bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of DNA · RNA Hybrids in Eukaryotes
Authors: WYERS Françoise , HUET Janine , SENTENAC André , FROMAGEOT Pierre
Published: March 2005

Ribonucleases H1 and H2, purified in homogeneous form from yeast cells, specifically degrade the RNA moiety of RNA · DNA hybrids. The two enzymes are physically and functionally distinct proteins. Gel electrophoresis with sodium dodecylsulfate and gel filtration revealed that RNAase H2 is a small very basic protein composed of a single polypeptide chain of molecular weight 21000. RNAase H1, also a basic protein, is made of a polypeptide chain of molecular weight 48000 which shows a marked tendency to aggregate when subjected to polyacrylamide gel electrophoresis, gel filtration or zone sedimentation. RNAase H1 and RNAase H2 require different conditions for activity. RNAase H2 digests hybridized RNA at alkaline pH and shows an absolute requirement for divalent cations, preferably Mg2+, and for ‐SH groups. The enzyme liberates a mixture of oligonucleotides with 5′‐phosphate termini and only a small amount of 5′‐mononucleotide. It is inhibited by the ‐ SH reagent N‐ethylmaleimide. RNAase H1 is active at acid pH (around pH 6), as well as at slightly alkaline pH. It is also stimulated by divalent cations but does not need to be supplemented with ‐SH groups and is not sensitive to N‐ethylmaleimide. With (rA)n ‐ (dT)n as substrate, the main degradation product is 5′‐AMP with some dinucleotide pApA, suggesting an exonucleolytic degradation process. The possible role of RNAases H in the cell is discussed.
Distribution of Protein Assembly Sites along the 23‐S Ribosomal RNA of Escherichia coli
Authors: CHEN‐SCHMEISSER Ursula , GARRETT Roger
Published: March 2005

The protein composition of the 13‐S and 18‐S ribonucleoproteins isolated after mild ribonuclease digestion of Escherichia coli ribosomal 50‐S subunits were examined and those proteins specifically assembled were identified. Many proteins were unspecifically hound but some could subsequently be reassembled specifically by high‐salt treatment and they were identified.

Three RNA compooents were isolated from these ribonucleoproteins, namely 13‐S RNA from the 5′ end and 11‐S and 18‐S RNA from the 3′ end of the 23‐S RNA. These RNA's were reconstituted with total 50‐S subunit proteins and single binding proteins and the assembled proteins were identified.

The results of each of these experimental approaches were in excellent agreement, and enabled us to position 22 proteins within three main sections of the 23‐S RNA.
The Neurohypophysial Hormone‐Binding Proteins: Complete Amino‐Acid Sequence of Ovine and Bovine MSEL‐Neurophysins
Authors: CHAUVET Marie‐Thérèse , CHAUVET Jacqueline , ACHER Roger
Published: March 2005

Ovine and bovine MSEL‐neurophysins, purified by hormone protein complex precipitation, molecular sieving and ion‐exchange chromatography, were subjected either to performic acid oxidation or to reduction and carboxamidomethylation. Cleavage by trypsin gave in each case eight fragments which were separated by peptide mapping. They were sequenced either directly by Edman degradation or after sub‐cleavage by subtilisin, chymotrypsin, thermolysin, pepsin or partial acid hydrolysis and characterization of the sub‐peptides.

Alignment of the tryptic fragments was determined by automated Edman degradation and by identification of overlapping peptides obtained after hydrolysis of the polypeptide chain by chymotrypsin, pepsin and subtilisin. Bovine tryptic peptides were arranged, in part, according to their homology with ovine peptides.

Both ovine and bovine MSEL‐neurophysins comprise 95 residues; in both cases, a three‐residue N‐terminal truncated form can be detected. Ovine and bovine MSEL‐neurophysins are nearly identical. Two positions are different: 48 (isoleucine in ovine and asparagine in bovine) and 89 which shows a microheterogeneity in the ox (isoleucine in ovine. isoleucine or valine in bovine). Porcine MSEL‐neurophysin presents four differences when compared with ovine homologue (positions 48, 89, 90 and 92) and an apparent three‐residue C‐terminal deletion.

MSEL‐neurophysins form a protein family which appears to be evolutionary stable in species of Artiodactyla. In this order of mammals, the rare variations seem to occur in the C‐terminal sequence. A second family of related neurophysins found in mammals. VLDV‐neurophysins, shows substitutions in the N‐terminal sequence when compared to MSEL‐neurophysins. There is a large invariant central part in the polypeptide chain which might include the neurophysial hormone‐binding domain of all neurophysins.
The Function of Menaquinone, Covalently Bound FAD and Iron‐Sulfur Protein in the Electron Transport from Formate to Fumarate of Vibrio succinogenes
Authors: KRÖGER Achim , INNERHOFER Anna
Published: March 2005

The anaerobic bacterium Vibrio succinogenes contains a system of electron transport phosphorylation in which formate functions as the donor and fumarate as the terminal acceptor. The electron transport chain of this system has been investigated.

The formate‐fumarate reductase is localized in the membrane fraction of the bacterium, together with acid‐extractable and covalently‐bound FAD, menaquinone, iron‐sulfur protein and b and c cytochromes. The soluble fraction contains FMN, acid‐extractable FAD, iron‐sulfur protein and c cytochromes.

The formate‐fumarate reductase is fully inhibited by 2‐(n‐nonyl)‐4‐hydroxyquinoline‐N‐oxide and 4‐chloromercuriphenylsulfonate in amounts which correspond to half the content of the b cytochromes and to the content of iron‐sulfur protein respectively. 2‐(n‐noryl)‐4‐hydroxyquinoline‐N‐oxide specifically inhibits the reduction of menaquinone by formate, 4‐chloromercuriphenylsulfonate the oxidation of reduced menaquinone by fumarate. Formate dehydrogenase, when measured with benzylviologen as acceptor, and fumarate reductase, measured with reduced benzylviologen as the donor, are not affected by the inhibitors.

The prosthetic group of fumarate reductase is covalently bound FAD. The specific activity of fumarate reductase is increased to the same extent (about 8‐fold) as the content of the covalently bound FAD when the membrane is fractionated with cholate and ammonium sulfate. The acidextractable FAD is removed by this procedure.

The iron‐sulfur protein is the donor for fumarate reductase. This is concluded from the finding that the succinate‐ferricyanide reductase, which is copurified with the fumarate reductase, requires only the covalently bound FAD and the iron‐sulfur protein for activity. The 4‐chloromercuriphenyl‐sulfonate inhibition curve of the succinate‐ferricyanide reductase of the membrane fraction is identical with that of the overall electron transport.

Menaquinone is an obligatory redox mediator of formate‐fumarate reductase. The activity is fully inhibited on the extraction of the menaquinone from the membrane fraction, and is reactivated on reincorporation of menaquinone into the membrane. The activities of formate dehydrogenase and succinate‐ferricyanide reductase are independent of the presence of menaquinone. The redox response of membrane‐bound menaquinone confirms participation in electron transport.
The Function of the b Cytochromes in the Electron Transport from Formate to Fumarate of Vibrio succinogenes
Authors: KRÖGER Achim , INNERHOFER Anna
Published: March 2005

The reduction of fumarate by formate of the anaerobic Vibrio succinogenes is catalyzed by an electron transport chain which involves menaquinone and an iron‐sulfur protein as redox mediators between the formate dehydrogenase and the fumarate reductase. The electron transport from formate to menaquinone is specifically inhibited by 2‐(n‐nonyl)‐4‐hydroxyquinoline‐N‐oxide and that from menaquinone to fumarate by 4‐chloromercuriphenyl sulfonate which reacts specifically with the iron‐sulfur protein (Kröger and Innerholer (1976), accompanying paper). The function of the cytochromes in this electron transport chain is investigated.

V. succinogenes contains b and c cytochromes, whereas cytochromes of the a, d and o type are missing.

The molar absorption coefficients of the b and c cytochromes are determined using the pyridine hemochromogens of heme IX and heme C respectively.

By redox titration it is shown that the b and c cytochromes consist of two species each. The midpoint potentials of the b cytochromes are −200 and −20 mV and those of the c cytochromes are −160 and +70 mV. The latter values hold also for the c cytochromes present in the soluble fraction of the cell.

The oxidation of cytochrome b (−200 mV) by fumarate is inhibited by the extraction of men aquinone, whereas the reduction by formate is unaffected. In contrast, the reduction of cytochrome b (−20 mV) by formate, but not the oxidation by fumarate is dependent on the presence of menaquinone.

The electron transport inhibitor 2‐(n‐nonyl)‐4‐hydroxyquinoline‐N‐oxide affects the redox reactions of the two b cytochromes in the same way as the extraction menaquinone.

The oxidation by fumarate of both the b and the c cytochromes is blocked by 4‐chloromercuriphenyl sulfonate, whereas the reduction of the cytochromes by formate is unaffected.

Cytochrome b (−20 mV) is reduced by succinate via the fumarate reductase. This reaction is independent of the presence of menaquinone and is inhibited by 4‐chloromercuriphenyl sulfonate.

It is concluded that the two b cytochromes participate in the reduction of fumarate by formate. Cytochrome b (−200 mV) interacts between the formate dehydrogenase and menaquinone and cytochrome b (−20 mV) between menaquinone and the iron‐sulfur protein.
Coenzyme‐Dependent Conformational Properties of Rat Liver Ornithine Aminotransferase
Authors: SANADA Yukihiro , SHIOTANI Taiichi , OKUNO Etsuo , KATUNUMA Nobuhiko
Published: March 2005

Two apoenzymes of crystalline ornithine aminotransferase from rat liver were separated by gel filtration. They could also be separated by ultracentrifugation and sucrose density gradient centrifugation. These enzymes have different molecular weights, specific activities and absorption spectra. Their molecular weights are 177 100 ± 7700 (form I) and 105000 ± 6300 (form II) and their specific activities are 11.7 and 23.3 U/mg respectively. The native ornithine aminotransferase and the reconstituted holo‐form I and holo‐form II have similar Km values for l‐ornithine (0.9 to 1.1 mM), Km values for 2‐oxo glutarate (1.4 to 1.8 mM) and Ki; values for l‐valine (4.0 to 6.5 mM). In the absence of coenzyme both enzymes are inert, but form I has an absorption shoulder at 330 nm, like the pyridoxamine 5′‐phosphate form of enzyme. Apo‐form II associates forming a molecule with the same molecular weight as native enzyme of 177000, on addition of the coenzyme, pyridoxal 5′‐phosphate. The molecular weights of each enzyme protomer of the native ornithine aminotransferase and of the two apo‐forms were all found to be 45000 ± 2000 by dodecylsulfate acrylamide gel disc electrophoresis. The amino acids compositions of form I and form II are very similar, and the total number of SH groups/monomer of the native enzyme, apo‐form I and apo‐form II were all 5.
Inhibition of Poxvirus‐Specific Functions Induced in Chick‐Embryo Fibroblasts by Treatment with Homologous Interferon
Authors: OSTERHOFF Julia , JÄGER Margarethe , JUNGWIRTH Christoph , BODO Gerhard
Published: March 2005

Poxvirus‐infected primary chick embryo fibroblasts have been used as a model system to study the mechanism of the antiviral activity of homologous interferon. The effect of pretreatment of cells with 10–300 units/ml of interferon on the synthesis of cowpox and vaccinia WR virus‐specific proteins was tested in the ‘early’ and ‘late’ phase of infection. [35S]Methionine‐labelled virus‐specific proteins were examined after separation from host cell proteins by sodium dodecylsulfate acrylamide gel electrophoresis followed by autoradiography. Synthesis of individual poxvirus‐specific proteins was affected to varying degrees by interferon treatment of the cells. Some ‘late’ viral proteins were synthesized at a reduced rate even if the cells had been treated with only 10 units/ml of interferon. On the other hand, even after pretreatment with high doses of interferon (1000 units/ml) synthesis of certain virus‐specific proteins could not be completely inhibited. In the late stage of infection with strain vaccinia WR or cowpox virus, a protective effect of interferon treatment on host cell protein synthesis could be detected.

Vaccinia‐WR‐specific DNA synthesis is fairly resistant to interferon pretreatment, thereby making it possible to analyze the transcription program of virus‐specific RNA in the ‘late’ phase of infection in cells treated with low doses of interferon. All vaccinia‐WR‐specific RNA species transcribed in the ‘late’ phase of infection were detectable in chick embryo fibroblasts pretreated with 100 units ml of interferon which resulted in strong inhibition of the synthesis of several ‘late’ viral proteins.

Cowpox‐virus‐infected primary chick embryo fibroblasts developed a distinct cytopathic effect at late stages of infection but vaccinia‐WR‐infected cells did not. Morphologically this cytopathic effect was reduced in interferon‐treated cowpox‐infected chick embryo fibroblasts.
Interferon‐Mediated Inhibition of Cell‐Free‐Protein Synthesis in Response to Double‐Stranded RNA
Authors: KERR Ian M. , BROWN Ronald E. , CLEMENS Michael J. , GILBERT Christopher S.
Published: March 2005

The inhibition by double‐stranded RNA (dsRNA) of the translation of encephalomyocarditis virion RNA (EMC RNA) in extracts from interferon‐treated mouse L‐cells has been investigated. In particular, we have found that the addition of small amounts of dialysed cell sap from interferon‐treated cells to cell‐free systems from control cells renders them more sensitive to inhibition by dsRNA. This has provided an assay for the component(s) involved in the inhibition. The translation of both viral and non‐viral messenger RNAs is inhibited by dsRNA in the presence of interferon cell sap. The concentration of dsRNA required for inhibition, however, varies with the salt concentration used. The lower sensitivity of the control cell‐free system to dsRNA does not reflect a higher level of dsRNA‐specific nuclease, nor is the inhibition by interferon cell sap and dsRNA reversed by the addition of tRNA. There is an enhanced breakdown of the EMC RNA message in the inhibited systems. It is not yet clear, however, whether this reflects the activity of a dsRNA‐activated single‐strand‐RNA‐specific nuclease, or merely the degradation of unused message in the dsRNAinhibited system. Interestingly, the abnormal distribution of polypeptide products synthesised in response to EMC RNA in systems inhibited by interferon cell sap and dsRNA is apparently identical to that obtained on translation of this RNA in cell‐free systems from interferon‐treated, vacciniavirus‐infected L‐cells.
l‐Glutamate Dehydrogenase. Enhancement of the Reactivity of the p‐Chloromercuribenzoate‐Binding Site in Presence of the Competitive Label Methyl Mercuric Chloride
Authors: COSSON Marie‐Paule , PANTALONI Dominique
Published: March 2005

Modification of l‐glutamate dehydrogenase with p‐chloromercuribenzoate leads to the incorporation of one molecule per subunit of enzyme. Correlation between spectrophotometric measurements and p‐chloromercuri[carboxyl‐14C]benzoic acid binding argues for the formation of one cysteinylmeteurial bond. Kinetic studies indicate that the slow reactivity of the ‐SH group is due to an inherent property of the enzyme.

In presence of methylmercuric chloride, there is a big decrease of the labelling of the enzyme by p‐chloromercuribenzoic acid. However kinetic studies demonstrate the existence of a ternary complex enzyme‐p‐chloromercuribenzoate‐methylmercuric chloride which is characterized by a high p‐chloromercuribenzoic acid dissociation rate constant. The possible significance with respect to the protein structure is discussed.
The Mechanism of C‐5(6) Double Bond Introduction in the Biosynthesis of Cholesterol‐by Rat Liver Microsomes
Authors: REDDY Vangala V. R. , CASPI Eliahu
Published: March 2005

The dehydrogenation of cholest‐7‐en‐3β‐ol to cholesta‐5,7‐dien‐3β‐ol by rat liver microsomal acetone powder has been known to involve the abstraction of 5α and 6α hydrogens, which were found in the water, of the medium.

It was proposed by other investigators that an enzyme‐oxygen‐metal complex may be involved in this dehydrogenation. This could then result in the reduction of the molecular oxygen to hydrogen peroxide. We have evaluated the possibility of the formation of hydrogen peroxide in the course of the transformation of 5α‐[3α‐3H]cholest‐7‐en‐3β‐ol to [3α‐3H]cholesta‐5,7‐dien‐3β‐ol by a rat liver microsomal acetone powder in the presence of NAD+ and O2.

Hydrogen peroxide was not detected in the reaction mixture even though the sensitivity of the H2O2 detection method exceeded four times the calculated amount of hydrogen peroxide which could be produced in this reaction.

We have determined that the reaction is mitigated by a metal‐dependent enzyme complex and the process requires the presence of EDTA. The involvement of a metal‐enzyme complex is supported by the. observation that the desaturation is inhibited by 2,2′‐dipyridyl, 1,10‐phenanthroline and diethyldithiocarbamate. The reaction was also inhibited by horse radish peroxidase which suggested the participation of an enzyme‐metal‐oxygen complex. It was found that NADH promoted the dehydrogenation better than NAD+.

Attempts to dehydrogenate enzymatically 5α‐[3α‐3H]cholestan‐3 β‐ol failed. This tends to indicate the specificity of the enzyme system for 5α‐cholest‐7‐en‐3β‐ol.

The free radical inhibitors diphenylamine, N‐methylaniline and p‐cresol exhibited a stimulatory effect on the reaction.

Based on these results, a tentative mechanism for the dehydrogenation of cholest‐7‐en‐3β‐ol is proposed.
Phenyl β‐d‐Galactopyranoside as an Acceptor Substrate for the Blood‐Group H Gene‐Associated Guanosine Diphosphate l‐Fucose: β‐d‐Galactosyl α‐2‐l‐Fucosyltransferase
Authors: CHESTER M. Alan , YATES Alan D. , WATKINS Winifred M.
Published: March 2005

Phenyl β‐d‐galactopyranoside was found to be an efficient acceptor of l‐[14C]fucose when guanosine diphosphate l‐[14C]fucose was used as the donor substrate and human serum, submaxillary glands or stomach mucosa were the sources of l‐fucosyltransferase. The enzyme utilising phenyl β‐D‐galactoside was present in the serum of donors of all ABO blood‐groups examined, except those of the rare Oh (Bombay) and Bh phenotypes, but was clearly demonstrable in submaxillary gland preparations only when the glands came from individuals who were secretors of ABH blood‐group substances. This distribution coincides with that previously established for the blood‐group H gene‐specified α‐2‐l‐fucosyltransferase. The product of l‐[14C]fucosyl transfer to phenyl β‐d‐galactoside is separable from the other radioactive components in the enzyme digest by chromatography for 4 h in ethyl acetate/pyridine/water (10/4/3, by vol.); it was characterised as phenyl 2‐O‐(α‐l‐[14C]fucopyranosyl)β‐d‐galactopyranoside by (a) the liberation of l‐[14C]fucose by a specific α‐2‐l‐fucosidase, (b) its resistance to degradation by alkali and (c) the identification of tritiated glycerol as a product of periodate oxidation after a 3H label had been introduced into the galactosyl moiety. The use of phenyl β‐d‐galactopyranoside as acceptor substrate thus provides a simple and relatively rapid method for the assay of the blood‐group H gene‐specified α‐2‐l‐fucosyltransferase.
Calf‐Spleen Nicotinamide‐Adenine Dinucleotide Glycohydrolase Kinetic Mechanism
Authors: SCHUBER Francis , TRAVO Pierre , PASCAL Marc
Published: March 2005

Steady‐state analysis of product inhibition indicates that the most probable minimum kinetic mechanism for soluble calf spleen NAD glycohydrolase is ping‐pong bi‐bi, reducing to an ordered uni‐bi for hydrolysis alone. In the hydrolysis of NAD nicotinamide is released first. Isotope exchange, transglycosidation reaction and nucleophilic competition with methanol show that the formation of the intermediary enzyme · adenosine‐diphosphoribosyl complex (E‐ADP‐Rib) is rate limiting.

The NAD hydrolysis is subject to inhibition by excess of substrate, which is markedly accentuated by increasing concentrations of nicotinamide. Kinetic analysis of this behaviour is in favour of the formation of an apparent dead‐end complex of NAD with the Michaelis complex.

The reactivies of methanol and nicotinamide compared to water with the intermediate E‐ADP‐Rib suggest the occurrence of an intermediary oxocarbonium ion in the reaction catalyzed by NAD glycohydrolase.
Uridine‐Diphosphate‐Glucose 4‐Epimerase from Saccharomyces fragilis
Authors: RAY Manju , BHADURI Amar
Published: March 2005

UDP‐glucose 4‐epimerase from Saccharamyces fragilis is rapidly inactivated by heating at 42 °C for 7 min and at 45 °C for 4 min. The effector site. specific for sugar phosphates, is destroyed still earlier. The enzyme is inactivated by the dissociation of NAD from it leaving the dimeric structure unaffected. It call be reactivated by mercaptoetlianol and NAD, both of which are essential for reactivation, and NAD becomes associated with the dimeric protein moiety.
Purification and Properties of Adductor Muscle Phosphofructokinase from the Oyster, Crassostrea virginica
Authors: STOREY Kenneth B.
Published: March 2005

Phosphofructokinase from oyster (Crassostrea virginica) adductor muscle occurs in a single electrophorectic form at an activity of 8.1 μmol of product formed per minute per gram wet weight. The enzyme was purified to homogeneity by a novel method involving extraction in dilute ethanol and subsequent precipitation with polyethylene glycol. Oyster adductor phosphofructokinase has a molecular weight of 3400000 ± 20000 as measured by Sephadex gel chromatography. Mg2+ or Mn2+ can satisfy the divalent ion requirement while ATP, GTP, or ITP can serve as phosphate donors for the reaction. Oyster adductor phosphofructokinase displays hyperbolic saturation kinetics with respect to all substrates (fructose 6‐phosphate, ATP, and Mg2+) at either pH 7.9 or pH 6.8. The Michaelis constant for fructose 6 phosphate at pH 6.8. the cellular pH of anoxic oyster tissues, is 3.5 mM. In the presence of AMP, by far the most potent activator and deinhibitor of the enzyme, this drops to 0.70 mM. Many traditional effectors of phosphofructokinase including citrate. NAD(P)H, Ca2+, fructose 1,6‐bisphosphate, 3‐phosphoglycerate, ADP, and phosphoenolpyruvate do not alter enzyme activity when tested at their physiological concentrations. Monovalent ions (K+, NH4+) are activators of the enzyme. ATP and arginine phosphate are the only compounds found to inhibit the adductor enzyme. The inhibitory action of both can be reversed by physiological concentrations of AMP (02–1.0 mM) and to a lesser extent by high concentrations of P, (20 mM) and aqdenosine 3′:5′‐monophosphate (0.1 mM). The two inhibitors exhibit very different pH versus inhibition profiles. The Ki(ATP) decreases from 5.0 mM to 1.3 mM as the pH decreases from 7.9 to 6.8. whereas the Ki for arginine phosphate increases from 1.3 mM to 4.5 mM for the same pH drop. Of all compounds tested, only AMP, within its physiological range, activated adductor phosphofructokinase significantly at low pH values.

The kinetic data support the proposal that arginine phosphate, not ATP or citrate, is the most likely regulation of adductor phosphofructokinase in vivo under aerobic, high tissue pH, conditions. In anoxia the depletion of arginine phosphate reserves and the increase in AMP concentrations in the tissue, coupled with the increase in the Ki for arginine phosphate brought about by low pH conditions, serves to activate phosphofructokinase to aid maintenance of anaerobic energy production.
Template Activity of Synthetic Deoxyribonucleotide Polymers in the Eukaryotic DNA‐Dependent RNA Polymerase Reaction
Authors: SASAKI Yukiko , GOTO Hideyuki , OHTA Hiromi , KAMIKUBO Tadashi
Published: March 2005

Template specificities of the eukaryotic DNA‐dependent RNA polymerases A and B from rat liver, pea, and cauliflower have been investigated using synthetic polydeoxyribonucleotides. Polymerases A and B from the three species exhibit different specificities for single‐stranded homopolymers: polymerase A preferentially reads poly(dT) and poly(dC), and polymerase B poly(dC). This preferential reading appears to be a property of eukaryotic DNA‐dependent RNA polymerases.

Polymcrases A and B transcribe synthetic polyribonucleotides also, but at a reduced rate. The polyribonucleotides which can be read by DNA‐dependent RNA polymerases have a base sequence similar to that of the polydeoxyribonucleotides, which are efficiently transcribed, suggesting that the base sequence of the template rather than its conformation is crucial in the template specificity for synthetic polymers. Competition experiments with polydeoxyribonucleotides indicate that the enzymes have different binding specificities, which are not the same as their template specificities
The Cytochrome bc1, Complex of Yeast Mitochondria
Authors: KATAN Martijn B. , VAN HARTGN‐LOOSBROGK Nel , GROOT Gert S. P.
Published: March 2005

Yeast cells were labelled with radioactive amino acids in the presence of cycloheximide and the cvtochrome bc1 complex was isolated from them as described in the preceding paper [Katan, M. B., Pool. L. & Groot, G. S. P. (1976) Eur. J. Biochem. 65, 95–105]. After analysis of this preparation by sodium dodecylsulphate polyacrylamide gel electrophoresis only one band, with an apparent Mr of 32000, was found to have incorporated radioactivity. The amount of label in the band was low, but could be increased approximately 5‐fold by preincubating the cells in erythromycin before the labelling period.

Cells were labelled in the presence of chloramphenicol and the cytochrome bc1 complex was isolated by (NH4)2SO4 fractionation. Upon electrophoresis in the presence of sodium dodecylsulphate only four of the six bands that belong to the complex were found to have incorporated radioactivity; no radioactivity was found in the bands with an Mr of 40000 and 17000. The same result was obtained after labelling in the presence of acriflavin. If, however, the cytochrome bc1 complex was isolated by immunoprecipitation, all bands were found to have incorporated radioactivity in the presence of chloramphenicol. The amount of radioactivity in the Mr 32000 band was now clearly depressed.

It is concluded that of the seven polypeptides of the cytochrome bc1 complex of yeast only one is trade on mitochondrial ribosomes. This polypeptide has an Mr of 32000 and is probably associated with cytochrorne b.
Kinetics of Thyroglobulin Iodination and of Hormone Synthesis Catalyzed by Thyroid Peroxidase
Authors: DÉME Daniéle , POMMIER Jacques , NUNEZ Jacques
Published: March 2005

The kinetics of tyrosine iodination and of thyroxine synthesis in thyroglobulin, different reactions catalyzed by the sane enzyme (thyroid peroxidase), have been compared.

Thyroxine synthesis always began after a lag period of 3–5 min. This lag was constant whatever the rate of iodination; this rate of iodination was increased either by increasing the concentration of iodide or enzyme or by decreasing the concentration of thyroglobulin. Increasing the rate of iodination resulted in increasing the number of iodine atoms incorporated during the lag period. Thus the lag observed for thyroxine synthesis was constant and did not depend on the fact that free iodide or non‐iodinated tyrosine residues of thyroglobulin were exhausted before thyroxine synthesis occurred. Finally, it appeared that, whatever the explanation of the lag, the enzyme catalyzes thyroid hormone synthesis at a slower rate than iodination.

The existence of a lag also allowed us to prepare thyroglobulin samples with different iodine contents but without thyroid hormones. Thus iodination and thyroxine synthesis could be studied independently and the following results were obtained.

Iodotyrosine residues which can couple to form thyroxine are made considerably before coupling occurs.

H2O2 is required for coupling of these hormonogenic residues; thus the coupling reaction requires enzymic oxidation of the iodotyrosine residues.

In addition a strict requirement for iodide was needed for coupling: the requirement was dependent on the concentration of iodide. Thus iodide, a substrate of the iodination reaction, may also have other effects on the activity of thyroid peroxidase.
Purification of Poly(ADP‐ribose) Polymerase from Ehrlich Ascites Tumor Cells by Chromatography on DNA‐Agarose
Authors: KRISTENSEN Tom , HOLTLUND Jostein
Published: March 2005

Poly(A DP‐ribose) polymerase with a high specific activity was obtained from Ehrlich aseites tumor cell, by extraction of nuclei With 175 mM potassium phosphate followed by chromatography on DNA‐agarose. Electrophoretic analysis indicated that the preparation contained two proteins, one of which was shown to catalyze the synthesis of poly(ADP‐ribose). As expected from results obtained by other workers the synthesis was inhibited by nicotinamide and thymidine, and stimulated by DNA. Addition of histones gave inhibition of the synthesis, unless DNA was present in the reaction mixture.
RNA Sequences Associated with Proteins L1, L9, and L5, L18, L25, in Ribonucleoprotein Fragments Isolated from the 50‐S Subunit of Escherichia coli Ribosomes
Authors: BRANLANT Christiane , KROL Alain , SRIWIDADA Johanes , BRIMACOMBE Richard
Published: March 2005

32P‐labelled 50‐S subunits from Escherichia coli ribosomes were hydrolysed under conditions known to give rise to two specific ribonucleoprotein fragments, containing proteins L1, L9, and L5, =L 18 and L 25 respectively. RNA corresponding to these ribonucleoproteins was isolated and purified, and the various RNA fragments obtained were subjected to oligonucleotide analysis.

The results showed that the RNA associated with proteins L1and L9 was very similar to the RNA found with protein L1 after controlled digestion of 23‐S‐RNA. L1 complexes (described elsewhere); this RNA lies within a region 550–1000 nucleotides from the 3′ terminus of 23‐S RNA. The RNA associated with proteins L5, L18, and L25 consisted predominantly of two species of similar size. One was 5‐S RNA, and the other a fragment of 23‐S RNA, lying within the region 450–1000 nucleotides from the 3′ terminus.
Studies on 3‐Deoxy‐d‐arabinoheptulosonate‐7‐phosphate Synthetase(phe) from Escherichia coli K12
Authors: SIMPSON Richard J. , DAVIDSON Barrie E.
Published: March 2005

3‐Deoxy‐d‐arabino heptulosonate‐7‐phosphate synthetase(phe) from Escherichia coli K12 has been purified to near homogeneity. The purified enzyme has a specific activity of 67 units mg which is about 1000 times that found in cell‐free extracts of wild type E. coli K12.

The minimum molecular weight of the enzyme was estimated by dodecylsulphate‐gel eleetrophoresis to be 33000. Re‐estimation of the native molecular weight by gel filtration confirmed the previously determined value of 110000.

Amino acid analysis and tryptic fingerprints indicated that the subunits of the enzyme are very similar and possibly identical.

The purified enzyme does not contain Co2+
Studies on 3‐Deoxy‐d‐arabino heptulosonate‐7‐phosphate Synthetase(phe) from Escherichia coli K12
Authors: SIMPPSON Richard J. , DAVIDSON Barne E.
Published: March 2005

Co2+ is not a cofactor for 3‐deoxy‐d‐arabinoheptulosonate‐7‐phosphate synthetase(phe).

The following analogues of phosphoenolpyruvate were tested as inhibitors of 3deoxy‐d‐arabinoheptolosonate‐7‐phosphate synthetase(phe): pyruvate, lactate, glycerate, 2‐phosphoglycerate, 3‐methylphosphoenolpyruvate, 3‐ethylphosphoenolpyruvate and 3,3‐dime‐thylphosoenolpyruvate. The results obtained indicate that the binding of phosphoenolpyruvate to the enzyme requires a phosphoryl group on the C‐2 position of the substrate and one free hydrogen atom at the C‐3 position.

The dead‐end inhibition pattern observed with the substrate analogue 2‐phosphoglycerate when either phosphoenolpyruvate or erythrose 4‐phosphate was the variable substrate is inconsistent with a ping‐pong mechanism and indicates that the reaction mechanism for this enzyme must be sequential. The following kinetic constants were determined: Km for phosphoenolpyruvate, 0.08 ± 0.04 mM: Km for erythrose 4‐phosphate, 0.9 ± 0.3 mM; Kis for competitive inhibition by 2‐phosphoglycerate with respect to phosphoenolpyruvate, 1.0 ± 0.1 mM.

The enzvmc was observed to have a bell‐shaped pH profile with a pH optimum of 7.0. The effects of pH on V and V/(Km for phosphoenolpyruvate) indicated that an ionizing group of pKa.8.0–8.1 is involved in the catalytic activity of the enzyme. The pKa of this group is unallected by the binding of phosphoenolpyruvate.
Replication of M‐13 DNA in Plasmolysed Escherichia coli Cells
Authors: KESSLER‐LIEBSCHER Barbara E. , STAUDENBAUER Walter L.
Published: March 2005

The replication of the double‐stranded replicative‐form DNA of bacteriophage M‐13 was studied in a cellular system in vitro prepared by plasmolysis of M‐13‐am5‐infected Escherichia coli cells. Newly synthesized DNA was density‐labelled with bromodeoxyuridine triphosphate and analysed by equilibrium centrifugation in neutral CsCI. After a 60‐min incubation at 30°C 15 – 20% of the radioactive label incorporated from [32P]dGTP was found in fully synthetic (duplex DNA, corresponding to 7–9 replicative form molecules cell. The plasmolysed cell system is therefore capable of re‐initiating new rounds of replicative form replication in vitro. The kinetics of labelling indicate that molecules are selected for replication at random from an intracellular pool of approximately 150 replicative form molecules. Rifampicin and nalidixic acid, which interfere with the semiconservative replication of replicative form DNA, completely prevent the formation of fully synthetic duplex DNA.
Isolation and Characterization of a Cat 2+‐Binding Polysaccharide Associated with Coccoliths of Emiliania huxleyi (Lohmann) Kamptner
Authors: DE JONG Elisabeth W. , BOSCH Leendert , WESTBROEK Peter
Published: March 2005

Coccolithophoridae, a group of mostly unicellular algae, possess a cell wall containing calcified plates, called coccoliths. The coccoliths from the species Emiliania huxleyi (Lohmann) Kamptner contain a water‐soluble acid polysaccharide. In this paper we describe the isolation and some characteristic properties of the polysaccharide, in particular its Ca2+ ‐binding capacity.

A large‐scale cultivation of the Coccolithophoridae was worked out and a new procedure for isolating coccoliths was developed. The polysaccharide obtained from the coccoliths contained two types of monobasic acid groups in a total amount of 1.8 μmol/mg polysaccharide.

One type consisted of weakly acid groups which were identified as uronic acids. The nature of the stronger acid groups remains to be established. The ratio between the respective groups was 1:0.8. Studies with; 45Ca2+ demonstrated that the isolated polysaccharide is capable of binding Ca2+. Equilibrium dialysis revealed that the maximum amount of Ca2+ which can be bound is 0.92 ± 0.05ltmol/mg polysaccharide. Flow‐rate dialysis experiments strongly suggested the presence of two classes of Ca2+‐binding sites differing in affinity for Ca2+. High‐affinity sites (dissociation constant Kd for Ca2+: 2.2 + 1.0 × 10−5 M) were found to be present in amounts (0.38 ± 0.04 μmol/ mg polysaccharide) approximately equivalent to the strongly acid monovalent groups mentioned above (0.8 μmol/mg polysaccharide).

Low‐affinity sites (Kd for Ca2+: 111 ± 39 × 10−s M) were estimated at 0.74 ± 0.11 μmol/ mg polysaccharide. Although this figure could be determined less accurately, it is suggested that the uronic acids (1.0 μmol/mg polysaccharide) are identical to the low‐affinity sites. Preferential binding of Ca2+ occurred in a 100‐fold excess of Na+ and Mg2+ as was shown by gel filtration. A 100‐fold excess ofSr2+ inhibited Ca2+ binding to a great extent while no Ca2+ was bound in the presence of an equimolar amount of La3+. The dissociation constants of the high‐affinity sites for Na+, Mg2+, Sr2+ and La3+ (in the presence of Ca2+) were determined with the flow‐rate dialysis technique. They confirm the order of binding preference found with gel filtration.

A polysaccharide with similar properties could be isolated from subfossil coccoliths of E. huxieyi (about 1000 years old).

The possible role of the polysaccharide as a heterogeneous matrix in coccolith formation is discussed.
Isolation and Characterization of Poly(glycosyl)ceramides (Megaloglycolipids) with A, H and I Blood‐Group Activities
Authors: KOŚCIELAK Jerzy , MILLER‐PODRAZA Halina , KRAUZE Robert , PIASEK Andrzej
Published: March 2005

Very complex glycosphingolipids with A, H and I blood‐group activities were isolated from human erythrocyte membranes. The membranes were obtained from erythrocytes of blood group A, A2 and O respectively. A general formula for the antigens is:

(Fuc)3–4(Gal)n(GlcNAc)n–2(Glc)1(Sphingosine)1

(where Fuc is fucose, Gal is galactose, GlcNAc is N‐acetylglucosamine and Glc is glucose) with values of n ranging from 10–27. A‐active preparations contain additionally 2–3 residues of N‐acetyl‐galactosamine. In view of the unusual complexity of these compounds they were designated poly(glycosyl)ceramides (formerly megaloglycolipids). Individual poly(glycosyl)ceramide fractions were isolated from A erythrocytes and were found to differ by about 8 glycosyl residues per molecule forming a series of compounds with 22, 30, 38, 51 and 59 glycosyl residues per mole. Structural studies indicate that the main sequence of poly(glycosyl)ceramides consists of the residues of galactopyranose and 2‐deoxy‐2‐acetamidoglucopyranose substituted at 3 and 4 position respectively. These residues are probably alternating. N‐Acetylglucosamine substituted at 3 position was not found in poly‐(glycosyl)ceramides. Branches of poly(glycosyl)ceramides originate from 3 and 6 position of galacto‐pyranosyl residues. The number of branches is proportional to the degree of molecular complexity. In poly(glycosyl)ceramides isolated from A and A2 erythrocytes the branches are terminated with the following structures GalNAc α 1→3 [Fuc α 1→2] Gal; Fuc α 1→2 Gal and Gal (presumably Gal β 1→4 GlcNAc). In poly(glycosyl)ceramides from A cells the total number of A and H‐active structures per average molecule of 30–35 glycosyl residues amounts to 2.1 and 1.2 respectively while the number of terminal galactose structures is 1.8. For poly(glycosyl)ceramides from A2 erythrocytes the corresponding figures are 0.75, 3.5, and 2.1 respectively.

Poly(glycosyl)ceramides from O cells comprise about 3.8 H‐active structures and 1.8 terminal galactopyranosyl residues. In poly(glycosyl)ceramides with high “n” values the number of terminal galactose structures is increased. These fractions display high blood‐group I activity. However, the removal of terminal galactose with β‐galactosidase affects I‐activity only slightly.
Deoxyribonucleotide Pools in Mouse‐Fibroblast Cell Lines with Altered Ribonucleotide Reductase
Authors: MEUTH Mark , AUFREITER Eva , REICHARD Peter
Published: March 2005

Mutant cells lines of 3T6 mouse fibroblasts, resistant to thymidine and deoxyadenosine, have an altered allosteric regulation of the enzyme ribonucleotide reductase (Meuth, M. and Green, H., Cell, 3, 367, 1974). Compared to 3T6, these lines contain larger pools of deoxynucleoside triphosphates, in particular deoxycytidine triphosphate, but show a normal rate of DNA synthesis. Addition of thymidine or deoxyadenosine to 3T6 cells results in large accumulations of the corresponding triphosphates and a dramatic decrease in the dCTP pool, concomitant with inhibition of DNA synthesis. Addition of thymidine to the mutant cell lines also leads to an increase in the dTTP pool but does not result in a depletion of dCTP or inhibition of DNA synthesis. Addition of deoxyadenosine only leads to a small increase of the dATP pool. In general the change in the allosteric regulation of ribonucleotide reductase is reflected in the deoxynucleotide pools.
A Spin‐Label Study of the Chromaffin Granule Membrane
Authors: MARSH Derek , RADDA George K. , RITCHIE Gillian A.
Published: March 2005

The structure of the chromaffin granule membrane has been probed using a number of different spin labels. Both the effect of temperature and high levels of calcium have been studied.

The results from three positional isomers of the stearic acid spin label demonstrate that a substantial part of the membrane lipid (that is sensed by the probe) is in a bilayer structure which undergoes a structural transition at 32–36 °C, characterized by an increase in the population of gauche isomers in the lipid chains. A possible mechanism for this transition would be the preferential segregation of cholesterol.

The covalently bound iodoacetamide spin label reveals a transition within the protein component of the membrane or its immediate lipid environment at 32°C. This transition corresponds to an increased degree of motional freedom of the spin label above the transition temperature.

The lipid‐soluble spin label 2,2,6,6‐tetramethyl‐piperidine‐1‐oxyl exhibits a break at 34°C in the temperature‐dependence of its partitioning into the membrane. This could correspond to the onset of a lateral separation in the membrane lipid, again possibly involving a re‐distribution of cholesterol.

Calcium abolishes, diminishes or shifts the transition observed by the spin label and decreases the amplitude of motion of the stearic acid spin labels, again possibly involving a restribution of cholesterol and also lysolecithin.

The temperatures of the structural transition agree well with the changes in the enzymic activity of the membrane ATPase and NADH oxidase functions and also with the results from fluorescent probes [Bashford et al., Eur. J. Biochem. 67, 105–114 (1976)]. It is possible that triggering of the transition either by calcium or some other stimulus may play a role in catecholamine release and membrane fusion.
Poly(A)‐Containing RNA in Tetrahymena pyriformis
Authors: RODRIGUEZ‐POUSADA Claudina , HAYES Donal H.
Published: March 2005

Poly(A)‐containing RNAs with sedimentation coefficients in the range 10–45 S and 10–35 S (Mr 0.2–3.3 × 106 and 0.2–2.2 × 106 approximately) are present in the nucleus and cytoplasm of T. pyriformis respectively. The poly(A) segments present in both nuclear and cytoplasmic poly(A)‐containing RNAs have a chain length of 80–150 nucleotides. Significant amounts of high‐molecular‐weight poly(A)‐containing RNAs (35 S, Mr 2.2 × 106) are found in the post‐polysomal fraction of the cytoplasm (messenger ribonucleoprotein particles?) and also in large polysomes. In order to exclude the possibility that these high‐molecular‐weight species are products of nonspecific aggregation, RNA preparations were submitted to severe denaturing conditions before analysis and all electrophoreses were carried out in the presence of 95% (v/v) formamide.
The Virtual Absence of Antigenic Cross‐Reactivity between Functionally Distinct Trout Hemoglobins
Authors: TAN‐WILSON Anna L. , REICHLIN Morris , BRUNORI Maurizio , NOBLE Robert W.
Published: March 2005

The antigenic properties of the hemoglobins of two species of rainbow trout, Salmo irideus and Salmo gairdneri, have been compared. Each of these species possess two different classes of hemoglobins, the members of the first of which have no Bohr effect while those of the second exhibit a pronounced pH dependence of their ligand affinities. The hemoglobins from these two species are antigenically indistinguishable. However, between the two classes of hemoglobins there is almost no immunological cross‐reactivity.
Human Coagulation Factor IX
Authors: SUOMELA Hannu
Published: March 2005

Human coagulation factor IX was purified by two ion‐exchange chromatographies on DEAE‐Sephadex A‐50, heparin‐Sepharose chromatography, hydroxyapatite chromatography and immuno‐adsorbent technique. Factor IX was homogeneous by ordinary and sodium dodecylsulphate disc electrophoresis, N‐terminal amino acid analyses and ultracentrifugation and by immunological criteria. The following molecular data were observed:

Sedimentation equilibrium indicated a molecular weight of 66 100 and sedimentation velocity gave S20, w= 3.97 S. A partial specific volume of v̄= 0.712 ml/g was calculated from the amino acid and carbohydrate composition.

Sodium dodecylsulphate disc gel electrophoresis suggested a molecular weight of 65 000.

Gel filtration indicated a Stokes radius of 4.08 nm, and ‘a molecular weight’ of 72 000, as well as a diffusion coefficient D20, w= 5.15 × 10−7 cm2 s−1 and a frictional ratio f/f0= 1.54.

Tyrosine was the N‐terminal amino acid. The amino acid composition is described. Factor IX contained approximately 17.5% carbohydrate, which includes 4.7% hexose, 6.8%N‐acetylhexos‐amine and 6% sialic acid.

Microheterogeneity of pure factor IX was demonstrated by isoelectric focusing. The isoelectric points of the major components lay within the range of pH 4.0 to 4.6.

The antibody raised in rabbits against the pure factor IX did not react with the other vitamin‐K‐dependent coagulation factors measured by coagulation factor assays or immunodiffusion in gels.
Further Studies on the Binding of Maltose to the Maltose‐Binding Protein of Escherichia coli
Authors: SCHWARTZ Maxime , KELLERMANN Odile , SZMELCMAN Sevec , HAZELBAUER Gerald L.
Published: March 2005

Extensive binding studies, performed by equilibrium dialysis, suggest that the periplasmic maltosebinding protein of Escherichia coli is homogeneous with respect to its affinity towards maltose. This finding contradicts an earlier conclusion, which was based on insufficient data.
Microtubular Proteins in Pigeon Erythrocyte Membranes
Authors: ZENNER Hans P. , PFEUFFER Thomas
Published: March 2005

[3H]Colchicine binds in a concentration and temperature dependent, saturatable and noncooperative manner to cytoplasmic proteins from pigeon erythrocytes: Kd= 3.5 × 10−7 M at 37 °C. Binding of [3H]colchicine at 0 °C and of [3H]lumicolchicine at 37 °C was significantly reduced. Hence microtubular proteins are present in the cytoplasm of pigeon erythrocytes.

Antibody against bovine brain tubulin was raised in rabbits and confirmed by immunodiffusion, passive immunohaemolysis and in radioimmunoassay. Pigeon erythrocyte membrane proteins solubilized with 2% sodium cholate competed with 125I‐labelled tubulin in the radioimmunoassay although much higher concentrations of membrane proteins than of purified bovine brain tubulin were required for effective competition. No binding to antibody occurred with boiled solubilized membrane preparations. Similar results were obtained with antitubulin‐dependent passive immunohaemolysis of tubulin‐coated sheep erythrocytes in the presence of complement. The presence of tubulin in membranes was verified by binding intact pigeon erythrocytes to colchicine‐Sepharose beads at 37 °C. Free colchicine (5 mM) or incubation at 0 °C prevented binding. Lumicolchicine‐Sepharose beads did not attach to erythrocytes at 37 °C. Thus pigeon erythrocyte membranes contain microtubular proteins.
Esterolytic Activities of Rat Intestinal Mucosa
Authors: NÉGREL Raymond , SERRERO Ginette , AILHAUD Gérard , FERNANDEZ‐LOPEZ Victor
Published: March 2005

The preferential cellular distribution in the villus tip and the subcellular localization in the endoplasmic reticulum of an intestinal glycerol‐ester hydrolase from rat mucosa are described. The enzyme is shown not to be from either pancreatic or bacterial origin; it catalyzes the hydrolysis of short‐ and medium chain triglycerides and of ρ‐nitrophenylacetate. Contrarily to the specificity found for the pig intestinal lipase (Serrero, Négrel and Ailhaud, 1975), no activity is detectable against acyl‐CoA; a thiolester hydrolase different from the glycerol‐ester hydrolase was demonstrated after differential solubilization and chromatographic separation. A high proportion of glycerol‐ester hydrolase is present in the intestinal lumen; its possible complementary role in lipid degradation is discussed.