bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemical Modifications of tyrosyl Residue(s) and Action of Human‐Fibroblast Interferon
Authors: BERTHOLD Wolfgang , TAN Celine , TAN Y. H.
Published: March 2005

Partially purified and purified human fibroblastoid interferon were iodinated and nitrared under experimental conditions in which the most likely amino acyl residue modified was tyrosine(s). The modification resulted in a loss of human interferon activity suggesting that tyrosyl residue(s) is associated with the antiviral action of human interferon.
Polyamine‐Induced Actin Polymerization
Authors: ORIOL‐AUDIT Christine
Published: March 2005

Muscle actin has been found to polymerize reversibly upon addition of low concentrations of polyamines. This polymerization, studied by centrifugation, has shown a linear relationship between the actin polymerization yield and the chain lingth of the polyamine. Aming the biological polyamines tested, spermidine and spermine are the most efficient.

The polymerization of actin can also be induced by the corresponding mono of diguanidine derivatives of these polyamines but monoamines of amino acids are inactive at the same concencentration.

The transformation of actin from a globular form upon addition of spermidine is also demonstrated by the changes in the near‐ultraviolet circular dichroic spectrum of this protein.

Moreover, the polyamine‐induced F‐actin exhibits the same properties as the salt‐induced F‐actin: it strongly activates the Mg2+‐ATPase of myosin, its specific viscosity is enhanced to the same extent and electron micrographs show homogeneous thin filaments.
Subcellular Metabolite Concentrations
Authors: SOBOLL Sibylle , SHOLZ Roland , HELDT Hans W.
Published: March 2005

Mitochondrial and cytosolic contents of adenine nucleotides and phosphate were measured in perfused rat livers empolying a technique of fractionation of freeze‐fixated tissue in non‐aqueous solvents. From the subcellular contents the mitochondrial and cytosolic concentrations of ATP. ADP, AMP and phosphate and the phosphorylation potentials of the subcellular ATP systems were calculated. An attempt was made to elucidate the relationship between mitochnodrial and cytosolic adenine nucleoide systmes and the dependency on the metabolic state of the liver.

The following results were obtaines.

Under all mtabolic conditions studied the mitochnodrial ATP/ADP ratios were considerably lower than the cytosolic ratios (mitochondria: 0.1–0.7); cytosol: 2–11).

The ATP/ADP ratios calculated from overall tissue contents refflect mainly the cytosolic rations.

An inverse relationship was found between mitochondrial and cytosilic ATP/ADP ratios, i.e. when the mitchondrial ratios tended to increase, the cytosolic ratios decrease and vice versa.

The phosphorylation potentials calculated from the subcellular concertratopms were higher in the cytosol that in the mitochondria. The potential difference varied between 11 and 3 kJ/mol in livers from fed and starved rats, respectively.

In the presence of mitochondrial inhibitors, i.e. amytal, dinitrophenol and carboxyatractyloside the potential difference between the subcellular ATP systems decreased predominantly due to an increase in the mitocnodrial ATP/ADP ratios.

A correlation between mitochondrial ATP/ADP ratios and the respiratory rates was not obsrved, but the subcellular ratios appeared to correlate with the rate of glycolysi. When the rate of lactate + pyruvate production was increased, the cytosolic ATP/ ADP ratios were increased, too, wheres the mitochondrial ratios tended to decrease.

The adenine nucleotides in the cytosol appear to be in near equilibrium catalysed by the adenylate kinase. In the mitochondria, the AMP concentration is much lowe than to be expected under equilibrium conditions.

These results were discussed with respect to rate control of processes involved in ATP generation, i.e. Oxidative phosphorylation, adenine nucleotide translocation and glycolysis.
Effect of High Pressure, Detergents and Phaospholipase on the Break in the Arrhenius Plot of Azotobacter Nitrogenase
Authors: CEUTERICK Francis , PEETERS Jozef , HEREMANS Karel , DE SMEDT Humbert , OLBRECHTS Henri
Published: March 2005

It is shown that lipids are responsible for the breaks in the Arrhenius plots of Azotobacter nitrogenase. The physical evidence is that temperature at which the break occurs increasaes with increasing pressure by 20 K/1000 atm. This is in agreement with the pressure dependence of the transition temperature of several synthetic phospholipids. We also find the same pressure dependence for the broad transitions observed in Escherichia coli phosphatidylethanolamine and in the membrane lipids from Azotobacter itself. Detergents and phospholipase remove the break. Reconstruction can be performed only with specific phospholipids.
Purification and Properties of 5′‐Nucleotidase from Lymphocyte Plasma Membranes
Authors: DORNAND Jacques , BONNAFOUS Jean‐Claude , MANI Jean‐Claude
Published: March 2005

5′‐Nucleotidase is purified from lymphocyte plasma membranes by teo affinity chromatog‐raphies. The first one, on Lens culinaris lectin‐Sepharose 4B yields a fraction of twelve lectin‐binding alyvoproteins (lectin‐receptor fractin). The second one on 5′‐AMP‐Sepharose 4B leads to pure enzyme. This enzyme is a glycoprotein with a molecular weigtht of 30000; it gives a single band in polyacrylamide/dodecylsulfte electrophoresis and displays a very high specific activity (2500–3000 μmol Pi h−1 mg−1). Some propertes of purified 5′‐nucleotidase are similar to those of membrance‐bound enzyme: substrate speciticity, temperature dependence, effects of ions and SH‐blocking reagents. Others are completely differnt for the teo systems and these differences result form an interaction between the enzyme molecule and other Lens culinarid lectin binding proteins.
Dissociation and Oxygen‐Binding Behaviour of β‐Hemocyanin from Helox pomatia
Authors: ZOLOLA Lello , KUIPER Harry A. , VECCHINI Paola , ANTONINI Eraldo , BRUNORI Maurizio
Published: March 2005

The dissociation behaviour of Helix pomatiaβ‐ hemocyanin as influenced by pH, calcium‐iopn concentraion and ionic strength, and the oxygen vinging properiotes were investigated. The dissociation behaviour of β‐hemocyuanin and α‐hemocyanin differs distinctly in a number of properties: (a) they show a different response to high salt concentration, (b) the stabilizing influence of calcium ions on the whole molecules of β‐hemocyanin is smaller than that on α‐hemocyanin, (c) half molecules of β‐hemocyanina are stablized to a large extent in teh presence of calcium ions and (d) at low ionic strength no peak separation between one‐tenth and one‐twentieth molecules of β‐hemocyanin is observed in the ultracentrifuge, which indicates an equilibrium between the species.

The oxygen binding behaviour of β‐hemocyanin shows the typical features of proteins with a large hnumber of binding sites. The Hill plot is characterized by a high value of the Hill coefficient (maxm nH=7), coupled to a low value for the interatrion free energy per site. The oxygen binding behaviour can bacisally be described by a mocdified tewo‐state allosteric model with the variation, imposed by teh expeimental data, taht KT is pH‐dependent (negative Bohr effect), while KR is pH‐indepenmdent. Regulation of the cooperativity by teh alloosteric effector H+ is mainly due to changes in the allosteric equilibrium constant.

In order to describe the cooperativity as a function of pH, the minimum number of interacting binding sites has to be varied, indicating that the size of the ‘functional constellation’ is variavle. This finding may have important implications on the relationship between the so‐called ‘structural’ and ‘functional constellation’.

A comparison fo the oxygen binding properties of β‐and α‐hemocyanin shows that these proteins are complementary in their behaviour, with respect to the responses to changes in H+ anmd Ca2+ concentrations. This may be of importance for the animal in order ito meet specific physiological and enviromental requirements.
Evidence for Catabolite Degradation in the Glucose‐Dependent Inactivation of Yeast Cytoplamic Malate Dehydrogenase
Authors: NEEFF Jürgen , HÄGELE Edger , NAUHAUS Jutta , HEER Ursual , MECKE Dieter
Published: March 2005

The cytoplamic malate dehydrogenase of Sacchoromyces cerevisiae was radioactively labeled during its synthesis on a glucose‐free derepression medium. After purification a sensitive radio‐immunoassay for this enzyme could be developed. The assay showed that after the physiological, glucose‐dependent ‘catabolite inactivation’ of cytoplasmic malate dehydorgenase an inactive enzyme protein is immunologically not detchtable. Together with the irreversiblity of this reaction in vivo this finding strongly suggests a proteolytic mechanism of enzyme inactivation. For this process the term ‘catabolite degradation’ is used.
Chemical Structure and Biochemical Activity of Cord Factor Analogs. A Comparative Study of Esters of Methyl Glcoside and Non‐Hydroxylated Fatty Acids
Authors: KATO Masahiko , TAMURA Toshihide , SILVE GAby , ASSELINEAU Jean
Published: March 2005

6‐Monoesters of methyl α‐D‐glucopyranoside and the even‐numbered normal chain fatty acids C14toC22 or 2‐tetradecyloctadecanoic acid (DL‐deoxy‐corynomycolic acid) have been prepared. All the normal chain fatty acid esters were devoid of toxicity for mice at the dose of 2 mg per animal, whereas the branched chain ester (deoxy‐corynomycolate) exhibited a low, bnut characteristic, toxicity.

Preincubation of rat liver mitochondria with aqueous suspensions of these synthetic esters gave the following results. The shorter chain (C14 to C20) esters affected the mitochondrial respiration and coupled phosphorylation in both succinate and NADH pathways at both coupling sites II and III. The same effects were obtained by using methyl 2‐hexad3ecanoyl‐α‐D‐glucopyranoside or 6‐hexadecanoyl‐l‐asorbic acid, showing that the position of the ester linkage on the sugar residue or the conformation of the sugar moiety are not essential. Deoxycorynomyucolate induced an impairmant of mitochondrial oxidative phosphoryl;ation mainly localized at couplilng site II. Methyl 6‐do‐cosanoyl and 2,6‐sisoxoanoyl α‐D‐glucopyranosides affected only coupling site II. Inbtroductionof a double bnonmd in the fattyacid chain (docos‐13‐cis‐enote) gave results similar to those obtained with shorter chain esters.

Two classes of glucoside esrters can thus be distinguished: those with a shosrtchain fattyacid part (C14 to C20), devopid of toxicitgy and affectomg pxodfatove phosphorylzation at both coupling II and III;ester of the C32‐branched chain acid exhibniting toxicity in vivo and specific inhibnition of oxidative phosphorylation at coupling site IIin vitro. Mono and diesters of docosanoic acid have an intermediate position (no toxicity, but a specific effect on mitochondria)

The absence of toxicity of the short cahin ester (C14 to C22)seems to be expplained by their neutralization bty serum albumin.
Regulation of Mammary Gland Metabolism
Authors: GREENBAUM A. Leslie , SALAM Aziza , SOCHOR Milena , MCLEAN Patricia
Published: March 2005

A method for the opreparation of isolated mammary gland cells of fthe rat is described.

The procedure involves disaggregation of the tissue in a collagenase‐hyaluronidase mixture and subsequent purification of the heterogeneous population of cells bycentrifugation in discontinuous Ficoll‐400 gradients; the preparation takes 60 minutes. The yield of cells is approximagtely 14%.

The cell as prepared have high rates of metabolism and synthetic capacity and exhibitmetabolic characteristics comparable to intact tissue.

Measurements of the co9ntent of metabnolic intermediates show cells to have, and retain, outstandingly hjigh levels of ATP and to have an energycharge close tgo 0.9. Levels of other intermediatesapproximate to those found in the int6acttissu, The level of glycolytic intermedites kbelow the triose phosphate stage indicatethe highly aerobic state of the cells.

The patytern and scale of glucose utilization, measusred using specifically labelled glucose incorporation into 14CO2 and 14C‐labelled lipid production, approximates closelyhin isolated cells at 5 and 20mM glucose and in tissue slices at 20mM glucose concentration. Mammary gland slices incubated with 5 mM glucose have a considerablylower rate of metabolism. Isolated cells exhibnit a higher proportionate rate of glucose utilization by way of the pentose phosphate pathway.

The isolated cells are hormone responsive. Insulin increases the oxication of glucose by the pentose phosphate pathway and stimulates lipid synthesis. Addition ofprogesterone and cortisonein vitro (10μM) leads to a marked and rapid decreasein the rate of glucose oxisdation and conversion to lipid.
Purification of Aspartate Transcabamylase from Drosophila melanogaster
Authors: JARRY Bruno P.
Published: March 2005

A purification procedure is described by which aspartate transcarbamylase was obtained from cultured cells of DDrosophila melanogaster as part of a high‐molecular‐weight enzyme complex. The complex is shown to contain several polypeptides.

An antiserum directed against the complex enzyme nihibited in vitro the activity of aspartate transcarbamylase, carbamylphosphate synthetase and dihyro‐orotase which were shown to copurify on a sucrose gradient and by gel electrophoresis. A fast preparation procedure using this antiserum yielded a 220000‐molecular‐weight protein in addition to the polypeptides present in the complex.

A purification procedure is also described to obtain aspartate transcarbamylase from second instar larvae of Drosophila. At this stage, the enzyme is not complexed with carbamylphosphate synthetase and dihydro‐orotase but exhibits the same molecular weight as the aspartate transcarbamylase moiety found in the high‐molecular‐weight complex of cultured cells.
Sequence of 129 Nucleotides at the 3′‐Terminus of Encephalomyocarditis Virus RNA
Authors: PORTER Alan G. , MERREGAERT Josef , VAN EMMELO John , FIERS Walter
Published: March 2005

The sequence of 129 nucleotides next to the poly(A)tail of encephalomyocarditis virus RNA has been determined by rapid gel sequencing of cDNA synthesized with DNA polymerase I or reverse transcriptase and a rapid transcriptase and a phaisng primer, [5′‐32P]p(dt)8dC. the sequence is in accored with (a) the pyrimideine tracts shich were mapped in blocks along the cDNA, (b) the sequences of seven characteristic T1 RNase oligonucleotides in the RNA transcribed form the cDNA with RNA polymersase, and (c) a limited amount of sequences deduced by parital spleen phosphodiesterase digestion and depurination of endouclease IV oligonucletodes.

The 3′ end shows little secondary strucutre on it own. Ten nonsense condons block all three reading frames such that at least 26 nucleotides do not code for protein. The possible function of a homology A‐A‐U‐A‐A‐A with other poilyadenylated RNAs is discussed.
Reassociation and Reactivation of Yeast Glyceraldehyde‐3‐Phosphate Dehydrogenase after Dissociation in the Presence of ATP
Authors: BARTHOLMES Peter , JAENICKE Rainer
Published: March 2005

Tetrameric d‐glyceraldehyde‐3‐phophate dehydrogenase from yeast in the presence of 5 mM ATP and 0.1 M 2‐mercaptoethanol at pH 8.5 can be reversibly inactivated and dissocited by changing the temperature from 25 to 0°C. Using ultracentrifugation and gel chromatography, the inactive low‐temperature dissociation product is found to be a compact monomer (Mr= 36000, s20,w= 3.2S) which is still capable of binding on molecule of NAD+ per subunit.

Deactivation caused by dissociation at 0°C is a slow process with a first‐order rate constant of k= 0.007 min −1 (τ1/2= 180 min); it follows the instantaneous competitive inhibition of the ezyme by ATP which is characterized by Ki= 2.3 mM.

Reactivation at high enzyme concentration (c > 10 μg/ml) obeys first‐order kineics. At low enzyme concentration a shift to a higher reaction order is obseved. Alimiting value of n= 2 is reached at 0.9 μg/ml. The data are in accordance with a uni‐bi molecular mechanism involving the association of (inactive) monomers as a prerequisite of enzymatic of enzymatic activity. The kinetic constants. k1= 9 ± 2 × 10−1 and k2= 7 ± 1 × 104 M−1× s−1, correspond to previous data reported for the reactivation after full denaturation and dissociation of the enzyme in 6 M guanidine · HCI at pH 2.3 [Rudolph et al. Eur. J. Biochem. 81, 563–570 (1977)]. This suggests a common intermediate in the reactivation process after deactivation and disscoiation under widely differing conditions.
Introduction of Hidden Breaks during rRNA Maturation and Ageing in Tetrahymena pyriformis
Authors: ECKERT Werner A. , KAFFENBERGER Walter , KROHNE Georg , FRANKE Werner W.
Published: March 2005

The stability of Tetrahymena pyriformis cytoplasmic rRNAs and nuclear rRNA precursors has been studied by polyacrylamide gel electrophoresis under partly and completely denaturing conditions. Cytoplasmic 17‐S rRNA (Mr= 0.66 × 106) consists of a continuous polynucleotide chain throughout its lifetime, whereas the bulk of 26‐S rRNA (Mr= 1.27 × 106) dissociates upon denaturation. Two large fragments (F1, F2) of somewhat differnt molecular weights (Mr 0.63 × 106 and 0.58 × 106) and the small 5.8‐S rRNA fragment (Mr about 50 000) are regularly observed. Some additional distinct minor fragments (F3–F6) are noted under certain preparastive condiontions, suggestive of artifactual origin. The following conclusions were made from the data obtained (a) Newly synthesized 26‐S rRNA molecules do not contain the ‘central’ hidden break (separating F1 and F2) until about 15 min after their appearance in the cytoplasm; however, they release during denaturation the 5.8‐S and/or a short‐lived 7‐S fragment (Mr about 75 000) which might represnt a direct precursor to the 5.8‐S rRNA. (b) The immediate nuclear precursor to the 26‐S rRNA (Mr 1.39 × 106) releases a small fragment of similar size (7 S). (c) The largest stable transcription product of the rDNA (pre‐rRNA) does not contain any hidden break.
Interation of DNA with DNA‐Binding Proteins
Authors: GEIDER Klaus
Published: March 2005

The Competition of the DNA‐binding proteins I and II of Escherichia coli and of the phage fd DNAbinding protein for single‐stranded DNA was investigated. Their roles in cells might be judged from thier binding affinited to DNA and their mutual exchange in the DNA ‐binding protein II displaced half of the protein I in the complex with singel‐stranded DNA when no double stranded DNA was present. SProtein‐complexd single strands were protected against degraddaiton.

The protection is less pronuounced for protein II which can increase the stability of the fd DNA complex with DNA‐binding protein I against nucleolytic celevage.
The Biosynthesis of the Mitochondrial ADP, ATP Translocator
Authors: HACKENBERG Heinz , RICCIO Paolo , KLINGENBERG Martin
Published: March 2005

The biosynthesis of the ADP, ATP carrier was studied in mitochondria of Neurospora crassa. The carrier was isolated as the carboxyatractylate‐protein complex and characterized in dodecylsulphate/polyacrylamide gel electrophoresis to have a Mr= 33000. Applying the inhibitors chloramphenicol for the intramitochondrial translation and cycloheximide for extramitochondrial translation, the site of synthesis of this polypeptide was found to be extramitochondrially located.
Alkylation of 6‐Phosphogluconate Dehydrogenase from Candida utilis with Coenzyme Analogues
Authors: BIELLMANN Jean‐François , GOULAS Philippe R. , DALLOCCHIO Franco
Published: March 2005

The mechanism of the inactivation of 6‐phosphogluconate dehydrogenase from Candida utilis with two coenzyme analogues can be differentiated on the basis of kinetic studies and of the properties of the inactivated enzyme. 3‐Chloroacetylpyridine–adenine dinucleotide phosphate is clearly an affinity label and 3‐chloroacetylpyridine–adenine dinucleotide a second‐order reagent. For 3‐chloroacetylpyridine–adenine dinucleotide phosphate, there is a loss of one thiol per subunit at complete inactivation whereas for 3‐chloroacetylpyridine–adenine dinucleotide 2.7 thiol groups are lost. The fluorescence of the protein is quenched after alkylation by 3‐chloroacetylpyridine–adenine dinucleotide phosphate and there is no quenching after the inactivation with 3‐chloroacetylpyridine–adenine dinucleotide.
Poly (ADP‐ribose) Polymerase from Ehrlich Ascites Tumor Cells
Authors: KRISTENSEN Tom , HOLTLUND Jostein
Published: March 2005

Poly(ADP‐ribose) polymerase from Ehrlich ascites tumor cells, partially purified by chromatography on DNA‐agarose, was obtained as a more than 80% homogeneous preparation by isoelectric focusing in a sucrose gradient. The polymerase activity was shown to be associated with the major protein in the preparation. Results obtained by electrophoresis in the presence of sodium dodecyl sulfate indicated that poly(ADP‐ribose) polymerase consists of a polypeptide chain with a molecular weight of 130000. Ultracentrifugation at non‐denaturating conditions indicated that the active enzyme may be an oligomeric form of this polypeptide chain. The isoelectric point of the polymerase was 9.40. The effects of various additions to the assay mixture on the synthesis of poly(ADP‐ribose) as well as some kinetic data, are given. It is shown that poly(ADP‐ribose) is a highly efficient inhibitor of its own synthesis, and results are presented which suggest that the well‐known stimulatory effect of DNA on the synthesis is due to reduction of this inhibitory effect of the product.
Presence of Non‐histone Proteins in Nucleosomes
Authors: DEFER Nicole , KITZIS Alain , LEVY Florence , TICHONICKY Lydie , SABATIER Marie‐Madeleine , KRUH Jacques
Published: March 2005

It has been established that nucleosomes are made of histones and DNA fragments. The purpose of this work was to establish whether some non‐histone proteins are also present in these chromatin subunits.

We have found that nucleosome preparations contain phosphorylated non‐histone proteins and protein kinases by sucrose gradient analysis. In order to establish whether these proteins are actually bound to nucleosomes or if they represent unbound or aggregated proteins, the following experiments were performed. (a) Free non‐histone proteins and proteins released from chromatin by DNase overdigestion were analyzed by sucrose gradient centrifugation. No phosphoproteins but some phosvitin kinase activity was found in the part of the gradient which contained the nucleosomes. It could be assumed that part of the phosphoproteins are bound to nucleosomes. (b) A digestion of nucleosomes with DNase I suppressed the phosvitin kinase activity in the 11‐S region of the gradient. (c) High ionic strength, which extracted non‐histone proteins, suppressed the phosvitin kinase activity in the nucleosome region.

Part of phosvitin kinase and of nuclear phosphoproteins are therefore bound to nucleosomes and are released by nuclease digestion and by high ionic strength.
Biosynthèse de Mannosyl‐Phosphoryl‐Dolichol dans la Membrane Externe des Mitochondires
Authors: GATEAU Odile , MORELIS Renée , LOUISOT Pierre
Published: March 2005

Biosynthesis of Dolichol Phosphate Mannose in the Mitochondrial Outer Membrane

Previous studies have shown that mannose incorporation into mitochondrial glycoproteins is catalysed by a mannosyl‐transferase, which is located in the mitochondrial inner membrane. It seems important to study the presence of mannosyl‐transferases which are able to catalyse the transfer of mannose into other acceptors, such as polyprenic intermediates.

Mouse liver mitochondria catalyse the incorporation of mannose from GDP‐[14C]mannose into three products: most of the radioactivity is in a product (A) soluble in chloroform/methanol (2/1, by vol.); mannose is also incorporated into product (B) soluble in chloroform/methanol/water (10/10/3, by vol.), and into a product which is insoluble in organic solvents and trichloroacetic acid and which is a glycoprotein. We have tried to localize in mitochondria the mannosyl‐transferases implicated in these reactions.

Mitochondrial outer membranes were prepared by swelling purified mitochondria in phosphate buffer, and were purified on a discontinuous sucrose gradient. The purified outer membranes catalyse the incorporation of mannose from GDP‐[14C]mannose in very large quantities into product A and in small quantities into product B.

Different procedures (purification on silicic acid and DEAE‐cellulose, hydrolytic procedures and chromatographic properties) demonstrated the identity of product A with authentic dolichol phosphate mannose.

Product B is lipid‐linked oligosaccharides.

The biosynthesis of dolichol phosphate mannose depends on Mn2+. When ethyleneglycol bis(2‐aminoethyl)N,N′‐tetracetic‐acid (EGTA) is added to the mixture, the reaction is inhibited. When EGTA is added to a mixture containing preformed endogenous dolichol phosphate [14C]mannose, the biosynthesis of the product stops, and the product is hydrolysed, with a concomitant apparition of lipid‐linked‐oligosaccharides. These results and the kinetics of the reactions suggest that dolichol phosphate mannose might be the mannosyl donor for the lipid‐linked‐oligosaccharides. The synthesis of dolichol phosphate mannose is reversed by the addition of GDP.

The role of these products as intermediates in mitochondrial glycoprotein biosynthesis is discussed.
RNA‐Protein Neighbourhoods of the Ribosome Obtained by Crosslinking
Authors: BÄUMERT Hans G. , SKÖLD Sven‐Erik , KURLAND Charles G.
Published: March 2005

It is possible to crosslink protein to nucleic acid with diepoxybutane which reacts mainly with the N‐7 of guanine as well as the thiol and aminogroups in protein; the resulting crosslinked complexes are cleavable. When this reagent is applied to ribosomes, even under conditions that minimize the extent of reaction, at least half of the ribosomal proteins can be recovered crosslinked to one or other of the major ribosomal RNA species. In addition, one fourth of the proteins can be recovered crosslinked to the RNA of the heterologous ribosomal subunit. Some of the remaining proteins may also be crosslinkable to the RNA, but additional experiments are required to confirm this.
A 1H Nuclear‐Magnetic‐Resonance Study of the Solution Conformation of the Isoinhibitor K from Helix pomatia
Authors: WAGNER Gerhard , WÜTHRICH Kurt , TSCHESCHE Harald
Published: March 2005

The isoinhibitor K from Helix pomatia was investigated by high‐resolution 1H nuclear magnetic resonance at 360 MHz. Detailed studies of the labile protons and the resonances of the aromatic residues indicated extensive homologies between the spatial structures of the snail inhibitor, the basic pancreatic trypsin inhibitor (Kunitz) from bovine tissue and the cow colostrum trypsin inhibitor. Comparison of these three homologous inhibitors indicated that the overall flexibility of the globular protein conformation is reduced and its stability with respect to thermal denaturation raised when the content of amino acids with charged side chains is increased. It is suggested that this relation between amino acid composition and stability of the globular solution structure might be valid also for other classes of homologous proteins.
Fluorescence Quenching and Energy Transfer in Complexes between Horse‐Liver Alcohol Dehydrogenase and Coenzymes
Authors: ABDALLAH Mohammed A. , BIELLMANN Jean‐François , WIGET Peter , JOPPICH‐KUHN Regula , LUISI Pier Luigi
Published: March 2005

The fluorescence properties of horse‐liver alcohol dehydrogenase were investigated with the aim of separating the contribution of Trp‐15 (which is close to the protein surface) from that of Trp‐314 (buried in the interior of the protein). Quenching of the protein fluorescence by iodide involves, to a larger degree, the longer wavelength region of the protein emission spectrum and is interpreted to involve only Trp‐15 (λmax at 340 nm and a quantum yield of 0.19). It is shown that quenching by NAD and NADH involves both types of tryptophan, but the ‘blue’ one to a larger extent. In the case of NADH, radiationless energy transfer between enzyme and reduced nicotinamide ring accounts for less than one half of the total protein fluorescence quenching. Energy transfer between the tryptophan and adenine rings is also possible, but it cannot account for the rest of the protein quenching. Thus, it is suggested that protein conformational changes, following NADH binding, are the cause of part of the fluorescence quenching. The extent to which quenching by NAD can be ascribed to radiationless energy transfer processes is also calculated. It is shown that despite the small spectral overlapping between coenzyme absorption and protein emission, the energy transfer contribution cannot be neglected. However, it is very likely that also in this case a sizeable part of the protein fluorescence quenching comes from protein conformational changes following coenzyme binding. The possible nature of these conformational changes is discussed, taking into account recent X‐ray data of enzyme‐coenzyme complexes.
Pyrimidine Metabolism in Microplasmodia of Physarum polycephalum
Authors: FINK Kirsten , NYGAARD Per
Published: March 2005

If microplasmodia of Physarum polycephalum are exposed to 14C‐labelled pyrimidine nucleosides or bases, an unusual pattern of metabolism is found. Only the nucleosides are taken up. Analysis of the distribution of the radioactivity in the cells revealed that ribonucleosides and deoxyribonucleosides are incorporated into nucleotides; however, a substantial catabolism takes place. Thus incubation with [2‐14C]pyrimidine nucleosides readily gives rise to [14C]O2, particularly in the case of [2‐14]thymidine. Due to this a significant part of the trichloroacetic‐acid‐insoluble radioactivity from exogenously supplied [2‐14C]thymidine is not associated with DNA.

The pattern of labelling of nucleoside triphosphates from exogenously supplied nucleosides indicated that the de novo synthesis of nucleotides was only partly repressed. An unusual conversion of deoxycytidine into cytidine was noted. Enzyme analysis on cell‐free extracts revealed that pyrimidine nucleosides can be salvaged by kinases and that their initial catabolism is initiated by hydrolases.

Incubation of microplasmodia with pyrimidine analogues showed that only nucleoside analogues are toxic.

The experimental results have led us to propose a scheme for the metabolism of pyrimidine nucleosides and bases in Physarum polycephalum.
Involvement of the Escherichia coli Phosphoenolpyruvate‐Dependent Phosphotransferase System in Regulation of Transcription of Catabolic Genes
Authors: BOLSHAKOVA Tatyana N. , GABRIELYAN Tatyana R. , BOURD Genrich I. , GERSHANOVITCH Vladimir N.
Published: March 2005

Synthesis of catabolite‐sensitive enzymes is repressed in mutants defective in the general proteins (enzyme I and HPr) of the Escherichia coli phosphoenolpyruvate‐dependent phosphotransferase system (ptsI and ptsH mutations). To elucidate the mechanism of this phenomenon we constructed isogenic strains carrying pts mutations as well as different lesions of regulation of the lac operon or mutations affecting adenylate cyclase activity (cya mutation) and synthesis of cyclic‐AMP‐receptor protein (crp mutation) Measurements of the differential rate of β‐galactosidase synthesis in these strains showed that the repressive effect of pts mutations was revealed in lac+, lacI, lacOc and cya bacteria, but it was lost in lacP and crp strains.

It was concluded that mutational damage to the general components of the phosphoenolpyruvate‐dependent phosphotransferase system diminishes activity of the lac promoter.

The results obtained led to the conclusion that pts gene products (apparently phospho ∼ HPr) are necessary for the initiation of transcription of catabolite‐sensitive operons in E. coli.
Analysis of Phosphorylation of Protein Synthesis Initiation Factor eIF‐2 by Two‐Dimensional Gel Electrophoresis
Authors: FARRELL Paul J. , HUNT Tim , JACKSON Richard J.
Published: March 2005

We describe a method for examining the state of phosphorylation of initiation factor eIF‐2 in reticulocyte lysates. The procedure involves incubation of the lysate with iodo[1‐14C]acetate in 8.5 M urea, and fractionation of the labelled proteins by two‐dimensional acrylamide gel electrophoresis. This approach has been used to show that haem deficiency, double‐stranded RNA, and oxidised glutathione, which all inhibit the initiation of protein synthesis in an analogous manner, all cause a net increase in the level of phosphorylated eIF‐2 in the complete lysate protein synthesis system.
Nuclear‐Magnetic‐Resonance Study of Aggregations and Conformations of Melanostatin and Related Peptides
Authors: HIGASHIJIMA Tsutomu , TASUMI Mitsuo , MIYAZAWA Tatsuo , MIYOSHI Muneji
Published: March 2005

The 1H and 13C nuclear magnetic resonance spectra of melanostatin (Pro‐Leu‐Gly‐NH2) and related peptides (Pro‐Leu‐Gly, Z‐Pro‐Leu‐Gly, Z‐Pro‐Leu‐Gly‐NH2 and Z‐Pro‐Leu‐Gly‐OCH3, where Z = benzyloxycarbonyl) were analysed in a variety of solvents. At physiological pH, the melanostatin molecule is N‐protonated in aqueous solution. The concentration dependences of the chemical shifts of amide‐proton and carbonyl‐carbon resonances and of proton spin‐lattice relaxation times were observed in relation to molecular aggregations. In dimethylsulfoxide solution, aggregations were observed for N‐protonated melanostatin and Pro‐Leu‐Gly prepared with HCl and for the Na salt of Z‐Pro‐Leu‐Gly but not for N‐protonated melanostatin prepared with HClO4 or HNO3, unprotonated melanostatin, Z‐Pro‐Leu‐Gly‐NH2, or Z‐Pro‐Leu‐Gly‐OCH3. The leucine NH and glycine CO groups of N‐protonated melanostatin are involved in the intermolecular hydrogen bonds of aggregates. The leucine NH group of N‐protonated Pro‐Leu‐Gly also forms the intermolecular hydrogen bond. The solvent and temperature dependences of the chemical shifts of amide‐proton and carbonyl‐carbon resonances were measured to determine intramolecular hydrogen bonding. In dimethylsulfoxide solution, N‐protonated melanostatin molecules in part take the β‐turn structure and the trans carboxamide NH proton and carbonyl oxygen of the proline residue form an intramolecular hydrogen bond.
The Structure of Chromatin Replicated in vitro
Authors: SCHLAEGER Ernst‐Jürgen , KLEMPNAUER Karl‐Heinz
Published: March 2005

Nuclei from concanavalin‐A‐activated lymphocytes were used to study the replication of chromatin in vitro. Micrococcal nuclease was employed to obtain information about the structure of the replicated chromatin. The nuclease digestion products were examined by sucrose gradient sedimentation and by gel electrophoresis. Experiments are presented which indicate that DNA replicated in vitro is organized into chromatin whose structure is similar to that of bulk chromatin. This conclusion is based on the following observations: (a) DNA replicated in vitro is associated with typical chromatin subunits (nucleosomes) even after short replication times, when the newly replicated DNA consists almost entirely of Okazaki fragments; (b) the length of internucleosomal spacer DNA in part of the replicated chromatin corresponds to that in bulk chromatin. Evidence which suggests that the structure of nucleosomes is transiently altered in the vicinity of the replication fork is presented.
Histidine in the Nucleotide‐Binding Site of NADP‐Linked Isocitrate Dehydrogenase from Pig Heart
Authors: EHRLICH Robert S. , COLMAN Roberta F.
Published: March 2005

Incubation of pig heart NADP‐dependent isocitrate dehydrogenase with ethoxyformic anhydride (diethylpyrocarbonate) at pH 6.2 results in a 9‐fold greater rate of loss of dehydrogenase than of oxalosuccinate decarboxylase activity. The rate constants for loss of dehydrogenase and decarboxylase activities depend on the basic form of ionizable groups with pK values of 5.67 and 7.05, respectively, suggesting that inactivation of the two catalytic functions results from reaction with different amino acid residues. The rate of loss of dehydrogenase activity is decreased only slightly in the presence of manganous isocitrate, but is reduced up to 10‐fold by addition of the coenzymes or coenzyme analogues, such as 2′‐phosphoadenosine 5′‐diphosphoribose (Rib‐P2‐Ado‐P). Enzyme modified at pH 5.8 fails to bind NADPH, but exhibits manganese‐enhanced isocitrate binding typical of native enzyme, indicating that reaction takes place in the region of the nucleotide binding site. Dissociation constants for enzyme · coenzyme‐analogue complexes have been calculated from the decrease in the rate of inactivation as a function of analogue concentration. In the presence of isocitrate, activating metals (Mn2+, Mg2+, Zn2+) decrease the Kd value for enzyme. Rib‐P2‐Ado‐P, while the inhibitor Ca2+ increases Kd. The strengthened binding of nucleotide produced by activating metal‐isocitrate complexes may be essential for the catalytic reaction, reflecting an optimal orientation of NADP+ to facilitate hydride transfer. Measurements of ethoxyformyl‐histidine formation at 240 nm and of incorporation of [14C]ethoxy groups in the presence and absence of Rib‐P2‐Ado‐P indicate that loss of activity may be related to modification of approximately one histidine. The critical histidine appears to be located in the nucleotide binding site in a region distal from the substrate binding site.
Human Serum Galactosyltransferase: Distinction, Separation and Product Identification of Two Galactosyltransferase Activities
Authors: BERGER Eric G. , KOZDROWSKI Irene , WEISER Milton M. , van den EIJNDEN Dirk H. , SCHIPHORST Wietske E. C. M.
Published: March 2005

Two different galactosyltransferase activities have been found in normal sera from A and O donors. Galactosyltransferase A incorporated galactose from UDP‐Gal into sialic‐acid‐free ovine submaxillary mucin (asialo‐mucin), whereas galactosyltransferase B transferred galactose from UDP‐Gal to free N‐acetylglucosamine or N‐acetylglucosamine‐glycoproteins. Specificity, kinetic and stability differences permitted the distinction of the activity of galactosyltransferase A from that of galactosyltransferase B; the only substrate found for galactosyltransferase A was asialo‐mucin, whereas galactosyltransferase B showed only low activity towards asialo‐mucin and free N‐acetylgalactosamine, but had a main specificity for either free N‐acetylglucosamine or N‐acetylglucosamine‐protein. Galactosyltransferase B was more stable on heat inactivation than galactosyltransferase A; galactosyltransferase B could be separated from galactosyltransferase A by affinity chromatography on N‐acetylglucosamine‐derivatized agarose.

The products of both enzyme activities have been analyzed. The galactosyltransferase A product was cleaved from asialo‐mucin by alkaline‐borohydride treatment. The acceptor used to identify the galactosyltransferase B product was free N‐acetylglucosamine. Periodate oxidation studies performed on the reduced disaccharides indicated the linkage type of the products. The anomeric configuration of the respective galactosyltransferase products were determined with specific galactosidases. Using these methods, galactosyltransferase A was found to form a Galβ(1→3)GalNAc‐protein linkage and galactosyltransferase B was found to form a Galβ(1→4)GlcNAc‐linkage.
The Biosynthesis of Linoleate from Oleoyl‐CoA via Oleoyl‐Phosphatidylcholine in Microsomes of Developing Safflower Seeds
Authors: STYMNE Sten , APPELQVIST Lars‐Åke
Published: March 2005

Microsomes prepared from developing safflower seeds rapidly desaturated added [14C]oleoyl‐CoA in the presence of NADH. The distribution of [14C]oleate and [14C]linoleate between different lipid classes revealed that phosphatidylcholine was labelled with [14C]linoleate before any other lipid class investigated. Considerable desaturation continued after the disappearance of [14C]oleoyl‐CoA in the reaction mixture. No [14C]linoleoyl‐CoA could be detected. Incubation in the absence of added NADH caused a rapid disappearance of [14C]oleoyl‐CoA by incorporation into triacylglycerols and phosphatidylcholine and also by release as [14C]oleic acid. Upon subsequent addition of NADH, [14C]linoleate was formed to the same extent as when NADH was present at the onset of the incubation.

These data are contradictory to the previously assumed pathway for linoleic acid biosynthesis in developing safflower seeds, claiming oleoyl‐CoA as the direct substrate and linoleoyl‐CoA as the primary product. However, our data corroborate published results on Chlorella, Candida, Torulopsis and on developing pea leaves, which strongly suggest that oleoyl‐CoA is first incorporated into phosphatidylcholine and then desaturated to linoleoyl‐phosphatidylcholine.
Primary Structure of Histone H2A from Gonad of the Sea Urchin Psammechinus miliaris
Authors: WOUTERS Danièle , SAUTIERE Pierre , BISERTE Gérard
Published: March 2005

The complete amino acid sequence (125 residues) of sea urchin histone H2A has been established by structural studies of peptides derived from tryptic and chymotryptic cleavage of the maleylated protein and from thermolysin cleavage of the intact protein. By comparison with calf homologous histone, the basic amino‐terminal and carboxy‐terminal parts of the protein show 11 substitutions and 4 deletions. The remainder of the sequence, mostly hydrophobic, is almost completely unchanged.
Anthracycline Antitumor Antibiotic · Nucleic‐Acid Interactions
Authors: PATEL Dinshaw J. , CANUEL Lita L.
Published: March 2005

The helix‐to‐coil transition of the synthetic DNA poly(dA‐dT) in the presence of the anthracylcline antitumor antibiotic daunomycin has been investigated by high‐resolution proton nuclear magnetic resonance (NMR) spectroscopy in 1 M salt solution. The dissociation of the complex, containing molar ratios of phosphate to daunomycin (pi/drug) of 50, 25, 9 and 5, with increasing temperature can be monitored independently at the nucleic acid and the antibiotic resonances under conditions of fast exchange. The antibiotic complex formation shifts suggest that either ring B and/or C of the intercalated anthracycline chromophore of daunomycin overlaps with adjacent nucleic acid base pairs. Ultraviolet/visible melting studies of daunomycin complexes with a series of synthetic DNAs substituted with halogen atoms (Br, I) at position 5 of the pyrimidine ring suggest that intercalation of the antibiotic into poly(dA‐dU) is not perturbed by bulky substituents at this position. A comparison of the melting curves for the daunomycin · poly(dA‐dT) complex with an analog of the antibiotic where the NH3+ group is replaced by dimethylglycine demonstrates the important contributions of electrostatic interactions between the amino sugar and backbone phosphates to the stability of the complex in low salt solution. The ultraviolet/visible and NMR studies monitor biphasic melting transitions at the nucleic acid markers in the daunomycin · poly(dA‐dT) complexes, Pi/drug = 50–9, so that antibiotic‐free base‐pair regions and those centered about bound daunomycin can be independently sutudied at the synthetic DNA level in solution.
Studies on the Occurrence of Disialosyl Groups in Glycoproteins of Salivary Glands
Authors: SLOMIANY Bronislaw L. , SLOMIANY Amalia , HERP Anthony
Published: March 2005

The major glycoprotein of pig submandibular and rat sublingual glands were investigated for the presence of disialosyl groups. The content of (C‐8)‐substituted sialic acid in the glycoproteins was determined by three different procedures: colorimetric, periodate oxidation and permethylation. Approximately 13% of sialic acid in pig submandibular glycoprotein, but none in rat sublingual glycoprotein, contained a substituent at C‐8. This substituent was identified as sialic acid from its susceptibility to neuraminidase treatment. From our estimation, it appears that one out of four sialic acid residues in pig submandibular glycoprotein occurs in an N‐acylneuraminyl‐α(2 → 8)‐N‐acylneuraminyl grup.
On the Mechanism of Assembly of the Aspartate Transcarbamoylase from Escherichia coli
Authors: CHAN William W.‐C.
Published: March 2005

The mechanism of subunit assembly of aspartate transcarbamoylase from Escherichia coli was studied by following the kinetics of reassociation. The isolated trimetric catalytic subunit (c3) and dimeric regulatory subunit (r2) were mixed together and formation of the dodecameric native enzyme (c6r6) was monitored by measuring changes in activity. Under appropriate conditions the reassociation was second order with respect to the c3 concentration and the effects of varying r2 concentration on the second‐order rate constant were examined. An optimum r2 concentration of about 0.07 μM was observed.

A scheme of the assembly pathways is proposed and is based on the reversible formation of c3r2n (n= 0, 1, 2 or 3) as intermediates. Various combinations of two such c3r2n species are considered as possible rate‐limiting steps. This model yields an expression which relates the experimentally determined (overall) second‐order rate constant to the equilibrium constant (Kd) governing the formation of c3r2n, the r2 concentration, and four coefficients which reflect the contribution of different types of assembly processes. Using previously determined values of Kd, the above expression for each r2 concentration reduces to a linear equation with four unknowns. The experimental data were subjected to multiple linear‐regression analysis and values for the four coefficients were found which gave an excellent fit.

Our results show that reassociation of the subunits is a fast bimolecular reaction with rate constants in excess of 106M−1s−1. Our analysis also suggests that interactions involving a total of more than three r2 subunits (e.g. the combination of c3r2 with c3r6) might contribute significantly to the overall assembly.

The influence of various ligands on the reassociation rate profile was also studied. All ligands examined were partially inhibitory to the formation of native enzyme. The effects of substrates were similar to those of CTP whereas the effects of ATP were substantially different. These observations can be readily interpreted by postulating different conformational changes induced by the ligands. These changes should alter the relative orientation of the subunit contacts which must be formed in the reassociation process. The interpretation is consistent with our previous model of the allosteric mechanism.
Comment on the Use of Linear Free‐Energy Slopes as Criteria for Distinguishing Hydride‐Ion from Hydrogen‐Atom Transfer in Reactions of Pyridine‐Nucleotide Coenzymes
Authors: KURZ Linda C. , KURZ Joseph L.
Published: March 2005

For reductions by dihydropyridines, the slopes of plots of log k vs the standard reduction potential of the dihydropyridine are not direct indicators of whether the rate‐determining step is hydride‐ion or hydrogen‐atom transfer. When there is no substituent effect on the reverse rate, those slopes are independent of mechanism and equal to about (30 mV)−1.
The N‐Terminal Sequence Protein of L7/L12 Is Responsible for Its Dimerization
Authors: GUDKOV Anatoli T. , BEHLKE Joachim
Published: March 2005

Ultracentrifuge studies of intact protein L7/L12, of its fragments 27–120, 1–74 and 74–120 and protein L7/L12 with oxidized methionine residues, indicate that the N‐terminal sequence of the protein L7/L12 is responsible for its dimerization. The symmetry model of the dimer is discussed.