bluuebunny/crossref_metadata_2025 huggingface dataset:
Fast Stages of Photoelectric Processes in Biological Membranes
Authors: DRACHEV Lel A. , SEMENOV Alexey Yu. , SKULACHEV Vladimir P. , SMIRNOVA Irina A. , CHAMOROVSKY Sergey K. , KONONENKO Alexander A. , RUBIN Andrey B. , USPENSKAYA Natalya Ya.
Published: July 2010

Chromatophores of photosynthetic bacteria Rhodospirillum rubrum, Rhodopseudomonas sphaeroides and Chromatium minutissimum were associated with a collodion film impregnated with a decane solution of asolectin. A very short light flash inducing a single turnover of the chromatophore photosynthetic redox system was found to induce the formation of an electric potential difference amounting to 60 mV, directed across the film as measured with an orthodox electrometer technique. The main phase of the photoelectric response had a τ value of < 200 ns. Addition of menadione and some other redox mediators increases the main phase amplitude and induces a slower phase (τ= 200 μs). In Ch. minutissimum chromatophores that retained their endogeneous cytochrome c pool, one more electrogenic phase was revealed (τ= 20 μs). Redox titrations of the electric response and bacteriochlorophyll absorption at 430 nm as well as measurements of the kinetics of cytochrome c oxidation have indicated that the fastest electrogenic phase is due to electron transfer from bacteriochlorophyll to Feubiquinone, the 20‐μs phase to cytochrome c2+ ‐bacteriochlorophyll+ oxidoreduction, and the 200‐μs phase to Fe‐ubiquinone− oxidation by a secondary quinone. In the decay of the photoelectric response, a 30‐ms phase was identified which was explained by a reverse electron transfer from reduced Fe‐ubiquinone to oxidited bacteriochlorophyll. The difference in the fast kinetics of photoelectric generation by the bacteriochlorophyll system from those by bacterial and animal thodopsins has been discussed.
Purification, Properties and Assembly of the Neck‐Appendage Protein of the Bacillus subtilis Phage φ 29
Authors: VILLANUEVA Nieves , LÁZARO José M. , SALAS Margarita
Published: July 2010

The purification of the neck appendage protein of φ29, p12*, which is involved in the adsorption of the phage to Bacillus subtilis, is described. The purified native protein is in a dimeric form and can be assembled, in vitro, onto purified 12− particles that lack the neck appendages, suggesting that the incorporation of p12* to the rest of the phage structure is a self‐assembly process. The assembly of protein p12*in vitro follows cooperative kinetics and it occurs with an efficiency of about 4%.
Detection of Proton‐Acceptor Sites of Hydrogen Bonding in Adenine · Uracil Base Pairs by the Use of 15N Magnetic Resonance
Authors: WATANABE Mayumi , SUGETA Hiromu , IWAHASHI Hideo , KYOGOKU Yoshimasa , KAINOSHO Masatsune
Published: July 2010

2′,3′‐5′‐Tri‐O‐acetyl[1,3,7,9,amino‐15N]adenosine(ac3Ado) and its 8‐2H and 8‐bromo derivatives (ac3[8‐2H]Ado and ac3br8Ado) were synthesized from 95%15N‐enriched adenosine which was obtained by microbial fermentation. The chemical shifts and nuclear Overhauser effects of 15N NMR of the adenosine derivatives were measured by changing the concentration of the mixed 1‐cyclohexyluracil (cHxUra) in chloroform. The limiting shift of each 15N resonance was calculated by using the association constants obtained from proton magnetic resonances of the ac3Ado/cHxUra and ac3br8Ado/cHxUra systems. From the quantitative analysis of the 15N chemical shifts of the N‐1 and N‐7 atoms it could be concluded that ac3Ado · cHxUra dimers prefer the Hoogsteen‐type pair while the Watson‐Crick‐type pairs are predominant in the ac3br8Ado · cHxUra dimers. The N‐3 atom, which does not locate at the interaction site, showed a fairly large induced shift. Thus the induced shift of 15N resonance by association does not always mean the involvement of the corresponding nucleus to the interaction site. The effect of intermolecular hydrogen bonding on the nuclear Overhauser enhancement of the 15N‐7 resonance was observed in the ac3[8‐2H]Ado/cHxUra mixture. The presence of the Hoogsteen‐type hydrogen bonding was proposed by the use of 15N NMR spectra. The present information will be useful for the elucidation of non‐Watson‐Crick‐type base‐pair interaction in transfer RNA and oligonucleotides.
A Synthetic Ionophore for Ca2+: Studies with Model and Biological Systems
Authors: GOMPERTS Bastien D. , BENNETT Jonathan P. , ALLAN David
Published: July 2010

We show that the relatively simple β‐diketone, 1,1,1.2,2,3,3‐heptafluoro‐7,7‐dimethyloctane‐d,6‐dione, known as ‘fod’, will solvate Ca2+ ions in an organic phase. In consequence it will transport Ca2+ across phospholipid bilayers. It acts as predicted for a Ca2+‐transporting ionophore in two biological systems: it will stimulate Ca2+‐dependent K+ efflux from human red blood cells, and Cat+‐dependent histamine secretion from rat peritoneal mast cells.
Induction of Casein Synthesis by Prolactin and Inhibition by Progesterone in the Pseudopregnant Rabbit Treated by Colchicine without any Simultaneous Variations of Casein mRNA Concentration
Authors: TEYSSOT Bertrand , HOUDEBINE Louis‐Marie
Published: July 2010

Prolactin injected into pseudopregnant rabbits induced casein synthesis and it provoked an accumulation of casein mRNA and of ribosomal RNA. Colchicine, which has been shown to block the prolactin signal, totally prevented the accumulation of β‐casein mRNA, when injected with the hormone. However, the drug did not hamper the initiation of casein synthesis and the accumulation of ribosomal RNA. The effect of prolacdn injected with colchicine on casein synthesis was totally abrogated by progesterone administered simultaneously and it was essentially unmodified by glucocorticoids. These results suggest that prolactin controls casein gene expression by supporting the accumulation of casein mRNAs and by stimulating the translation of these mRNAs, through independent mechanisms. Progesterone which is known to prevent the accumulation of casein mRNAs is also a potent inhibitor of casein mRNA translation whereas glucocorticoids exert their effect essentially by favouring the accumulation of casein mRNA but not by modifying their translation efficiency.
Immunochemical Evidence for Phytochrome Regulation of the Specific Activity of Ascorbate Oxidase in Mustard Seedlings
Authors: NEWBURY H. John , SMITH Harry
Published: July 2010

A monospecific antiserum to a Cucurbita ascorbate oxidase was shown to cross‐react with the mustard enzyme. Purified antiserum was used in rocket immunoelectrophoresis plates to measure the amount of ascorbate oxidase protein in mustard cotyledon and hypocotyl extracts. On transfer from darkness to far‐red light the activity of the enzyme in expanding cotyledons increased but the amount of enzyme protein remained the same. Unimbibed mustard seeds were shown to contain the same amount of antigenic protein as expanding cotyledons although in the seeds the enzyme was inactive. In hypocotyls continuous far‐red light treatment also led to an increase in ascorbate oxidase activity; in this case there was also an increase in enzyme protein although this was relativelv small. The results are discussed in relation to the mechanism by which phytochrome may control the activity of this enzyme.
Identification and Purification of Distinct Isomerase and Decarboxylase Enzymes Involved in the 4‐Hydroxyphenylacetate Catabolic Pathway of Escherichia coli
Authors: GARRIDO‐PERTIERRA Amando , COOPER Ronald A.
Published: July 2010

The possible involvement of an isomerase in the 4‐hydroxyphenylacetate meta‐cleavage pathway has been studied. 5‐Carboxymethyl‐2‐hydroxymuconate has been shown to undergo both spontaneous and enzymecatalysed isomerisation to give 5‐carboxymethyl‐2‐oxo‐hex‐3‐ene‐1,6‐dioate, a compound with an absorbance maximum at 246 nm. The latter compound rather than the former is the substrate for a decarboxylase that produces 2‐hydroxyhepta‐2,4‐diene‐1,7‐dioate. The isomerase and decarboxylase enzymes have been purified to over 90% homogeneity. Mg2+ is required for the decarboxylase reaction but not for the isomerase.
Substrate Specificity of the Nuclear Protein Kinase NII from Porcine Liver
Authors: HOPPE Jürgen , BAYDOUN Hassan
Published: July 2010

The substrate specificity of the nuclear protein kinase NII from pig liver was studied using the purified casein variants αs1B and βA2 both in the phosphorylated and dephosphorylated form. Chymotryptic peptides of the phosphorylated casein were analyzed by ascending thin‐layer chromatography followed by thin‐layer electrophoresis. Phosphorylated peptides were detected by autoradiography. The determination of a phosphorylated site in a cluster of three serines was performed by solid phase Edman degradation of the whole βA2 casein.

Phosphorylated βA2 casein was by far the best substrate. A threonine at position 41 in the amino acid sequence was found to be predominantly phosphorylated. Dephosphorylated βA2 casein became rephosphorylated at serine‐17 whereas phosphorylation at threonine‐41 was greatly reduced. In both sites acidic amino acid side chains are present at the C‐terminal side though at different positions suggesting that acidic residues in the vicinity of the serine or threonine residue to be phosphorylated play an important role in the recognition by the nuclear protein kinase NIL
The Complete Amino‐Acid Sequence of the K88 Antigen, a Fimbrial Protein from Escherichia coli
Authors: KLEMM Per
Published: July 2010

The complete primary structure of the fimbrial protein of the K88 antigen has been elucidated. This protein, which makes up the building block for the macromolecular structure that comprises a fimbria, consists of 264 amino acid residues in a single polypeptide chain. The K88 antigen was fragmented by chemical cleavage with cyanogen bromide, and by subsequent enzymatic sub‐cleavage of resulting fragments with trypsin and chymotrypsin, and was additionally cleaved with o‐iodosobenzoic acid. Peptides were sequenced by manual Edman degradation.

The carboxy‐terminal part of the molecule is remarkable in being almost devoid of charged amino acid residues and is highly hydrophobic. Furthermore, this part of the structure could have a specific function as a molecular anchor.
Oxidation of meso‐Diaminosuccinic Acid, a Possible Natural Substrate for d‐Aspartate Oxidase
Authors: RINALDI Augusto , PELLEGRINI Mariagiuseppina , CRIFÒ Carlo , DE MARCO Carlo
Published: July 2010

meso‐Diaminosuccinic acid, a natural antagonist of aspartic acid, is a good substrate for beef kidney d‐aspartate oxidase. The oxygen consumption and the ammonia production are in good agreement with the stoichiometry of a typical oxidative deamination.The deamination involves only one of the two amino groups of diaminosuccinate, and is followed by decarboxylation of the first reaction product, 2‐amino‐3‐oxosuccinic acid. By condensation of the compounds thus originating from this latter, pyrazine 2,5‐dicarboxylic acid and pyrazine 2,6‐dicarboxylic acid are formed. dl‐Diaminosuccinic acid is not oxidized by d‐aspartate oxidase, nor does it inhibit the enzyme activity.
DNA Synthesis Catalyzed in vitro by Yeast Extracts
Authors: BADARACCO Gianfranco , GINELLI Enrico , SACCHI Nicoletta , SORA Silvio , PLEVANI Paolo
Published: July 2010

A procedure to prepare crude extracts from single colonies of Saccharomyces cerevisiae is described. Partially purified extracts catalyzed DNA synthesis directed by single‐stranded fd DNA. Maximum activity requires the presence of ribonucleoside triphosphates although extensive DNA synthesis is observed in the presence of only deoxynucleoside triphosphates. Alkaline sucrose gradient analysis demonstrated that initiation of new DNA chains occurs in vitro and the DNA products synthesized are heterogeneous in size. Isopycnic analysis of the products of ribonucleotide‐initiated fd DNA replication showed covalent linkage between the initiator RNA and the newly synthesized DNA. The fd‐replicating capacity of extracts prepared from cell‐division‐cycle mutants, defective in events controlling DNA initiation or elongation, showed an increased thermosensitivity in the DNA replicatioi: reaction in vitro.
Isolation and Characterization of Xenopus laevis Albumin mRNA
Authors: DIMITRIADIS Giorgos J.
Published: July 2010

The messenger RNA for albumin was isolated from the liver of male frogs. Purification was achieved using oligo(dT)‐cellulose chromatography and sucrose gradient centrifugation under denaturing conditions. As judged by translational activity in a cell‐free protein‐synthesizing system derived from rabbit reticulocytes, albumin mRNA was enriched 259‐fold as compared to the total ribonucleic acid of the liver cells. Purified albumin mRNA migrated after sucrose gradient centrifugation as a single symmetrical peak of approximately 17 S and also moved as a single band following denaturing agarose gel electrophoresis. Albumin mRNA possesses properties compatible with the presence of a poly(adenylic acid) sequence. Translation in vitro yielded a product which is immunoprecipitable with anti‐(frog albumin) and which showed a single radioactive peak having a molecular weight of about 74000 in sodium dodecylsulfate/polyacrylamide gel electrophoresis. Complementary DNA was synthesized using reverse transcriptase and, as a template, purified albumin mRNA. Following hybridization under conditions of excess RNA, the r0t1/2 of albumin mRNA was found to be 1.8 × 10−3 mol · s · 1−1. This result also confirmed that albumin mRNA had been isolated in a highly purified form.
Interaction of Long‐Chain Acyl‐CoA Analogs with Pig Kidney General Acyl‐CoA Dehydrogenase
Authors: THORPE Colin , CIARDELLI Thomas L. , STEWART Charles J. , WIELAND Theodor
Published: July 2010

The interaction of two long‐chain acyl‐CoA analogs with pig kidney general acyl‐CoA dehydrogenase (EC 1.3.99.3) was examined. The effect of S‐heptadecyl‐CoA and heptadecan‐2‐onyl‐dethio‐CoA on the flavoprotein was observed spectrophotometrically using the flavin as an active‐site probe. The S‐heptadecyl thioether analog bound strongly to the enzyme (Kd= 17 nM) and was a powerful competitive inhibitor (Ki < 40 nM). In contrast to the thioether analog, the dethiocarba derivative, heptadecan‐2‐onyl‐dethio‐CoA, was a substrate in the standard assay system being dehydrogenated at about 60% of the rate shown by palmitoyl‐CoA. These results support the proposal that α‐carbanion formation is an early event in the dehydrogenation of acyl‐CoA substrates.
Gabaculine and m‐Carboxyphenyl‐Pyridoxamine 5‐Phosphate as Probes of the Catalytic Binding Sites of 4‐Aminobutyrate Aminotransferase
Authors: KIM Doo Sik , MOSES Udoudo , CHURCHICH Jorge E.
Published: July 2010

A homogeneous 4‐aminobutyrate aminotransferase isolated from pig brain exhibits a kcat of 9.6 s−1 and contains one mole of pyridoxal 5‐phosphate/mole of dimer. The reaction of the enzyme with gabaculine (5‐amino‐1, 3‐cyclohexadiene carboxylic acid) was studied by observing changes in the absorption spectrum of the bound cofactor and by monitoring loss of catalytic activity. The enzyme is completely inactivated by gabaculine, but the dialyzed inactive sample containing 0.5 mol of gabaculine/mol dimer is fully reconstituted by addition of pyridoxal 5‐phosphate. Stopped‐flow kinetic studies reveal that gabaculine reacts with the cofactor bound to the aminotransferase with a second‐order rate constant of 2.5 × 103 M−1s−1. Fluorometric titrations of the apoprotein with m‐carboxyphenyl‐pyridoxamine 5‐phosphate show the binding of two moles of inhibitor/mole of enzyme. The binding process is reversible and the affinity of the apoprotein for the inhibitor is at least 10‐fold higher than the affinity for the cofactor. It is postulated that the dimeric enzyme contains two potential active sites per dimer, but the binding site characterized by a weaker affinity constant for pyridoxal 5‐phosphate becomes functional only after specific chemical modification of the molecule of cofactor tightly bound to the protein.
Self‐Stabilization of the Energy Charge in a Reconstituted Enzyme System Containing Phosphofructokinase
Authors: SCHELLENBERGER Wolfgang , ESCHRICH Klaus , HOFMANN Eberhard
Published: July 2010

The self‐stabilization of the energy charge and of ATP was investigated in an open reconstituted enzyme system containing phosphofructokinase, pyruvate kinase, adenylate kinase, and glucose‐6‐phosphate isomerase. The experiments were performed in a stirred flow‐through reactor containing gel‐entrapped enzymes.

The dynamics of the system were analyzed theoretically by a model based on the kinetic properties of the individual enzymes. The energy charge was identified as one of the essential variables of the system.

According to the theoretical prediction, homoeostasis of the energy charge was observed experimentally when either the maximal activity of phosphofructokinase, the energy charge of the influx solution or the flow rate through the reaction chamber was varied. It is shown that the efficiency of stabilization of the energy charge is related to the occurrence of alternative stationary states.
1H NMR Studies of Natural and Synthetic Immunostimulating Peptides in Aqueous Solution
Authors: DELBARRE Alain , MIGLIORE‐SAMOUR Danièle , WERNER Georges H. , ROQUES Bernard P. , JOLLÈS Pierre
Published: July 2010

The peptides lAla‐dGlu (l‐alanyl‐d‐glutamic acid), lAladGlu[llA2pm(Gly)]NH2, i.e. N2‐(l‐alanyl‐d‐γ‐isoglutaminyl)‐N6‐glycyl‐ll‐2, 6‐diaminopimelic acid, and lAla‐dGlu[llA2pm(Gly)‐dAla], i.e. N2‐(l‐alanyl‐d‐γ‐glutamyl)‐N6‐glycyl‐ll‐α‐2,6‐diaminopimelyl‐d‐alanine, extracted from a Streptomyces strain and their immunostimulating synthetic lauroyl derivatives were studied by 1H NMR spectroscopy at 270 MHz. The chemical shift analysis confirms (a) the sequence of these compounds, (b) the occurrence of a γ bond between Glu (or GluNH2) and A2pm, (c) the presence of a carboxamide group on the backbone of the tetrapeptide lAla‐dGlu[llA2pm (Gly)]. Temperature and pH studies strongly suggest that the tetrapeptide and pentapeptide exist under several folded conformations characterized by a proximity between the NH+3 group of Gly and the Glu (or GIuNH2) residue. The similarity between the chemical shifts of the corresponding protons in lAla‐dGlu, lauroyl‐lAla‐dGlu and muramyl‐lAla‐dGluNH2 shows that the coupling of the dipeptide with a lauroyl or a muramyl group does not induce significant changes in the structure of the peptide moiety. In the lipopeptide series the presence of the lauroyl chain leads to amphipathic substances which form micelles in aqueous solution as shown by the large increase of the proton linewidths. The immunostimulating properties of this kind of peptide manifest themselves at very low concentration and only after their coupling with a sugar or lipid moiety. These features can be interpreted by the formation of mixed complexes between the immunoadjuvants and cell membrane constituents.
Characterization of the Second Prosthetic Group in Methanol Dehydrogenase from Hyphomicrobium X
Authors: DUINE Johannis A. , FRANK Johannes , Jzn  , VERWIEL P. Eugène J.
Published: July 2010

Procedures are described for preparing 2,7,9‐tricarboxy‐1H‐pyrrolo[2,3‐ƒlquinoline‐4,5‐diol (pyrrolo‐quinoline quinol) from 2,7,9‐tricarboxy‐1H‐pyrrolo[2,3‐ƒ]quinoline‐4, 5‐dione (pyrrolo‐quinoline quinone).

When methanol dehydrogenase is denatured, two compounds are liberated which have the same properties as the quinone and quinol mentioned above. On analysing the extract by high‐performance liquid chromatography, one molecule of the quinone and one molecule of the quinol per enzyme molecule are found.

Mixtures of pyrrolo‐quinoline quinone and pyrrolo‐quinoline quinol at high pH produce the semiquinone form and, under certain conditions, a diamagnetic complex. Since electron spin resonance (ESR) shows that methanol dehydrogenase contains the semiquinone and the absorption spectrum suggests the presence of a diamagnetic dimer, it is tentatively concluded that the two prosthetic group molecules in the enzyme interact with each other.

NMR experiments of pyrrolo‐quinoline quinone in 2H2O demonstrate that it is partly hydrated, most prohably at the C‐5 position. Although methanol adds in the same way, it is still questionable whether the product of this addition plays a role in the mechanism of the enzymic reaction.

Potentiometric titrations show a midpoint potential of the quinone/quinol couple of + 90 mV at pH 7.0 and the information of the semiquinone as an intermediate in the titration at pH 13.0.
DNA‐Dependent RNA Polymerases of Thermoacidophilic Archaebacteria
Authors: PRANGISHVILLI David , ZILLIG Wolfram , GIERL Alfons , BIESERT Lothar , HOLZ Ingelore
Published: July 2010

The component compositions of the DNA‐dependent RNA polymerases of the extremely thermophilic. anaerobic sulfur‐respiring archaebacteria Thermoproteus tenax and Desu/furococcus mucosus strongly resemble each other but also that of the RNA polymerase of Suljolobus acidocaldarius suggesting that both organisms belong to the same novel order Thermoproteales, which together with the order represented by Sulfolobus, forms the thermoacidophilic branch of archaebacteria.

The component pattern of the RNA polymerase of T/iermoplasmu acidophilum, which does not belong to this branch, also appears homologuous. The archaebacterial type of the DNA‐dependent RNA polymerase is thus characterized by 9–10 components yielding a characteristic pattern which resembles that of yeast RNA polymerase A(1). In contrast to the α subunit of eubacterial RNA polymerases, the third largest component of archaebacterial RNA polymerases, although similar in size, is present only once per enzyme monomer.

The polymerases of T. tenax and D. mucosus, like those previously isolated from other archaebacteria, are completely resistant against 100 μ/ml rifampicin and streptolydigin.

The RNA polymerases of both organisms are highly thermostable. The enzyme from D. inucosus transcribes selectively and almost completely the H strand of phage T7 DNA.
Efficient Translation of the Coat Protein Cistron of Tobacco Mosaic Virus in a Cell‐Free System from Escherichia coli
Authors: GLOVER James F. , WILSON T. Michael A.
Published: July 2010

Translation of tobacco mosaic virus (TMV) RNA in a cell‐free system derived from Escherichia coli (MRE 600) reveals several discrete polypeptides in the Mr range of 10000–50000. The major product is a polypeptide of Mr 17500 which comigrates with authentic TMV coat protein on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Structural investigations by peptide‐mapping techniques and differential radiolabelling confirm that the major product is TMV coat protein with an N‐terminal methionine. The major polypeptide product can be assembled in vitro into virus‐like ribonucleoprotein particles.

The structural and evolutionary implications of this observation, and the values of TMV in elucidating eukaryotic mRNA interactions with the prokaryotic protein‐synthesizing machinery, are discussed.
‘Artificial’ Myosin Isozymes; Preparation and Characteristics
Authors: CARDINAUD Robert
Published: July 2010

Using precisely monitored proteolytic digestion conditions rabbit fast skeletal muscle myosin could be selectively modified in different ways. A myosin isozyme with a 20‐kDa alkali light chain 1 (Al) could be obtained by digesting with papain in the presence of Ca2+. Under these conditions alkali light chain 2 (A2) was cleaved at Lys‐17 and lost a 2.3‐kDa N‐terminal fragment including the strongly basic N terminus and about half of the characteristic (Ala‐Pro) sequence. The Nbs2‐[5,5′ dithiobis(2‐nitrobenzoic acid)‐]light chain and A2 were left unmodified and less than 5% of the myosin heavy chain presented a break in the subfragment‐2 region. EDTA and Ca2+ ATPase activities were unchanged.

A myosin isozyme with an 18‐kDa Nbs2‐light chain was obtained by limited digestion with trypsin in the presence of Ca2+. The 18.9→18‐kDa conversion was nearly 100% whereas less than 10% of the heavy chain was fragmented and only about 5% of A1 was converted to A1′. The Nbs2‐light chain was cleaved at Arg‐7 preserving Ser‐15 and consequently a phosphorylated modified myosin could be obtained. A quasi‐elastic light‐scattering study showed that this modified myosin in high‐ionic‐strength solutions exhibited physicochemical characteristics quite similar to those of unmodified myosin.
Biosynthesis of Vitamin B12 in Anaerobic Bacteria
Authors: LAMM Lydia , HECKMANN Gernot , RENZ Paul
Published: July 2010

[1 ‐13C]Glycine, [2‐13C]glycine, and [15N]glycine were added to fermentations with the strictly anaerobic vitamin B12 producer Euhacterium limosum. The vitamin B12 isolated was almost exclusively labeled in its 5,6‐dimethylbenzimidazole moiety. It was methylated and degraded to 1,5,6‐trimethylbenzimidazole. The position of the 13C label was determined from the 13C NMR spectrum, using the natural‐abundance 13C NMR spectrum of 1,5,6‐triinethylbenzimidazole as reference. Thus it was found that C‐1 of glycine is incorporated into position 7a, C‐2 of glycine into position 3a of the 5,6‐dimethylbenzimidazole moiety of vitamin B12.

In order to show whether the nitrogen atom of glycine is also incorporated, the vitamin B12 from the experiment with [15N]glycine was degraded to 5,6‐dimethylbenzimidazole. The H NMR spectrum of this compound exhibited in the region of the C‐2 proton a doublet from the 15N‐labeled part of the molecules (35 %) and a singlet from the unlabeled molecules. This doublet indicates that only one of the two nitrogen atoms of 5,b‐dimethyl‐ benzimidazole is labeled. From the incorporation pattern of 13C‐labeled glycine it must be deduced that this is N‐3 of the 5,6‐dimetliylbenzimidazole moiety of vitamin B12.

These experiments reveal that one molecule of glycine is incorporated into 5,6‐dimethylbenzmidazole moiety of vitamin B12 in a regiospecific manner.
A Mitochondria1 Protease that Cleaves the Precursor of Ornithine Carbamoyltransferase
Authors: MIURA Satoshi , MORI Masataka , AMAYA Yoshihiro , TATIBANA Masamiti
Published: July 2010

Ornithine carbamoyltransferase of rat liver mitochondrial matrix (subunit Mr= 36000) is synthesi/ed extramitochondrially as a larger precursor (subunit Mr= 39400) which is transported into mitochondria, in association with post‐translational proteolytic processing. Rat liver mitochondria convert the precursor to the mature enzyme as well as to a 37000‐Mr product. a possible intermediate of the processing [Mori, M., Miura, S., Tatibana. M., and Cohen, P. P. (1980) Proc. Natl Acad. Sci. USA, 77, 7044‐7048]. A protease responsible for the conversion of the precursor to the 37000‐Mr product was purified 140‐fold from the matrix fraction of rat liver mitochondria. The protease had an estimated Mr of 108000 and an apparent pl of 5.5. Mature ornithine carbamoyltransferase (0.5 μg) did not inhibit the cleavage of the precursor by the protease and presumably the latter cleaves a specific site on the extrapeptide of the carbamoyltransferase precursor. The protease was inhibited by metal‐chelating reagents such as EDTA, o‐phenanthroline and zincon and by a high concentration (1 mM) of leupeptin. It did not cleave several of the protein and peptide substrates tested including the precursor of mitochondrial carbamoyl‐phosphate synthetase I. Apparently the same protease activity is widely distributed among mitochondria of rat kidney, spleen, heart and ascites tumor cells, all of which lack ornithine carbamoyltransferase. A possible physiological role of the protease in the processing of the mitochondria1 protein precursors is discussed.
Asymmetric Transbilayer Distribution of Sterol across Plasma Membranes Determined by Fluorescence Quenching of Dehydroergosterol
Authors: HALE John E. , SCHROEDER Friedhelm
Published: July 2010

A new method for measurement of transbilayer distribution of sterol in plasma membranes is reported. The procedure utilized a fluorescent sterol, dehydroergosterol, and a chemical quenching agent, trinitrobenzenesulfonic acid. Dehydroergosterol was useful as a probe molecule for sterols for the following reasons. (a) Dehydro‐ ergosterol contained no bulky side chains as reporter groups. (b) Dehydroergosterol structurally resembled cholesterol and desmosterol, the primary sterol synthesized by LM fibroblasts. (c) Dehydroergosterol interacted with digitonin, filipin, and served as a substrate for cholesterol oxidase. (d) The phase transition of dipalmitoylglycero‐phosphocholine was completely abolished by dehydroergosterol. (e) The native sterol of LM fibroblasts, desmosterol, was completely replaced by dehydroergosterol without effect on LM cell growth, cell doubling time, plasma membrane (Na +,K +)‐ATPase and 5′‐nucleotidase activity, microsomal NADPH‐dependent cytochrome c reductase activity, and mitochondria1 succinate‐dependent cytochronie c reductase activity. (f) Neither the phospholipid composition nor the sterol/phospholipid ratio of LM fibroblasts were altered by supplementation with dehydroergosterol. The trinitrophenyl group of trinitrophenylglycine or of surface membranes of LM fibroblasts or red blood cells treated with trinitrobenzenesulfonic acid was an excellent quencher of dehydroergosterol fluorescence. Fluorescence in mouse very‐low‐density lipoproteins, LM fibroblast plasma membranes, red blood cell surface membranes, and in rat red blood cell membranes was quenched 95 ± 3%, 20 ± 2% 75 ± 4% and 69 ± 4% respectively when the quenching agent was present on only the extracellular side of the membrane. Trinitrophenyl residues effectively quenched the dehydroergosterol fluorescence in the plasma membrane of LM cells by 20% when dehydroergosterol was present from 1–85 mol/l00 mol of the mcrnbrane sterol. When both sides of the plasma membrane were trinitrophenylated, greater than 95% of the dehydroergosterol fluorescence was quenched. In addition, when LM cells were cultured with dehydroergosterol, exposed to latex beads, and the endocytosed particles isolated as phagosomes and treated with trinitrobenzenesulfonic acid under non‐penetrating conditions, the fluorescence of the dehydroergosterol was quenched nearly 64%. From these and other results we deduced that the inner monolayer of the LM fibroblasts plasma membrane was enriched with dehydroergosterol. In contrast, the distribution of the sterol in red blood cell membranes indicated an enrichment in the outer monolayer
Small‐Angle X‐Ray Scattering Study of the Interaction between Human α2‐Macroglobulin and Trypsin
Authors: BRANEGÅRD Bo , ÖSTERBERG Ragnar , SJÖBERG Bo
Published: July 2010

The interaction between human α2‐macroglobulin and trypsin yields a complex of two trypsin molecules and one α2‐macroglobulin molecule as the main product. This is indicated from small‐angle X‐ray titration measure‐ ments in 0.02 M Tris/HCl buffer of pH 7.40 containing 0.2 M NaCl and 5 mM EDTA. The radius of gyration for the complex was determined to be 7.1 nm. The complex formation coincides with a decrease in the first side maximum of the scattering curve for α2‐macroglobulin. A comparison of the experimental data with those calculated from various models indicates that the data are consistent with the scattering from a hollow cylinder containing two sites, each of which is partly filled with one trypsin molecule.
Complete Amino Acid Sequence of Ostrich (Struthio camelus) Egg‐White Lysozyme, a Goose‐Type Lysozyme
Authors: SCHOENTGEN Francoise , JOLLÉS Jacqueline , JOLLÉS Pierre
Published: July 2010

The complete amino acid sequence (185 amino acid residues) of the goose‐type lysozyme from the egg white of ostrich (Struthio camelus) has been determined from the study of tryptic and Staphylococcus aureus V8 protease peptides as well as of CNBr and BNPS‐skatole fragments. A slight sequence homology between the goose‐type and chicken‐type lysozymes has been observed.
Accumulation of Decarboxylated S‐Adenosyl‐l‐Methionine in Mammalian Cells as a Consequence of the Inhibition of Putrescine Biosynthesis
Authors: MAMONT Pierre S. , DANZIN Charles , WAGNER Joseph , SIAT Mirlyse , JODER‐OHLENBUSCH Anne‐Marie , CLAVERIE Nicole
Published: July 2010

Biological transmethylation reactions and polyamine biosynthesis share the substrate S‐adenosyl‐l‐methionine.Under normal conditions, decarboxylated S‐adenosyl‐l‐methionine, the aminopropyl donor for polyamine biosynthesis, does not accumulate because of its rapid utilization in spermidine and spermine synthesis. Alteration of polyamine synthesis by DL‐αdifluoromethylornithine, an enzyme‐activated irreversible inhibitor of l‐ornithine decarboxylase, leads to a striking accumulation of decarboxylated S‐adenosyl‐l‐methionine in rat hepatoma cclls cultured in vitro and in rat ventral prostate. This increase is due both to lack of putrescine and spermidine for the aminopropyltransferase reactions and to the elevation of S‐adenosyl‐l‐methionine decarboxylase activity. The biological implications of accumulation of decarboxylated S‐adenosyl‐l‐methionine are discussed with regard to the regulation of S‐adenosyl‐l‐methionine decarboxylase activity and to the antiproliferative effects of dl‐α‐difluoromethylornithine.
Direct Control of Glycogen Metabolism in the Perfused Rat Liver by the Sympathetic Innervation
Authors: HARTMANN Heinz , BECKH Karlheinz , JUNGERMANN Kurt
Published: July 2010

The mode of action of hepatic nerves on the metabolism of carbohydrates was studied in the rat liver perfusedin sifu.

Electrical stimulation of the nerve bundles around the hepatic artery and the portal vein resulted in an increase of glucose and lactate output, an enhancement of phosphorylase a activity and a decrease of portal flow.

Sodium nitroprusside prevented the hemodynamic changes after nerve stimulation without affecting the metabolic alterations.

Phentolamine or an extracellular calcium level below 300 μmol × 1−1 abolished both hernodynamic and metabolic changes after nerve stimulation, while propranolol or atropine were without effect.

Norepinephrine infusion mimicked nerve stimulation only at the highly unphysiological concentration of 0.1 μM; it was not effective at a concentration of 0.01 μM, which might be reached in the sinusoidal blood due to an overflow from intrahepatic synapses.

The present results suggest that, in rat liver, glycogen breakdown is regulated by α‐sympathetic nerves directly rather than indirectly via hemodynamic changes or via norepinephrine overflow.
Non‐ionic Adsorptive Immobilization of Proteins to Palmityl‐Substituted Sepharose 4B
Authors: NEMAT‐GORGANI Mohsen , KARIMIAN Khashayar
Published: July 2010

Palmityl‐substituted Sepharose 4B prepared by the glycidyl either method of S. Hjertén et al. [J. Chromatogr. (1974) 101, 281–288] has been used as a non‐ionic matrix for protein adsorption. A number of proteins, some of which are catalytically active, may be immobilized on this adsorbent in the form of suspension or column.

Of the proteins examined, bovine serum albumin, hemoglobin, myoglobin, lysozyme, glutamate dehydrogenase, and β‐galactosidase were immobilized on the adsorbent irrespective of NaCl concentration. Trypsin, α‐chymotrypsin, papain, pepsin, and amyloglucosidase were totally adsorbed either in the absence of any additional salt or at high salt concentrations. Cytochrome c, used as a model protein, was totally immobilized only ar high ionic strength and low pH.

Immobilization normaly took place with an apparent in initial acitivity rates. In the case of trypsin using Nα‐benzoyl‐cl‐arginine p‐nitroanilide as substrate, adsorption resulted in anincrease in V (app).

Beef‐liver glutamate dehydrogenase, used as a model allosteric enzyme, was found to retain its allosteric properties towards ADP and GTP after immobilization.

Results are discussed in terms of specific interactions involving a smaller number of binding sites in protein molecules as compared to the multiple attachment to highly substituted adsorbents prepared with shorted ligands. Retention of the essential properties of the proteins examined in this study are ascribed to these characteristics of the adsorbent and to its non‐ionic nature. Relevance of these observations to in vivo processes and the potential use of the adsorbent for enzyme immobilization are also discussed.
Regulation of Glutamine Synthetase from Saccharomyces cerevisiae by Repression, Inactivation and Proteolysis
Authors: LEGRAIN Christiane , VISSERS Stéphan , DUBOIS Evelyne , LEGRAIN Michèle , WIAME Jean‐Marie
Published: July 2010

Glutamine synthetase activity is modulated by nitrogen repression and by two distinct inactivation processes. Addition of glutamine to exponentially grown yeast leads to enzyme inactivation. 50% of glutamine synthetase activity is lost after 30 min (a quarter of the generation time). Removing glutamine from the growth medium results in a rapid recovery of enzyme activity. A regulatory mutation (gdhCR mutation) suppresses this inactivation by glutamine in addition to its derepressing effect on enzymes involved in nitrogen catabolism. The gdhCRmutation also increases the level of proteinase B in exponentially grown yeast. Inactivation of glutamine synthetase is also observed during nitrogen starvation. This inactivation is irreversible and consistes very probably of a proteolytic degradation. Indeed, strains bearing proteinase A, B and C mutations are no longer inactivated under nitrogen starvation.
Dichlorobenzimidazole‐riboside Inhibition of Nuclear RNA Accumulation Initiated with γ‐Thio Analogues of ATP and GTP
Authors: WINICOV Ilga , BUTTON Jane D.
Published: July 2010

The γ‐thio analogues of ATP and GTP, ATP[S] and GTP[S], have been used as affinity probes to measure RNA synthesis initiated in vitro in L cell nuclei. 5,6‐Dichlororibofuranosylbenzimidazole was found to inhibit total RNA synthesis in vitro and the amount bound by mercury‐affinity chromatography. Initiation in vitro was also shown by [35S]ATP[S] and [35S]GTP[S] incorporation in RNA molecules with a large size distribution. Although 66% of RNA molecules labeled with [35S]GTP[S] and 74% of those labeled with [35S]ATP[S] were 3–12 S in size, the remainder of the label was recovered in RNA molecules larger than 12 S and a significant portion in RNA larger than 18 S. The specificity of the initiation process seems to be indicated by the finding of molecules of the size of pre‐tRNA on gel electrophoresis which were labeled with [35S]GTP[S] but not [35S]ATP [S] under our experimental conditions. 5,6‐Dichlororibofuranosylbenzimidazole severely inhibited the incorporation of [35S]GTP [S] and [35S]ATP [S] by all size classes, indicating that it can decrease accumulation of RNA initiated in vitro in the L cell nuclear system.
The Interaction of the A and A* Proteins of Bacteriophage φX174 with Single‐Stranded and Double‐Stranded φX DNA in vitro
Authors: VAN DER ENDE Arie , LANGEVELD Simon A , VAN ARKEL Gerard A. , WEISBEEK Peter J
Published: July 2010

The binding of the bacteriophage φX174‐coded A and A* proteins to single‐stranded (ssDNA) and double‐stranded (dsDNA) φX DNA was studied by electron microscopy. The interaction of the A* protein with ssDNA and dsDNA was also studied by sedimentation velocity centrifugation.

It was shown that the binding of the A and A* proteins to ssDNA occurs in a non‐cooperative manner and requires no or very little sequence specificity under the conditions used here. Both protein‐ssDNA complexes have the same compact structure caused by intrastrand cross‐linking through the interaction of protein molecules with separate parts of the ssDNA molecule.

The A protein does not bind to φX dsDNA in the absence of divalent cations. The A* protein does bind to dsDNA, although it has a strong preference for binding to ssDNA. The structure of the A* protein‐dsDNA complexes is different from that of the A* protein‐ssDNA complexes, as the former have a rosetre‐like structure caused by protein‐protein interactions. High ionic strengths favour the formation of large condenwi aggregates.
Primary Structure of the CFAl Fimbrial Protein from Human Enterotoxigenic Escherichia coli Strains
Authors: KLEMM Per
Published: July 2010

The complete amino acid sequence of the structural protein that constitutes the subunit of the CFAI fimbria has been elucidated. The protein was fragmented by cyanogen bromide cleavage, and by enzymatic cleavage with trypsin. Secondary cleavage of the resulting peptides was performed with chymotrypsin, Staphylococcus aureus protease, and thermolysin. Sequential Edman degradation was performed manually. The CFAI protein comprises 147 amino acid residues, with a molecular weight of 15058.
Human‐Liver Alanine Aminopeptidase
Authors: STARNES Willis L. , SZECHIŃSKJ Jacek , BĚHAL Francic J.
Published: July 2010

Human liver alanine aminopeptidase (EC 3.4.11.14; l‐α‐aininoacyl‐peptide hydrolasc) catalyzes the stepwisc hydrolysis of mcthionyl‐lysyl‐bradykinin to yield methionine, lysine, and the limit nonapeptide, bradykinin. which is resistant to further hydrolytic cleavage by this enzyme. Alanine aminopeptidase also catalyzes the hydrolysis of various neutral amino acid β‐naphthylamide This enzyme cleaves N‐terminal arginyl residues unless the adjacent penultimate residue is proline as is the case for bradykinin. The properties are consistent with requirements of a kinin converting enzyme. Human alanine aminopeptidase activity is reduced by several β‐lactani antibiotics, with the cloxacillin, oxacillin. and inethicillin Ki values being 0.51 mM, I.6 mM. and 2.4mM respectively. Our experiments with radioactively labelled penicillin indicate that two moles of antibiotic are bound per mole of enzyme. Neither chromatography of the penicillin‐treatcd enzymc on G‐25 Sephadex, treatment penicillin‐Ci‐treated enzyme with penicillinase, nor extensive dilution of cloxacillin‐treated enzyme diminished the degree of inactivation produced. Inhibition was obtained with 6‐aminopenicillanic acid, which indicated that penicillin nucleus itself was being bound, but substitutions, as in cloxacillin, could enhance the binding.
Uniformity of Glycans within Molecular Variants of αl‐Protease Inhibitor with Distinct Affinity for Concanavalin A
Authors: BAYARD Bernard , KERCKAERT Jean‐Pierre , LAINE Anne , HAYEM Annette
Published: July 2010

Human a,‐protease inhibitor contains four asparagine‐linked carbohydrate chains per molecule. Three types of carbohydrate chains were released from the polypeptide backbone by hydrazinolysis:(a) biantennary (80%),(b) biantennary with an intercalated N‐acetylglucosamine residue (14%), and (c) triantennary (6%). Using concanavalin‐A‐affinity chromatography, native and S‐carboxymethylated α1,‐protease inhibitor were fractionated into three distinctmolecular variants which were shown to contain only one type (a,b or c, respectively) of glycan per molecule. This and previous observations on other serum glycoproteins support the proposal of uniformity of glycan type within individual molecular variants of glycoproteins.
Formyltetrahydrofolate Synthase from Clostridum thermoaceticum
Authors: MAYER Frank , ELLIOTT James I. , SHEROD David , LJUNGDAHL Lars G.
Published: July 2010

Formyltetrahydrofolate synthetase (EC 6.3.4.3) from Clostridium thermoaceticum is a 240000‐molecular‐ weight, tetrameric enzyme composed of identical subunits. When negatively stained with uranyl acetate or sodium phosphotungstate the enzyme appears as two distinct projections when viewed in the electron micro‐scope. One of these, resembling the letter ‘H’, exhibits four flexible arms, while the other. called a ‘twin’. has a compact projection. The twin projection appears to represent the native enzyme in solution. It consists of two outstanding intensity maxima separated by a narrow bridge of dense stain, with each of these intensity maxima being divided into the two submaxima by a second bridge of stain running perpendicular to the first one. The outstanding intensity maxima represent dimers, whereas the submaxima represent a single subunit. The volume calculated from the electron microscopy projections for the dimer is 157 nm3, which corresponds to a Mr of 127000 or 254000 for the tetrameric enzyme. The H projections reflect enzyme particles artificially flattened and ‘opened’ during the drying of the negatively stained enzyme. The four arms of the H each represent a subunit. The appearance of H projections is prevented by either using a deep‐stain procedure, which offers a mechanical preservation of the native enzyme structure, or by the binding of ATP, ADP or adenosine 5′‐[β,γ‐methylene]triphosphate. These nucleotides, when binding to the enzyme, cause a conformational change. This was observed by ultraviolet and visible circular dichroism and absorption spectrometry with the native enzyme and an enzyme labeled with fluorescein mercuric acetate attached to a sulfhydryl group of each subunit.
Plasminogen Activator Released as Inactive Proenzyme from Murine Cells Transformed by Sarcoma Virus
Authors: SKRIVER Lars , NIELSEN Lars S. , STEPHENS Ross , DANØ Keld
Published: July 2010

We have previously reported the purification of a plasminogen‐activating serine pro1 ease with an approximate Mr of 48000 from sarcoma‐virus‐transformed murine cells. We now report that under serum‐free conditions the enzyme is released from the cells in an inactive form. After affinity chromatography with 4‐aminobenzamidine‐cellulose, ion‐exchange chromatography and gel filtration, the proenzyme could be obtained from culture fluid as a pure, homogeneous protein as evaluated by polyacrylamide gel electrophoresis with sodium dodecylsulphate. Proenzyme was quantitatively converted to active enzyme by incubation with catalytic amounts of plasmin. Analysis by polyacrylainide gel electrophoresis with sodium dodecylsulphate under reducing and non‐reducing conditions showed that the inactive form consisted of a single polypeptide chain with an Mr of approximately 48000, while the active form consisted of two chains with Mr values of approximately 18000 and 29000, held together by one or more disulphide bridges. The active‐site reagent diisopropylfluorophosphate in radiolabelled form was incorporated into the 29000‐Mr chain of thc active enzyme, but not into the inactive form. These findings provide conclusive evidence for the existence of an inactive proenzyme to this. plasminogen activator and thus demonstrate an additional step in a cascade‐like reaction leading to extrac ellular proteolysis. Regulatory as well as methodological implications of this finding are discussed.
Base‐Excision Repair in Carrot Cells
Authors: TALPAERT‐BORLÉ Myriam , LIUZZI Michel
Published: July 2010

Uracil‐DNA glycosylase and apurinic/apyrimidinic (AP) endodeoxyribonuclease hacc been purified from cultured carrot cells.The two enzymes, separated by affinity chromatography on Sepharosc‐poly(rU), were found to have properties similar to those of the homologous bacterial and mammalian enzymes. the action of AP endo‐ deoxyribonuclease on (dA)230· (dT, dU)230 partially depyrimidinated by uracil‐DNA glycosylase suggests that these two enzymes might act successively to initiate the repair of uracil‐containing DNA.
The Amino‐Acid Sequence of Histone H2A from Cuttlefish Sepia officinalis
Authors: WOUTERS‐TYROU Daniéle , MARTIN‐PONTHIEU Annie , BRIAND Gilbert , SAUTIERE Pierre , BISERTE Gérard
Published: July 2010

The amino acid sequence of cuttlefish testis histone H2A (124 residues) was establijhed from structural data obtained by automated sequencing of large peptides generated by the cleavage of the protein with V8 staphylococcal protease or by limited chymotryptic hydrolysis.

Compared to the calf thymus homologous histone, cuttlefish H2A shows 14 substirutions (most of them conservative) and 5 deletions.

Extensive evolutionary changes were mainly observed in the basic amino‐terminal and carboxy‐terminal regions of the molecule, which are the primary DNA‐binding sites. Few punctual changes are observed in the central region (residues 18‐ 118), which interacts strongly with histone H2B to form the dimer H2A‐H2B.