex with citrulline, leading to inefficient binding of phosphate and citrul‐line to the enzyme. Therefore the phosphorolytic cleavage of citrulline is equally inefficient. It should be mentioned that the affinity of the enzyme for citrulline at its catalytic site is low as compared to other transferases.
Nuclear‐Magnetic‐Resonance Studies of Pseudomonas aeruginosa Cytochrome c‐551
Authors: MOORE Geoffrey R. , PITT Robert C. , WILLIAMS Robert J. P.
Published: March 2005

Nuclear magnetic resonance (NMR) spectroscopy was used to study Pseudomonas aeruginosa cytochrome c‐551. Assignments of resonances to specific residues have been made. A low‐resolution X‐ray structure was used to aid assignments. A structural comparison was made between P. aeruginosa cytochrome c‐551 and mammalian cytochrome c, based on comparisons of NMR data.
X‐Ray Investigation of the Binding of 1,10‐Phenanthroline and Imidazole to Horse‐Liver Alcohol Dehydrogenase
Authors: BOIWE Torne , BRÄNDEN Carl‐Ivar
Published: March 2005

We have studied the binding of two inhibitor molecules, imidazole and 1,10‐phenanthroline, to liver alcohol dehydrogenase by crystallographic methods. X‐ray data for the imidazole complex were collected to 0.29‐nm resolution and for the 1,10‐phenanthroline complex to 0.45‐nm resolution. In both cases we found only one peak in the difference electron density maps close to the active zinc atom. The peak corresponding to 1,10‐phenanthroline overlaps the site of the density of the zinc‐bound water in the apoenzyme and the imidazole density partly overlaps this density. We can not discern any additional peaks close to the zinc atom which would correspond to new positions of bound water. We thus conclude that both these inhibitors bind to the catalytic zinc atom and that upon binding they displace the water molecule that is firmly bound to this zinc atom in the apoenzyme. We do not see any structural changes in the remaining part of the molecule.
Effects of Nucleoside Phosphates and Salts on the Activity of a Heart Phosphoprotein Phosphatase and Its Catalytic Subunit
Authors: LI Heng‐Chun , HSIAO Kwang‐Jen
Published: March 2005

A phosphoprotein phosphatase (phosphatase B, Mr= 161000) isolated from canine heart extract can be dissociated by ethanol into a catalytic subunit (phosphatase S, Mr= 34800). The dissociation of phosphatase B into phosphatase S is accompanied by an increase in the enzymic activity toward phosphohistone, phosphocasein and phosphorylase a and by a pronounced change in catalytic properties. Phosphatases B and S exhibit different substrate saturation kinetics. pH profile and respond differently to nucleoside phosphates and salts.

The increased activity toward phosphohistone accompanying the dissociation of phosphatase B reflects loss of substrate inhibition. Preincubation of either phosphatase B or S with ATP results in an inactivation of its activity and phosphatase S is much more susceptible to ATP inactivation than phosphatase B. ATP and various ionic compounds, however, can greatly stimulate the activity of phosphatase B by interacting with phosphohistone, indicating the modification at the substrate level represents an important regulatory mechanism. By contrast, the activity of phosphatase S is slightly stimulated by relatively low concentrations of salts, and is inhibited by nucleoside phosphates. The present data indicates that the activity of phosphatase B is more sensitive to the modification of the substrate, phosphohistone, by nucleoside phosphates and salts than is phosphatase S. These observations suggest that the regulatory properties of phosphatase B are lost following its dissociation.
Determination of the Chain Stoichiometries from the Number of Reactive Sulfhydryl Groups in the Pyruvate Dehydrogenase Complexes of Azotobacter vinelandii and Escherichia coli
Authors: DE ABREU Ronney A. , DE KOK Arie , DE GRAAF‐HESS Adriana C. , VEEGER Cees
Published: March 2005

The pyruvate dehydrogenase complex from Azotobacter vinelandii incubated with 0.05–0.7 mM [2‐14C]pyruvate, magnesium chloride and thiamine pyrophosphate under anaerobic conditions at 0°C, incorporates four [14C]acetyl groups per mole FAD which are bound to the high‐molecular‐weight lipoyl transacetylase. With 10 mM pyruvate, the low‐molecular‐weight lipoyl transacetylase is also labelled; to this enzyme 3–4 [14C]acetyl groups are bound per mole FAD. This enzyme is not labelled when pyruvate is used in concentrations lower than 0.7 mM. The complex of A. vinelandii pre‐labelled with non‐radioactive N‐ethylmaleimide did not incorporate label onto low‐molecular‐weight lipoyl transacetylase; a maximum of four acetyl groups are bound to the high‐molecular weight lipoyl transacetylase.

Maximum incorporation of four [14C]acetyl groups per mole FAD is found, within five seconds, on the lipoyl transacetylase component when the Escherichia coli complex is labelled under anaerobic conditions, also in the presence of 10 mM pyruvate.

In both complexes all incorporated acetyl groups are bound to sulfhydryl groups since they can be completely removed with hydroxylamine, by performic acid oxidation and by reaction with coenzyme A and arsenite. Under aerobic conditions a slow deacetylation is observed, which is blocked in the presence of N‐ethylmaleimide.

The multi‐enzyme complex from A. vinelandii pre‐labelled with non‐radioactive N‐ethylmaleimide and then labelled with N‐ethyl [2,3‐14C]maleimide in the presence of pyruvate, magnesium chloride and thiamine pyrophosphate incorporates under anaerobic conditions at 0°C, even at 10 mM pyruvate, a maximum of four N‐ethyl[14C]maleimide groups per mole of FAD. The label is almost exclusively bound to the high‐molecular‐weight lipoyl transacetylase of the A. vinelandii complex.

The E. coli complex binds only 2–3 N‐ethyl[14C]maleimide groups per mole of FAD under these conditions. Direct labelling and correction for labelling without pyruvate yields 4–5 N‐ethyl‐[14C]maleimide per mole FAD.

The low‐molecular‐weight lipoyl transacetylase can be removed by chromatography of the complex from A. vinelandii on a blue‐dextran–Sepharose 4B column [De Abreu et al. (1977) FEBS Lett. 82, 89–92]. The complex eluted from the column, consisting of the other three enzyme components, binds a maximum of four [14C]acetyl groups per mole of FAD, even with 10 mM pyruvate.

It is concluded that the lipoyl/FAD ratio is four in the complexes as isolated by us from both sources. The interpretation of this ratio with respect to the stoichiometries of the different enzyme components will be discussed.
Control of Teichoic Acid Synthesis in Bacillus licheniformis ATCC 9945
Authors: HUSSEY Helen , SUEDA Satoru , CHEAH Suan‐Choo , BADDILEY James
Published: March 2005

Analysis of cell walls of Bacillus licheniformis ATCC 9945 grown under phosphate limitation showed that teichoic acid could be replaced by teichuronic acid under these conditions. Teichuronic acid, however, was always present in the walls to some extent irrespective of the growth conditions. The enzymes involved in teichoic acid synthesis were investigated and the synthesis of these was shown to be repressed when the intracellular Pi level fell. CDP‐glycerol pyrophosphorylase was studied in some detail and evidence is presented to shown that the enzyme is inactivated under phosphate‐limited conditions. The mechanism of inactivation is unknown but it has been shown that it does not require protein synthesis de novo.
Metabolic Stability of the Two Forms of Initiation Factor IF‐3 in Escherichia coli during the Growth Cycle
Authors: MINKS Michael A. , SURYANARAYANA Tangirala , SUBRAMANIAN Alap R.
Published: March 2005

Possible alteration in the ratio of the long and short forms of initiation factor IF‐3 (FEBS Lett. 79, 264–275, 1977) during the growth cycle of Escherichia coli was examined. The ratio was found to remain unchanged between the exponential and stationary growth phases. Contrary to an earlier report (Eur. J. Biochem. 29, 319–325, 1972), the total amount of IF‐3 relative to the ribosome content in stationary phase cells was essentially the same as in midlogarithmic phase cells. The activity of IF‐3, assayed after its separation from other initiation factors by chromatography, was also the same in extracts from midlogarithmic and stationary phase cells. The data show that in Escherichia coli the ratio of IF‐3/ribosome is maintained constant.

The ribosomes themselves have been shown to retain virtually full activity in vitro during this transition indicating that growth‐cycle‐dependent biochemical modifications of the ribosome do not affect its protein synthetic capacity per se.
Evidence for the Participation of Cytosolic Protein Kinases in Membrane Phosphorylation in Intact Erythrocytes
Authors: PLUT David A. , HOSEY M. Marlene , TAO Mariano
Published: March 2005

The effects of adenosine 3′:5′‐monophosphate (cyclic AMP) on the phosphorylation of membrane proteins in intact rabbit and human erythrocytes were investigated. The addition of cyclic AMP to intact human or rabbit erythrocytes results in an increase in the incorporation of ortho[32P]phosphate into several membrane protein components which are known to serve as substrates for the cyclic‐AMP‐dependent protein kinases. Thus this increase in protein phosphorylation is probably due to the activation of either soluble or membrane‐bound cyclic‐AMP‐dependent protein kinases. Incubation of human erythrocytes in the presence of ortho[32P]phosphate and cyclic AMP also leads to the phosphorylation of a membrane protein component, band 7, which has not been previously detected in the autophosphorylation of isolated ghosts. Since rabbit erythrocyte membranes do not contain any cyclic‐AMP‐dependent protein kinase, the results suggest that cytoplasmic kinases also play a role in the phosphorylation of membrane proteins in intact cells.
The Specificity of Ribonucleoside Triphosphate Reductase
Authors: LUDWIG Walter , FOLLMANN Hartmut
Published: March 2005

Ribonucleotide reductase of Lactobacillus leichmanii, a monomeric enzyme requiring 5′‐deoxyadenosylcobalamin (coenzyme B‐12), catalyzes reduction of all common ribonucleoside 5′‐triphosphates but its substrate and effector specificity is not precisely known. Structure variations in the base, as in ATP analogs with N6‐dimethyladenine, unsubstituted purine, or even benzimidazole, are tolerated but lead to greatly reduced reaction rates. These substrates respond to the same effector nucleotide (dGTP) as ATP. br8ATP is completely inactive as a consequence of its abnormal molecular (syn) conformation. XTP is not reduced at pH 7.8 but reduction is observed at lower pH values, indicating that the species with an extra negative charge in the base (pK= 5.7) is inactive but uncharged xanthine results in substrate activity. dTTP stimulates reduction of XTP (and also of ITP).

The uncommon deoxyribonucleoside triphosphates dUTP, dITP, and dXTP act as allosteric effectors of ribonucleotide reduction like the common ones but they usually produce intermediate, or less specific types of stimulation, or weak inhibition. The same is true for br8dATP the activity of which is explained by a certain conformational flexibility of the aglycone. A complete range of deoxyribonucleotide effects from strong activation to inhibition of enzyme activity is thus observed, indicating that the apparently specific activation pattern among natural ribonucleotide/deoxyribonucleotide couples is in fact part of the protein's general capacity to interact with nucleotides. Inspection of the substituent structures has led us to postulate certain amino acid side chains, among them a carboxylate, and hydrogen bonding interactions at the substrate and effector sites. A two‐sided induced‐fit mechanism is suggested to explain the unusually dynamic allosteric behaviour of Lactobacillus reductase.

The activity of modified ribonucleotides and deoxyribonucleotides to promote enzyme‐catalyzed hydrogen exchange between 5′‐[5′‐3H]deoxyadenosylcobalamin and water has been tested. ATP analogs and XTP are active, but in contrast to all other deoxyribonucleotides known so far the effectors dXTP and br8dATP are not. In agreement with our earlier studies of sugar‐modified nucleotides, these results support the existence of two different deoxyribonucleotide sites for stimulation of substrate binding and for modulation of coenzyme action.

Because enzymic reduction of modified ribonucleotides by Lactobacillus reductase (and also by other reductases) is very slow these findings provide a rationale as to why such compounds (e.g. biosynthetic intermediates or phosphorylated antibiotics) will not interfere with DNA biosynthesis under normal conditions.
The Submicrosomal Localization of 3‐Hydroxy‐3‐Methylglutaryl‐Coenzyme‐A Reductase, Cholesterol 7α‐Hydroxylase and Cholesterol in Rat Liver
Authors: MITROPOULOS Konstantinos A. , VENKATESAN Soundararajan , BALASUBRAMANIAM Santhirasegaram , PETERS Timothy J.
Published: March 2005

In order to determine the submicrosomal distribution of cholesterol 7α‐hydroxylase, 3‐hydroxy‐3‐methylglutaryl‐coenzyme‐A reductase and of free cholesterol, the microsomal fraction and the digitonin‐treated microsomal preparation of rat liver were subjected to analytical isopycnic centrifugation on sucrose density gradients.

Cholesterol 7α‐hydroxylase, its substrate and hydroxymethylglutaryl‐CoA reductase are present in similar membranes originating from the endoplasmic reticulum with low density of ribosomes. Smooth membranes originating from Golgi apparatus and plasma membranes do not contain appreciable amounts of cholesterol 7α‐hydroxylase and of hydroxymethylglutaryl‐CoA reductase, whilst they contain a considerable portion of the free cholesterol present in microsomal preparations.

The addition of trace amounts of [14C]cholesterol to untreated and digitonin‐treated microsomal preparations resulted in heterogeneous labelling of cholesterol as seen from the variation of the specific radioactivity of free cholesterol in the microsomal subfractions. Both with untreated and with digitonin‐treated microsomes the specific radioactivity of cholesterol was minimal in the fractions of maximal contamination with plasma membranes and in those of maximal contamination with smooth membranes from Golgi apparatus. Therefore, the [14C]cholesterol added to the microsomal preparations did not label the plasma membrane and the Golgi membrane cholesterol pools as heavily as it labelled other pools of microsomal cholesterol. The ‘substrate pool’ for cholesterol 7α‐hydroxylase was heavily labelled by [14C]cholesterol added to untreated or digitonin‐treated microsomes and, since the specific radioactivity of 7α‐hydroxycholesterol obtained with various microsomal subfractions from the same preparation was very similar, the ‘substrate pool’ was uniformly labelled.
Homoserine Dehydrogenase of Rhodospirillum rubrum
Authors: EPSTEIN Carol Cherkis , DATTA Prasanta
Published: March 2005

A detailed physicochemical characterization of purified homoserine dehydrogenase of Rhodospirillum rubrum is presented. The enzyme has a molecular weight of 110 000 and consists of two subunits of identical molecular weight of 55 000. Depending on the ionic strength and protein concentration it is possible for the native enzyme to dimerize to produce an enzymatically active species of molecular weight 220 000.

Titrations of the native and detergent‐treated enzyme with a variety of sulfhydryl reagents show 2 mol free–SH groups per 110 000 g, one of which is buried in the protein interior. l‐Threonine and/or high concentrations of salt can expose the buried–SH group, and this–SH group is essential for the catalytic activity of the enzyme. Two independent lines of evidence show that extensive polymerization of the enzyme caused by l‐threonine and/or high concentrations of salt does not involve the formation of intermolecular disulfide bonds.