bluuebunny/crossref_metadata_2025 huggingface dataset:
The Activation of Brain Adenylate Cyclase and Brain Cyclic‐Nucleotide Phosphodiesterase by Seven Calmodulin Derivatives
Authors: THIRY Philippe , VANDERMEERS André , VANDERMEERS‐PIRET Marie‐Claire , RATHE Jean , CHRISTOPHE Jean
Published: March 2005

A comparison has been made of the ability of seven calmodulin derivatives to displace 125I‐labeled calmodulin and to activate adenylate cyclase in a brain particulate fraction. The activation of brain‐soluble cyclic‐nucleotide Phosphodiesterase by the same calmodulin derivatives was examined in parallel.

In general, the dose for half‐maximal inhibition of 125I‐labeled calmodulin binding and the apparent Km of adenylate cyclase activation were comparable in brain membranes. These concentrations were 20–40‐times higher than the corresponding apparent Km values of activation of cyclic‐nucleotide Phosphodiesterase.

Modifying the single histidine residue or both tyrosine residues exerted no influence on the biological properties of calmodulin. The carboxymethylation of two methionine residues or the amidation of several carboxyl groups reduced the activation properties of calmodulin on adenylate cyclase and cyclic‐nucleotide Phosphodiesterase. Altering seven lysine or four arginine residues resulted in two proteins whose activation properties on adenylate cyclase and Phosphodiesterase had been modified in a way suggesting that lysine and arginine residues play distinct roles in the interaction of native calmodulin with each enzyme.
A New Method for the Purification of DNA‐Binding Proteins with Sequence Specificity
Authors: WEIDELI Hansjörg , GEHRING Walter J.
Published: March 2005

We describe a technique for a rapid and efficient isolation and purification of proteins binding to defined DNA sequences. Cloned double‐stranded DNA was covalently coupled to m‐amino‐benzyloximethylcellulose in order to purify proteins which recognize and bind to specific sequences on the DNA. The purification of two DNA‐binding proteins from Drosophila melanogaster is demonstrated using the respective cloned DNA sequences.
The Role of the Codon and the Initiation Factor IF‐2 in the Selection of N‐Blocked Aminoacyl‐tRNA for Initiation
Authors: van der LAKEN Kees , BAKKER‐STEENEVELD Hanny , BERKHOUT Ben , van Knippenberg Peter H.
Published: March 2005

Poly(uridylic acid) [poly(U)] and poly(xanthidylic acid) [poly(X)] strongly stimulate the IF‐2‐dependent binding of fMet‐tRNA to 30‐S ribosomal subunits from Escherichia coli [Van der Laken et al. (1979) FEBS Lett. 100, 230–234]. The N‐formylmethionine moiety is incorporated into poly(phenylalanine) upon subsequent addition of other components required for protein synthesis when poly(U) is used as template.

This paper shows that N‐acetylated Phe‐tRNAPhe (AcPhe‐tRNA), but not Phe‐tRNAPhe or tRNAPhe, competes with fMet‐tRNA for binding to poly(U)‐programmed 30‐S ribosomal subunits. The two species of N‐blocked aminoacyl‐tRNA are bound to poly(U)‐programmed and poly(X)‐programmed 30‐S subunits in a ratio that is linearly dependent on the ratio of the two species added. With poly(U) as template there is no apparent preference for either fMet‐tRNA or AcPhe‐tRNA, whereas with poly(X) there is a 2–3‐fold preference for fMet‐tRNA.

The initiation factor IF‐2, which is strictly required for the binding of N‐blocked aminoacyl‐tRNAs, has a higher affinity for Met‐tRNA than for AcPhe‐tRNA.

It is concluded that (a) interaction of the 30‐S ribosomal subunit with poly(U) or poly(X) leads to IF‐2‐dependent binding of N‐blocked aminoacyl‐tRNA; (b) the initiation factor IF‐2 discriminates in favour of fMet‐tRNA; (c) the presence of the cognate codon discriminates in favour of the corresponding N‐blocked aminoacyl‐tRNA.
Aspartate‐β‐Semialdehyde Dehydrogenase from Escherichia coli
Authors: BIELLMANN Jean‐François , EID Pierre , HIRTH Christian , JÖRNVALL Hans
Published: March 2005

Aspartate‐β‐semialdehyde dehydrogenase, from an Escherichia coli mutant derepressed for the biosynthesis of l‐lysine, has been purified to homogeneity. Its isoelectric point is pH 4.3. This enzyme has a molecular weight of 77000 and is composed of two identical or highly similar subunits of molecular weight 38000 ± 2000.

Their N‐terminal amino‐acid sequence is Met‐Lys‐Asx‐Val‐Gly‐. Three cysteine residues per subunit were detected: two are reactive in the native enzyme and one is partially protected by the substrate. Formation of an acyl‐enzyme intermediate was also detected. Correlation of the 1H nuclear magnetic resonance spectrum of [4‐2H]NADPH produced from [4‐2H]NADP+ indicated that aspartate β‐semialdehyde dehydrogenase transfers the pro‐S hydrogen from NADPH (class B dehydrogenase). A short comparison with the corresponding yeast enzyme is given.
The Interaction of the EcoRI Restriction Endonuclease with its Substrate
Authors: GOPPELT Margarete , PINGOUD Alfred , MAASS Günter , MAYER Hubert , KÖSTER Hubert , FRANK Ronald
Published: March 2005

The interaction of the EcoRI restriction endodeoxyribonuclease with polynucleotides has been studied in a qualitative manner by an affinity adsorption technique using polynucleotides immobilized on cellulose. It is shown that EcoRI binds to single‐stranded and double‐stranded polyribonucleotides and polydeoxyribonucleotides. Mg2+ ions are not required for binding.

In order to define differences between specific and non‐specific binding we have investigated the interaction of EcoRI with d(T‐A‐A‐A‐T‐G), d(T‐T‐A‐C‐A‐T), d(G‐A‐A‐T‐T‐C) and d(G‐G‐A‐A‐T‐T‐C‐C). We have synthesized for this purpose d(T‐A‐A‐A‐T‐G), d(T‐T‐A‐C‐A‐T) and d(G‐A‐A‐T‐T‐C) by the diester approach. d(G‐A‐A‐T‐T‐C) and d(G‐G‐A‐A‐T‐T‐C‐C) are self‐complementary. Differential melting experiments show that the octanucleotide has a melting point of 28°C under ionic conditions where the hexanucleotide is single‐stranded even below 0°C. Correspondingly, the octanucleotide is cleaved by EcoRI, while the hexanucleotide is not.

The binding of oligonucleotides to EcoRI can be monitored by the circular dichroism of the enzyme. Titrations show that in the absence of Mg2+ ions all oligonucleotides are bound with similar magnitude: Ka∼ 107 M−1. Complex formation is weakened with increasing temperature, corresponding to a ΔH° of ‐21 kJ/mol and increasing ionic strength, corresponding to an involvement of two ion‐pair bonds between DNA and enzyme. Mg2+ ions have no significant influence on the binding of d(T‐A‐A‐A‐T‐G), d(T‐T‐A‐C‐A‐T) and d(G‐A‐A‐T‐T‐C) to the enzyme. The binding of d(G‐G‐A‐A‐T‐T‐C‐C) to EcoRI is strengthened by a factor of 50 in the presence of Mg2+ ions, as measured by cleavage experiments using d(G‐A‐A‐T‐T‐C) as a competing inhibitor in the enzymatic assay.
Initiation Factor eIF‐2 from Wheat Germ
Authors: BENNE Rob , KASPERAITIS Marcelle , VOORMA Harry O. , CEGLARZ Ewa , LEGOCKI Andrzej B.
Published: March 2005

Protein synthesis initiation factor eIF‐2 was isolated and purified 4300‐fold from wheat germ 23000 ×g supernatant. The procedure described resulted in a 70% pure factor and involves three chromatographic steps only: (a) on Sepharose‐heparin, (b) on Sepharose–concanavalin‐A and (c) on phosphocellulose. The Sepharose‐concanavalin‐A column was required for the removal of the bulk of the proteins present in the wheat germ supernatant, its use increased the speed of the procedure considerably. The factor consists of three non‐identical subunits in a 1:1:1 stoichiometric ratio with apparent Mr of 56000, 41000 and 36000, which results in an Mr of 133000 for the undissociated factor. The subunits were found to be distinct from those of the eIF‐2 from reticulocytes.

The factor was tested in the various model reactions of the initiation process such as ternary complex formation with Met‐tRNAfMet and GTP, methionyl‐puromycin synthesis and in unfractionated wheat germ 23000 ×g supernatant and rabbit reticulocyte 30000 ×g supernatant systems. The results support the idea of an universal mode of action of initiation factors in eukaryotic cells: i.e. the wheat germ factor stimulated the above‐mentioned systems in a manner similar to eIF‐2 from rabbit reticulocytes. The two factors are fully exchangeable in ternary complex formation (both factors are approximately 30% active, reach maximum levels of [3H]Met‐tRNAfMet binding at 0.6 μM GTP and show very little dependence on the Mg2+ concentration) and, provided that small amounts are used, in the assay for methionyl‐puromycin and in the crude rabbit reticulocyte lysate.

Both factor preparations act as a substrate for a cAMP‐independent protein kinase from reticulocytes (hemin‐regulated translational inhibitor) which is thought to be responsible for the shut‐off of protein synthesis in hemin‐deprived lysates from rabbit reticulocytes: the 41000‐Mr subunit of wheat germ eIF‐2 and the 38000‐Mr, subunit of reticulocytes eIF‐2 were stoichiometrically phosphorylated in vitro by this inhibitor in the presence of [γ‐32P]ATP. Indeed, large amounts of the inhibitor inhibited protein synthesis in crude wheat germ extracts to some extent (25%). However, such an inhibition could not be overcome by the addition of purified eIF‐2.

Furthermore, when crude wheat germ extracts were incubated with [γ‐32P]ATP, the eIF‐2 subunit of Mr 36000 (but not the others) became phosphorylated, although no decrease in the rate of protein synthesis could be detected.
Glycogen‐Synthase Phosphatase Activity in Rat Liver
Authors: DOPERÉ Frans , VANSTAPEL Florent , STALMANS Willy
Published: March 2005

Three subfractions of glycogen synthase b (termed b1, b2, b3) have been isolated from the glycogen fraction of dog liver on the basis of a different affinity for DEAE‐cellulose. Their kinetic properties and chromatographic behaviour are compatible with the presence of an increasing number of phosphorylated sites from synthase b1 towards b3.

Synthase phosphatase activity in rat liver stems from two heat‐labile and trypsin‐labile proteins. These components are conveniently prepared from the cytosolic fraction of glycogen‐depleted liver; the ‘G‐component’ of the phosphatase co‐sediments with added particulate glycogen, whereas the ‘S‐component’ remains in the supernatant. The G‐component alone did not convert any available synthase b to the a form. The synthase phosphatase activity of the S‐component was variable according to the actual type of substrate. When acting on synthase b2 and b3, the S‐component had a low phosphatase activity that was increased 7‐fold and 11‐fold, respectively, upon addition of the G‐component. Synthase b1, however, was efficiently activated by the S‐component, and only 35% faster in the presence of both components.

When the cytosolic fraction of glycogen‐depleted livers was analysed by sucrose‐gradient centrifugation a single peak of phosphatase activity (s20, w= 10.2 S; provisional Mr= 254000) was detected with synthase b2 as substrate. In addition to this peak, presumably an S‐G complex, synthase b1 also identified free S‐component of lower and heterogeneous molecular weight.

Our results illustrate in general the influence of the type of synthase b on the detection of synthase phosphatase activity, and specifically may provide an explanation for some discrepant reports on the subcellular distribution of the enzyme.
Sugar‐Lectin Interactions: How Does Wheat‐Germ Agglutinin Bind Sialoglycoconjugates?
Authors: MONSIGNY Michel , ROCHE Annie‐Claude , SENE Claude , MAGET‐DANA Régine , DELMOTTE Francis
Published: March 2005

The specific binding of N‐acetylneuraminic acid to wheat‐germ agglutinin is based on configurational similarities between N‐acetylneuraminic acid and N‐acetylglucosamine. The N‐acetamido group and an adjacent hydroxyl group, both in an equatorial position are shown to be the main determinants. The N‐acetylneuraminic acid–wheat‐germ agglutinin interaction is increased by the removal of the last two carbons C8 and C9. The interaction between wheat‐germ agglutinin and glycoconjugates containing N‐acetylneuraminic acid is shown to be dependent on a charge effect and on an avidity effect. Succinylated wheat‐germ agglutinin which is negatively charged at physiological pH, in contrast with wheat‐germ agglutinin which is positively charged, does not bind cell surface glycoconjugates containing N‐acetylneuraminic acid but does bind cell surface glycoconjugates containing N‐acetylglucosamine. The use of wheat‐germ agglutinin and of succinylated wheat‐germ agglutinin leads to the determination of the number of cell surface receptors containing N‐acetylneuraminic acid.
The Peroxidatic Reaction Catalyzed by Horse Liver Alcohol Dehydrogenase
Authors: FAVILLA Roberto , CAVATORTA Paolo , MAZZINI Alberto , FAVA Adriano
Published: March 2005

The results of steady‐state kinetic measurements on the initial rate of the peroxidatic reaction between β‐NAD+ and hydrogen peroxide, catalyzed by horse liver alcohol dehydrogenase, at pH 7 are described. A simple sequential mechanism is deduced from graphical analysis of the data plotted according to Eadie‐Augustinsson‐Hofstee primary plots and the values of the true kinetic parameters KNADm, KH2O2m and V are estimated from the corresponding secondary plots. Ethanol has been found to compete with hydrogen peroxide for the same enzyme active site. During the catalytic process a progressive inactivation of the enzyme occurs caused by H2O2. The rate law of this process is quantitatively described at pH 7 both in the absence and in the presence of NAD+. The coenzyme has been found to protect the enzyme against inactivation by H2O2, which oxidized essential cysteine residues. The results obtained from the study of both catalytic and inactivating processes are finally rationalized on the basis of a general mechanistic scheme.
The Peroxidatic Reaction Catalyzed by Horse Liver Alcohol Dehydrogenase
Authors: MAZZINI Alberto , DRADI Emanuele , FAVILLA Roberto , FAVA Adriano , CAVATORTA Paolo , ABDALLAH Mohamed A.
Published: March 2005

As previously reported [Favilla, R. & Cavatorta, P. (1975) FEBS Lett. 50, 324–329], the enzyme horse liver alcohol dehydrogenase catalyzes a reaction between NAD+ and H2O2. The final isolated product was then called NADX because of its unknown structure. In this paper the results of spectroscopic investigations on this compound are described. They indicate that only the nicotinamide moiety of the original NAD+ molecule was modified by the action of hydrogen peroxide.

From the 1H and 13C nuclear magnetic resonance spectra of NADX the following structure was deduced: adenosine(5′)diphospho(5)‐β‐d‐ribose‐NH–CH ‐ 6 C(CHO)–CONH2. This structure is consistent with both ultraviolet and reactivity measurements performed on NADX.

A tentative mechanism for the whole peroxidatic reaction pathway leading to NADX is finally proposed.
Metal Ion Binding to Yeast Phosphoglycerate Kinase
Authors: WROBEL Jean A. , STINSON Robert A.
Published: March 2005

Metal ion binding to yeast phosphoglycerate kinase was monitored with two systems that were sensitive to the conformation of the protein. One system used the enzyme labelled with the sulfhydryl reporter group 2‐chloromercuri‐4‐nitrophenol. Titrations of this complex with Mg2+, Mn2+, Co2+, Ca2+ or Cd2+ increased the absorbance of the bound chromophore. Dissociation constants for these metal ions to the complex were determined from Scatchard plots and ranged from 8 μM to 43 μM. A single class of binding site per 47000 g of enzyme was indicated. In contrast, two classes of Zn2+ binding sites were demonstrated and both appeared to be distinct from the other class of metal ion binding sites.

A second system involved the reaction between native phosphoglycerate kinase and 5,5′‐dithio‐bis(2‐nitrobenzoic acid). The rate constant for this reaction increased in the presence of each of the divalent metal ions examined. Analysis of the data gave dissociation constants which agreed with those determined from the mercurial system. Further, the same two classes of Zn2+ binding sites were apparent.

The affinity of the native enzyme for anions as previously reported by us [Eur. J. Biochem. 85, 345–350 (1978)] was not influenced by occupancy of the metal ion binding site with Mg2+ or Ca2+, or by saturation of one of the two classes of Zn2+ binding sites. Anion binding did not alter the affinity of the enzyme for Mg2+ or Ca2+, nor did it significantly weaken the binding of Zn2+ to one of its sites.
Effect of Ultraviolet Irradiation on 30‐S Ribosomal Subunits. Identification of the RNA Region Crosslinked to Protein S7
Authors: EHRESMANN Bernard , BACKENDORF Claude , EHRESMANN Chantal , MILLON Régine , EBEL Jean‐Pierre
Published: March 2005

The effects of ultraviolet irradiation on Escherichia coli 30‐S ribosomal subunits were studied. At the doses of radiation used in this work (0—4.5 × 105 quanta/30‐S subunit), only protein S7 was found to be significantly crosslinked to the 16‐S RNA. In conditions where 25% of the protein was covalently crosslinked, the ability of the irradiated 30‐S subunits to reassociate with 50‐S subunits and their activity in polyphenylalanine synthesis decreased strongly. Similar results were obtained by irrradiation with a germicide lamp (254 nm) or with a monochromatic ultraviolet light at 248 nm. No additional proteins were crosslinked to the 16‐S RNA by irradiating 30‐S subunits depleted in protein S1 or 70‐S ribosomes.

The covalent complex of 16‐S RNA and protein S7 was isolated and digested by T1 ribonuclease. The oligonucleotide remaining attached to the crosslinked protein was characterised as A‐C‐C‐U‐C‐G [position 1261–1266, see the sequence published by Carbon et al. (1979) Eur. J. Biochem. 160, 399–410]. Analysis of this fragment suggests that protein S7 was linked to the cytosine at position 1265 in the RNA sequence.
Post‐transcriptional Modifications of Oat Coleoptile Ribonucleic Acids
Authors: HAUGLAND Richard A. , CLINE Morris G.
Published: March 2005

Evidence is presented for the occurrence of 5′‐terminal capping structures in poly(A)‐rich RNAs from oat coleoptile tissue. Similar to the cap structures in mRNA from other eukaryotic organisms, the 5′ terminus of these oat coleoptile RNA molecules consists of 7‐methylguanosine joined 5′ to 5′ with the adjacent (penultimate) nucleoside by means of three phosphate groups in two pyrophosphate linkages. The penultimate nucleoside contains primarily purine bases, but small amounts of pyrimidines (cytidine) are also detectable. Some monophosphorylated 5′‐termini were also detected, however, they appear to occur as a result of RNA degradation. In addition to the 5′ cap, oat RNA molecules are also post‐transcriptionally modified with a low frequency of N6‐methylations of internal adenosines.
Structural Studies on Cutinase, a Glycoprotein Containing Novel Amino Acids and Glucuronic Acid Amide at the N Terminus
Authors: LIN Tzong‐Shin , KOLATTUKUDY P. E.
Published: March 2005

Cutinase I and cutinase II, two extracellular enzymes produced by Fusarium solani pisi, were shown to be glycoproteins containing 4.3% and 5.1% carbohydrates, respectively. Upon treatment with alkali both enzymes generated chromophores which absorbed at 241 nm. Treatment of both proteins with alkaline NaB3H4 gave labeled protein and labeled monosaccharides. Hydrolysis of the labeled protein followed by chromatographic and enzymatic analyses of the products showed that alanine, 2‐aminobutyrate, phenylalanine, tyrosine and l‐gulonic acid accounted for nearly all of the 3H contained in the protein. The four labeled amino acids were shown to be 1:1 mixture of d and l isomers and 3H was nearly equally distributed between α and β positions in each amino acid. The N‐terminal amino group of cutinase I did not react with either phenylisothiocyanate or dansyl chloride. This amino group was suggested to be in amide linkage with glucuronic acid because upon treatment of the protein with neutral NaB3H4, gulonic acid attached to the protein became labeled and only gulonic acid was labeled when the protein was deglycosylated with HF prior to alkaline NaB3H4 treatment. Furthermore, N‐gulonylglycine was isolated from the pronase digest of the labeled protein. Chromatographic identification and quantification of the labeled carbohydrates released from cutinase I by alkaline NaB3H4 showed that one mole of cutinase I has one mole each of mannose, arabinose, N‐acetylglucosamine, and glucuronic acid O‐glycosidically linked to serine, threonine, β‐hydroxyphenylalanine, and β‐hydroxytyrosine. In addition, the N‐terminal glycine is in amide linkage with glucuronic acid. Since almost identical experimental results were obtained with cutinase II this protein is also suggested to have the same structural features as those suggested above for cutinase I.
On the Role of Inorganic Phosphate in Divalent‐Cation Sequestration by Mitochondria
Authors: PUSKIN Jerome S. , GUNTER Thomas E. , COENE Mark T.
Published: March 2005

Rat liver mitochondria which have actively accumulated Mn2+ ions exhibit an electron paramagnetic resonance (EPR) spectrum dominated by a component E in which the usual hyperfine sextet has been collapsed to a single line through the process of spin exchange narrowing. When uptake occurs in the absence of added Pi, the line width of E shows considerable variation. By comparing experimentally observed spectra with computer‐generated simulations it is demonstrated however that, under these conditions, E can be as narrow as ∼ 190 Oe, considerably narrower than the spectrum from a manganese phosphate precipitate. Furthermore parallel quantification on a single mitochondrial sample shows the level of Pi to be several times lower than the amount of Mn2+ observable in E. It is concluded therefore that the spectral component E, obtained under these conditions, cannot be accounted for in terms of manganese phosphate precipitate. These findings further indicate that a substantial number of sites (∼ 100 nmol/mg protein), other than Pi, must exist inside the mitochondria which are capable of complexing divalent cations in regions of high local concentration.
The Non‐catalytic Nucleotide‐Binding Site of Mitochondrial ATPase Is Localised on the α‐Subunit(s)of Factor F1
Authors: KOZLOV Igor A. , MILGROM Yakov M.
Published: March 2005

The incubation of isolated factor F1 with the di‐aldehyde derivative of ADP (oxADP) which is formed as a result of ADP treatment by periodate, causes the covalent binding of 0.9–1 molecules of the oxADP with a molecule of the enzyme. This modification of factor F1 is not accompanied by any changes in the ATPase activity of the enzyme. The modification of factor F1 is preceded by the reversible binding of oxADP with the enzyme with a Kd of 80 μM. ADP partly prevents factor F1 from modification by oxADP. The electrophoresis of modified factor F1 in polyacrylamide gel in the presence of sodium dodecyl sulphate showed that oxADP binds with the α‐subunit(s) of factor F1.

When submitochondrial particles are incubated with [3H]oxADP, the main part of the radioactive label may be discovered in the polypeptide with a molecular weight of some 30000 which is probably the adenine nucleotides' translocase. The isolation of factor F1 from particles preincubated with [3H]oxADP showed that the membrane‐bound factor F1 covalently binds 0.2–0.3 mol of oxADP per mol of enzyme. Here again, all the oxADP is bound with the α‐subunit(s) of factor F1. The modification of membrane‐bound factor F1 by oxADP is accompanied by the partial inhibition of the particles' ATPase activity. The results obtained testify to the fact that the non‐catalytic site of mitochondrial ATPase located on the α‐subunit(s) of factor F1 may participate in the mechanism of ATP hydrolysis by membrane‐bound ATPase.
Properties of Biospecific Adsorbents, Obtained by Immobilization of Oestradiol 7α‐Derivatives, for Purification of Calf‐Uterine Cytosol Oestradiol Receptor
Authors: REDEUILH Gérard , RICHARD‐FOY Robert , SECCO Claude , TORELLI Vesperto , BUCOURT Robert , BAULIEU Etienne‐Emile , RICHARD‐FOY Hélène
Published: March 2005

The properties of three types of adsorbents obtained by coupling oestradiol 7α‐derivatives to agarose were compared. The adsorbents examined were: oestradiol 7α‐decamethylene‐agarose, ocstrndiol 7α ‐decamethylene‐poly(alanyl‐lysine)‐agarose and oestradiol 7α‐trimethylene‐poly(alanyl‐lysine)‐agarose. The following results were obtained.

(1) All these adsorbents are stable at 0°C for at least a year when stored in water. In the presence of cytosol they are stable for several hours and are reusable after a simple wash. (2) A new method allowing the calculation of the maximal receptor binding capacity of an absorbent was developed. (3) The geometry of the column and the dynamics of the loading have no influence on the binding capacity of the adsorbents. (4) Binding of the cytosol receptor to the adsorbent depends on whether the receptor had previously been partially purified by heparin‐Ultrogel chromatography or treated with low or high salt concentration or trypsin. It was demonstrated that aggregation decreases the binding of the receptor to the adsorbents. (5) A satisfactory recovery of receptor upon elution is possible only with biospecific adsorbents containing low concentrations of coupled steroid (≤ 0.2 mg/ml).

The use of these adsorbents for the purification of the trypsin‐treated receptor directly from cytosol allowed a 2500‐fold purification corresponding to 5% pure protein (assuming one oestradiol binding site per molecule of Mr, 60000). When starting from a low salt preparation containing the native 8‐S receptor, partially purified by heparin‐Ultrogel chromatography, preliminary experiments using affinity chromatography gave a further purification of 250–500‐fold and led to a 50–90% pure protein (assuming one oestradiol binding site per molecule of Mr 70000).
A Cross‐Linking Study of the Ca2+, Mg2+‐Activated Adenosine Triphosphatase of Escherichia coli
Authors: BRAGG Philip D. , HOU Cynthia
Published: March 2005

The solubilized Ca2+, Mg2+‐activated adenosine triphosphatase of Escherichia coli is composed of five subunits designated α, β, γ, δ and ɛ in order of decreasing molecular weight. The subunit structure of the enzyme has been investigated by the use of the cleavable cross‐linking agents dithiobis(succinimidyl propionate), methyl‐4‐mercaptobutyrimidate, dimethyl‐3,3′‐dithiobispropion‐imidate, disuccinimidyl tartarate, and cupric 1,10‐phenanthrolinate. The products of cross‐linking were analyzed by two different two‐dimensional gel electrophoresis systems. The following cross‐linked subunit dimers were observed: α2, β2, αβ, αδ, βγ, βδ, βɛ and γɛ. These results, together with other published data, are discussed in relation to a model of the arrangement of the subunits in the ATPase molecule.
Type‐IV Collagens
Authors: DIXIT Saryu N.
Published: March 2005

Two sets of structurally distinct collagen components resembling type IV collagen have been isolated and purified from the pepsin digest of kidney cortex homogenates. C and D chains with the size of α chains were separated from disulfide‐linked material on molecular sieves and were further purified by ion‐exchange chromatography. The molecular gel filtration of the starting material also gave two high‐molecular‐weight collageneous fractions. The reduction of these fractions with dithiothreitol followed by carboxymethyl‐cellulose chromatography resulted in the separation of two major peaks, one corresponding to C‐1 and the second peak corresponding to D‐1 and a 75000‐Mr components. These latter two components were further separated from each other by molecular sieve chromatography. In addition some smaller peptides (two 50000‐Mr and one 15000‐Mr peptides) were purified and analyzed. By amino acid composition, chromatographic properties and cyanogen bromide peptide patterns on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the C‐1 and C chains and one of the 50000‐Mr components appear to be derived from the same chain by pepsin cleavage at different sites. Similarly, the D1 and D chains, and the 75000‐Mr the second 50000‐Mr and the 15000‐Mr components are related to each other but are structurally distinct from the C‐1 and C components. The presented data are consistent with the presence of two structurally distinct chains in basement membrane collageneous molecules.
Folding Mechanism of the Triple Helix in Type‐III Collagen and Type‐III pN–Collagen
Authors: BÄCHINGER Hans Peter , BRUCKNER Peter , TIMPL Rupert , PROCKOP Darwin J. , ENGEL Jürgen
Published: March 2005

The kinetics of triple‐helix formation in type III pN‐collagen, type III collagen and a quarter fragment of type III collagen was followed by optical rotation and circular dichroism. Kinetic intermediates were detected by trypsin digestion and polyacrylamide gel electrophoresis. The end products of refolding at 25°C were identical to the native molecules according to their melting profiles, molecular weights and sedimentation behavior. Only at low temperatures (4–15°C) were mismatched structures of lower stability formed. At 25°C helix formation started exclusively at the set of three disulfide bridges which link the three chains at the carboxy‐terminal end. The growth of the triple helix proceeds from this single nucleus at a rather uniform rate in a zipper‐like fashion. This gives rise to zero‐order kinetics over a large fraction of the conversion. Consequently the time of half conversion is proportional to the length of the molecule. From the appearance and disappearance of intermediates the growth of the triple helix could be observed directly. The rate of helix propagation is determined by the rate of cis → trans isomerization of peptide bonds. A model mechanism was developed which quantitatively described the overall kinetics as well as the time course of the intermediates with a single set of parameters: the rate constant of cis → trans isomerization k= 0.015 s−1 and an average number of 30 tripeptide units in uninterrupted stretches of residues with all peptide bonds in trans configuartion.
Somatic Antigens of Pseudomonas aeruginosa
Authors: DMIITRIEV Boris A. , KNIREL Yuriy A. , KOCHAROVA Nyna A. , KOCHETKOV Nikolay K. , STANISLAVSKY Evgeny S. , MASHILOVA Galina M.
Published: March 2005

A loosely bound lipopolysaccharide‐protein complex was extracted from cells of Pseudomonas aeruginosa strain 170015 (O:7ab; Lanyi classification) by saline solution and purified from contaminant nucleic acid by Cetavlon treatment followed by precipitation in an ultracentrifuge. The saline‐treated cells were re‐extracted with hot aqueous phenol to give firmly bound lipopolysaccharide which was isolated from the phenol layer and purified by ultracentrifugation. The identity of both lipopolysaccharide preparations was proved by serological and chemical evidence.

Mild acid degradation of the lipopolysaccharide resulted in the splitting off of a lipid component and led to polysaccharide which was purified by gel‐filtration on a Sephadex G‐50 column.

The polysaccharide consisted of N‐acetyl‐d‐fucosamine, N‐acetyl‐l‐tucosamine and d‐glucose in the ratio 1:1:1. On the basis of nuclear magnetic resonance spectra, results of methylation analysis and two sequential Smith degradations, the following structure can be assigned to the repeating unit of the polysaccharide: – 3)lFucNAc(α1–3)DFucNAc(β1–2)DGlc(β1–.

The polysaccharide did not show serological activity whereas alkali‐treated lipopolysaccharide readily sensitised sheep erythrocytes and inhibited the passive haemagglutination reaction with anti‐(O:7a,b)serum. Evidence is presented that the oligosaccharide repeating units of the polysaccharide and alkali‐treated lipopolysaccharide are indistinguishable. ps. aeruginosa strain 170016 (O:7a, c) was shown to have the O‐specific lipopolysaccharide identical with that from strain 170015. The presented data show that subfactors 7b.and 7c in the Lanyi classification of Ps. aeruginosa O‐antigens seem to relate to components of the bacterial surface other than lipopolysaccharides.
Binding and Inhibitory Effect of 2‐Heptyl‐4‐hydroxyquinoline‐N‐oxide in the Presence of Ubiquinone‐3 in Saccharomyces cerevisiae
Authors: BURGER Gertraud
Published: March 2005

2‐Heptyl‐4‐hydroxyquinoline‐N‐oxide (HpHOQnO) binds to oxidized mitochondria of Saccharomyces cerevisiae with a dissociation constant of 5 × 10−8 M and with a ratio of 1 mol per mol cytochrome b.

After addition of 171 μM ubiquinone‐3 to oxidized mitochondria, 0.3 mol HpHOQnO bind to 1 mol cytochrome b. The binding strength of the inhibitor is not essentially affected.

Reduction of mitochondria with succinate results in a strong decrease of the HpHOQnO binding. The dissociation constant could not be calculated on the basis of the applied method.

On the basis of the inhibitory effect of HpHOQnO on reduced mitochondria, a dissociation constant of 5 × 10−6 M is calculated.

The respiratory electron flow to oxygen is about 100 times as sensitive to HpHOQnO as the electron flow to cytochrome c.

Ubiquinone‐3 reverses the inhibition by HpHOQnO and stimulates the electron flow.

The different influence of ubiquinone‐3 in binding and in inhibition experiments is discussed as a result of redox conditions.
The Effect of Chemical Modification of the CCA End of Yeast tRNAPhe on Its Biological Activity on Ribosomes
Authors: KRUSE Torben A. , SIBOSKA Gunhild E. , SPRINZL Mathias , CLARK Brian F. C.
Published: March 2005

Yeast tRNAPhe containing 2‐thiocytidine (s2C) at position 75 was alkylated specifically at this residue. The biological activities of alkylated and native tRNAPhe were compared in an Escherichia coli protein‐synthesizing system in vitro. The alkylated tRNAPhe proved to be active in all steps involved in the elongation phase but the rate of the peptide transfer reaction was somewhat lower when the alkylated tRNAPhe acted as an acceptor of peptidyl residues as compared to native tRNAPhe. These results raise the possibility for attaching spectroscopic or affinity labels at the s2C‐75 residue of tRNAPhe without impairing the activity of the tRNA.
Phenylalanyl‐tRNA, Lysyl‐tRNA, Isoleucyl‐tRNA and Arginyl‐tRNA Synthetases
Authors: FREIST Wolfgang , CRAMER Friedrich
Published: March 2005

Ten analogs of ATP have been tested in the ATP/PPi exchange reaction of Phenylalanyl‐tRNA, lysyl‐tRNA, isoleucyl‐tRNA and arginyl‐tRNA synthetases from baker's yeast. Three compounds are substrates for Phenylalanyl‐tRNA, seven for lysyl‐tRNA, two for isoleucyl‐tRNA and five for arginyl‐tRNA synthetase. Their Km and V values have been determined. No analog was an inhibitor.

(3′‐dATP), 3′‐Deoxyadenosine 5′‐triphosphate which is an inhibitor of the four enzymes in the aminoacylation reaction, becomes a good substrate in the PPi exchange. Additionally lysyl‐tRNA synthetase accepts two analogs with modifications at position 6 of the purine and three analogs modified at the ribose moiety as substrates in the PPi exchange, whereas these compounds are inactive or inhibitors in the aminoacylation reaction.

In general the enzymes are less specific in the ATP/PPi exchange and the results indicate a more sophisticated proof of the nucleotide moiety upon aminoacylation. This could occur with the amino‐acyladenylate intermediate as well as with any other intermediate.
A Bifunctional Enzyme from Glyoxysomes
Authors: FREVERT Jürgen , KINDL Helmut
Published: March 2005

Enoyl‐CoA hydratase and 3‐hydroxyacyl‐CoA dehydrogenase copurified when extracts from cotyledons of 5‐day‐old cucumber seedlings were fractionated by acetone precipitation, ion‐exchange chromatography on CM‐cellulose and affinity chromatography on blue‐dextran‐Sepharose. The protein was purified 600‐fold with 16% recovery.

Bifunctionality of the protein was substantiated by exactly coinciding activity profiles upon chromatography on hydroxyapatite, CM‐cellulose, or blue‐dextran‐Sepharose respectively. Analysis of the purified protein using isoelectric focusing as well as native electrophoresis confirmed the presence of a bifunctional protein. A monospecific antiserum could be raised against the homogeneous protein.

Further characterization of the protein revealed that the two enzyme activities are contained in a single peptide chain of Mr 75000. An extremely alkaline isoelectric point of pH 9.8 was determined.

The bifunctional protein was localized in glyoxysomes which were shown to be the exclusive site of β‐oxidation at this stage of germination. At least 10% of the enzyme were attributable to the organelle's membrane.
Metabolically Labile Nonhistone Proteins of Chromatin
Authors: DJONDJUROV Lalju , IVANOVA Emilia , PIRONCHEVA Ganka , TSANEV Roumen
Published: March 2005

In a previous paper in this journal [Djondjurov, L., Ivanova, E. and Tsanev, R. (1979) Eur. J. Biochem. 97, 133–139], we showed that a nuclear fraction released from chromatin under a mild nuclease digestion contained an increased amount of hnRNA and the bulk of nonhistone proteins with a high metabolic rate. The present investigation has revealed that the nonhistone proteins of this fraction could be divided into three distinct metabolic groups. The first group consists of proteins with a fast turnover rate (mean half‐life 30 min) which migrate into chromatin immediately after their synthesis. These proteins are predominantly acid‐soluble and have relatively high molecular weights. The second group includes proteins which migrate to the nucleus more slowly and metabolize with a moderate turnover rate (mean half‐life 5 h). The third group contains proteins with a more conservative metabolic behaviour. In experiments with actinomycin D it was found that the bulk of the nonhistone proteins of this fraction are not real components of the chromatin but belong to the protein moiety of heterogeneous nuclear ribonucleoprotein particles associated with chromatin.
Purification and Characterization of Esterase 2B of the House Mouse, Mus musculus
Authors: LEXOW Ullrich , RONAI Adam , von DEIMLING Otto
Published: March 2005

Esterase 2B was isolated from mouse kidney and purified about 1300‐fold by ion‐exchange chromatography and repeated molecular sieve chromatography. The resulting product is apparently homogeneous by the criteria of gel electrophoresis and immunodiffusion. The molecular weight was found to be 57000. The equivalent weight (54000) was estimated by titration of the catalytic site by bis(p > ‐nitrophenyl) phosphate. The Km constants of seven 4‐nitrophenyl esters of n‐fatty acids (C2–C8) were determined. The logarithm of Km decreases linearly with increasing number of C atoms of the fatty acid moiety. Concurrently, the free enthalpy ΔG′o of the enzyme‐substrate complex increases by about 2.46 kJ/mol CH2 group. Similar results are obtained when a series of esters of 8‐hydroxyquinoline were used as substrates.
Structural Studies on the O‐Specific Polysaccharide Side‐Chains of Yersinia pseudotuberculosis, Type III, Lipopolysaccharides
Authors: GORSHKOVA Raisa P. , KOMANDROVA Nadezhda A. , KALINOVSKY Anatoly I. , OVODOV Yury S.
Published: March 2005

Lipopolysaccharide from pathogenic Yersinia pseudotuberculosis type III, strain 209, has been isolated and characterized. Lipopolysaccharide was shown to contain lipid A (30%), protein (4.4%), 2‐keto‐3‐deoxyoctonate (7.8%) and a polysaccharide component (45%), which consisted of paratose, l‐fucose, d‐mannose, d‐glucose, d‐galactose (trace), l‐ and d‐glycero‐d‐manno‐heptose, 2‐deoxy‐2‐amino‐d‐glucose and 2‐deoxy‐2‐amino‐d‐galactose residues. Molecular‐sieve chromatography on Sephadex G‐50 afforded an O‐specific polysaccharide, being hydrolysed to yield equimolar amounts of paratose, l‐fucose, d‐mannose and 2‐deoxy‐2‐amino‐d‐galactose. The chemical repeating unit of the O‐specific polysaccharide has been suggested on the basis of methylation studies and periodate oxidation data.
Heterogeneity of Antigenic‐Side‐Chain Length in Lipopolysaccharide from Escherichia coli 0111 and Salmonella typhimurium LT2
Authors: GOLDMAN Robert C. , LEIVE Loretta
Published: March 2005

Lipopolysaccharide from Escherichia coli 0111, its galE derivative when grown in galactose, E. coli 086, and Salmonella typhimurium LT2 all contain antigenic side chains and separate into more than 40 components by electrophoresis in gradients of polyacrylamide containing sodium dodecylsulfate. These components from E. coli 0111 are not interconvertible and show a heterogeneous size distribution when fractionated with Sephadex G‐200. Isoelectric focusing of this mixture in pH 3.5–10 ampholines reveals a single component, ruling out extensive charge heterogeneity.

The relative antigenic side chain lengths for the components, estimated using ratios of galactose in antigenic side chain to phosphate in the lipid‐A–core oligosaccharide region, show that the size heterogeneity is due to differences in the number of antigenic side chain units per molecule and ranges from none to over 40. Preference for molecules of specific chain lengths, especially short ones, was observed.

In contrast, the galE mutant grown without galactose does not synthesize antigenic side chains, and more than 90% of its lipopolysaccharide migrates as a single band at a position corresponding to the lowest‐molecular‐weight component from the above preparations. Lipopolysaccharide from E. coli PL2, a K12 strain lacking antigenic side chain, separates into two low‐molecular‐weight components on electrophoresis. These results confirm that the heterogeneity which we observe in lipopolysaccharide containing antigenic side chains, is due to the side chain rather than the lipid‐A –core oligosaccharide region.
Effect of Tamoxifen on Oestradiol and Progesterone‐Induced Synthesis of Ovalbumin and Conalbumin in Chick Oviduct
Authors: CATELLI Maria Grazia , BINART Nadine , ELKIK François , BAULIEU Etienne Emile
Published: March 2005

Tamoxifen, a non‐steroidal antioestrogen, did not display any oestrogenic effects biochemically or histologically in the chick oviduct when 10 mg/kg were administered to oestrogen‐withdrawn animals each day and effects were monitored during periods ranging from a few hours to 10 days.

After a single injection of oestradiol benzoate, 1 mg/kg, to oestrogen‐withdrawn animals, the relative rate of ovalbumin synthesis was significantly elevated by 3 h, and reached a maximum by ∼ 16 h. Conalbumin synthesis increased immediately after oestrogen administration, and attained a maximum by ∼ 16 h. Between 16 and 24 h there was a decrease of ∼ 50% in the rate of synthesis of the two proteins and in the levels of nuclear oestrogen receptor. Administration of tamoxifen together with oestradiol benzoate inhibited the oestrogen effect on ovalbumin and conalbumin synthesis. This effect of tamoxifen was dose‐dependent; 50% inhibition of the maximum induction of both proteins was obtained with 1 mg of tamoxifen/kg.

A series of experiments indicated that tamoxifen given after oestradiol benzoate could rapidly inhibit the oestrogenic effect, and oestradiol benzoate administered subsequent to tamoxifen could overcome the antioestrogenic effect; that is, each ligand could produce its own characteristic activity, although in both cases, at the time of administration of the second drug, the level of cytoplasmic oestrogen receptor was low.

When tamoxifen was administered during the lag period of ovalbumin induction, it decreased the entire pattern of ovalbumin synthesis, but when given later, the inhibitory effect was retarded. On the other hand, the inhibition by tamoxifen of oestradiol‐dependent conalbumin synthesis was virtually immediate. The basal level of conalbumin synthesis, already significant in withdrawn chickens (1%), was unaffected by tamoxifen, suggesting that endogenous oestrogens are not required to maintain this level of synthesis.

Progesterone‐induced increases in ovalbumin and conalbumin synthesis were not inhibited by tamoxifen. On the contrary, the simultaneous injection of tamoxifen potentiated the progesterone‐increased synthesis of both proteins. These observations were supported by histological studies showing an increased accumulation of secretory granules in the magnum of chickens treated by tamoxifen plus progesterone as compared to progesterone alone.
Demonstration of Specific Receptors of the Rough Endoplasmic Membrane for the Signal Sequence of Carp Preproinsulin
Authors: PREHN Siegfried , TSAMALOUKAS Antonis , RAPOPORT Tom A.
Published: March 2005

Evidence is given for the cotranslational processing in a wheat germ cell‐free system of carp preproinsulin to proinsulin by dog pancreatic rough endoplasmic membranes stripped of ribosomes by EDTA. Concomitantly with processing there is a translocation of the protein across the membrane as shown by the accessibility to proteases. Conversion of preproinsulin to proinsulin occurs post‐translationally only in the presence of detergent.

Stripped rough endoplasmic membranes bind a significant proportion of the cell‐free translation products synthesized in a wheat germ system in response to poly(A)‐rich RNA from carp islets. Among the bound material there is completed preproinsulin. The bound material is accessible to proteases. It is located on the cytoplasmic side of the membranes. Peptides lacking a signal sequence, such as globin or proinsulin, are not bound. The binding can be abolished by pretreatment of the rough membranes with proteases. Neither smooth endoplasmic membranes from dog pancreas nor erythrocyte plasma membranes from rabbits are able to bind signal peptides. The binding sites on the rough membranes can be saturated with cell‐free products coded by carp islet RNA and are then no longer able to process preproinsulin to proinsulin in a cotranslational assay. The amount of membranes necessary to bind a certain amount of cell‐free products post‐translationally corresponds roughly to the amount required for complete conversion of preproinsulin to proinsulin cotranslationally. The binding phenomenon occurs independently of the presence of ribosomes and is not sensitive to high ionic strength. A hydrophobic peptide (Ac‐Lys‐Phe‐Phe‐Gly‐Leu‐Nle‐NH2) did not compete with signal peptides.

The results show that stripped rough endoplasmic membranes possess specific proteincontaining receptors for signal sequences on their cytoplasmic side. The recognition of the signal by the endoplasmic membranes occurs without participation of the ribosomes. The latter are only required for the translocation of the nascent peptide into the lumen of the membrane.
Competing Pathways in the Formation of Alkyl, Alk‐1‐enyl and Acyl Moieties in the Lipids of Mammalian Tissues
Authors: MUKHERJEE Kumar D. , WEBER Nikolaus , MANGOLD Helmut K. , VOLM Manfred , RICHTER Ingolf
Published: March 2005

The distribution of radioactivity from intravenously administered cis‐9‐[1‐14C]octadecenol into various tissues of the rat was studied as a function of time. The pattern of incorporation of radioactivity into alkyl, alk‐1‐enyl and acyl moieties of the lipids in heart, lungs, liver, intestine, kidney, brain and plasma revealed that oxidation of the long‐chain alcohol and esterification of the resulting fatty acid to a wide variety of lipids are by far the most predominant reactions. Acylation of the long‐chain alcohol is observed especially in liver, which appears to be the major site of biosynthesis of wax esters. Alkylation of the long‐chain alcohol to alkoxylipids occurs in most tissues, most predominantly in the heart.
Analysis of Semliki‐Forest‐Virus Structural Proteins to Illustrate Polyprotein Processing of Alpha Viruses
Authors: KALKKINEN Nisse , SÖDERLUND Hans , JÖRNVALL Hans , KÄÄRIÄINEN Leevi
Published: March 2005

The four structural proteins of Semliki Forest virus were purified in an amount of 30–50 nmol by preparative sodium dodecylsulfate/polyacrylamide gel electrophoresis. Each protein was subjected to N‐terminal structural analysis by degradation in a liquid‐phase sequencer. About 20 residues were determined for each of the two membrane glycoproteins E1 and E2. The amino acid sequence of E1 but not that of E2 showed extensive homology to the corresponding proteins of the closely related Sindbis virus. Both E1 and E2 seem to lack a signal sequence at the N terminus, since the proportion of polar amino acids in this region deviates from the proportion in the known hydrophobic signal sequences. The envelope glycoprotein E3 and the capsid protein did not yield any significant result on Edman degradation, suggesting that they have blocked N‐terminal amino groups.