bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigations on the Activation of Bovine Prochymosin
Authors: BARKHOLT PEDERSEN Vibeke , ASBÆK CHRISTENSEN Kurt , FOLTMANN Bent
Published: March 2005

Activation of prochymosin at pH below 2.5 results in formation of the active enzyme pseudochymosin by proteolytic cleavage of the bond 27–28. Pseudochymosin is 15 amino acid residues longer than chymosin. It is the final activation product at low pH, whereas chymosin is formed by activation between pH 4 and 5. Pseudochymosin is converted to chymosin when it is brought to pH 5.5.

Our present knowledge does not allow quantitative evaluation of the possible reactions involved in formation of pseudochymosin, but the course of activation at pH 2 is in accordance with an intermolecular reaction between two zymogen molecules as the predominat reaction. We find indications of an intramolecular reaction when intermolecular reactions are prevented by immobilization of the zymogen.
Recognition of Messenger RNA in Eukaryotic Protein Synthesis
Authors: KAEMPFER Raymond , HOLLENDER Rivka , SOREQ Hermona , NUDEL Uri
Published: March 2005

Equilibrium studies of the interaction between messenger RNA and the rabbit reticulocyte initiation factor that binds methionyl‐tRNAf, eIF‐2, were performed, using filtration through nitrocellulose membranes to measure complex formation. The formation of complexes between the initiation factor and messenger RNA is shown to be consistent with a simple, bimolecular equilibrium. Binding of native, translatable 125I‐labeled globin messenger RNA to the factor is tight, with an apparent dissociation constant of approximately 5 × 10 −10 M at physiological salt concentration. This binding does not involve the poly(A) sequence at the 3′ end of globin messenger RNA, nor the 90‐nucleotide sequence adjacent to it, for poly(A)‐free derivatives of globin messenger RNA prepared by controlled, processive phosphorolysis bind the initiation factor with an affinity essentially equal to that of native globin messenger RNA. In contrast to other cases of protein‐nucleic‐acid interaction, binding of initiation factor eIF‐2 to messenger RNA is as tight at 0.15 M KCI as it is at 0.01 M KCI and is consistent with a preference for the RNA conformation existing at physiological salt concentration. 32P‐labeled bacteriophage R17 RNA, a messenger RNA that can be translated, although at relatively low efficiency, in a mammalian cell‐free system, also forms an equimolar complex with eIF‐2, but binds 10 to 15‐fold less tightly than globin messenger RNA. These results show that the eukaryotic initiation factor that binds methionyl‐tRNAf recognizes a sequence in globin messenger RNA that does not include the poly(A) moiety or most of the untranslated sequence at the 3′ end of the molecule. The data suggest a relationship between the affinity of a given messenger RNA species for initiation factor eIF‐2 and its efficiency as a template in protein synthesis.
Transport and Metabolism of Bromosulfophthalein by Isolated Rat Liver Cells
Authors: SCHWARZ Leslie R. , SUMMER Karl‐Heinz , SCHWENK Michael
Published: March 2005

Kinetics of transport and metabolism of bromosulfophthalein have been studied in isolated liver cells in a dose‐dependent manner obtaining the following results.

The disposition of bromosulfophthalein in suspensions of isolated liver cells is similar to the turnover in the whole liver. The initial maximal rate of uptake of bromosulfophthalein is 2–3 times faster than intracellular conjugation with glutathione. Conjugation proceeds to an equilibrium between intracellular substrate (bromosulfophthalein) and product (bromosulfophthalein‐glutathione conjugate) which are both transiently accumulated in the cell. Formation of bromosulfophthaleinglutathione is accompanied by an equimolar decrease of glutathione. The bromosulfophthaleinglutathione conjugate is slowly released from the cells in an energy‐dependent and saturable transport process. The maximal velocity of excretion amounts to only 6% of the maximal velocity of uptake and to 20% of the maximal velocity of conjugation. Excretion, therefore, represents the slowest step in the overall turnover.
Compartmentation and Communication in Living Systems. Ligand Conduction: a General Catalytic Principle in Chemical, Osmotic and Chemiosmotic Reaction Systems
Authors: MITCHELL Peter
Published: March 2005

Chemical reactions, like osmotic reactions, are transport processes when looked at in detail. Chemical catalysis by enzymes or catalytic carriers, and osmotic catalysis by porters, may be conceived as occurring by specific ligand‐conduction mechanisms. In chemiosmotic reaction systems, the pathways of specific ligand conduction are spatially orientated through anisotropic enzyme and catalytic carrier complexes in which the reactions of chemical group transfer occur as vectorial diffusion processes of group translocation down gradients of group potential that represent real spatially‐directed fields of chemical force. Thus, it is easier to explain biochemistry in terms of transport than it is to explain transport in terms of biochemistry.
Degradation of the DNA‐Binding Domain of Wild‐Type and i−dlac Repressors in Escherichia coli
Authors: SCHLOTMANN Manfred , BEYREUTHER Konrad
Published: March 2005

It has been shown that 28 transdominant mutant lac repressors which have lost operator DNA‐binding ability in vivo and in vitro, but still bind inducer and are able to form tetramers (i−d repressors), could be divided into two groups by their capacity or incapacity to bind non‐specifically to the phosphate groups of the DNA backbone. All but one of 15 analysed i−d repressors with amino acid substitutions to the C‐terminal of residue 52 showed uneffected non‐specific DNA binding. All 13 tested i−d repressors with amino acid substitutions to the N‐terminal of residue 53 did not bind to double‐stranded DNA, and 11 of these repressors derived from missense mutations in the lacI gene were endogenously degraded. The degradation in vivo only affects the aminoterminal 50–60 residues producing a mutant‐specific pattern of stable repressor fragments. These fragments are tetrameric and capable of binding inducer in vivo and in vitro. The proteolytic attack presumably takes place during synthesis of the i−d repressors, since the resulting fragments are stable, both in vivo (as shown by a pulse‐chase experiment) and in vitro. The proteolysis in vivo depends on the growth conditions of the bacteria and is higher in cells grown in minimal media than in rich media. Wild‐type repressor is only susceptible to limited proteolysis in cells grown in minimal media but not in cells grown in rich media. The results suggest that the majority of the sequence alterations before residue 53 in missense mutant i−dlac repressor proteins affect the three‐dimensional structure of the amino‐terminal DNA‐binding domain of the repressor protein, making it susceptible to proteolytic attack by one or several intracellular proteases.
Expression of External‐Surface Membrane Proteins in Differentiated and Undifferentiated Mouse Neuroblastoma Cells
Authors: ZISAPEL Nava , LITTAUER Uriel Z.
Published: March 2005

Murine neuroblastoma cultures were labeled externally with the cationic reagent N,N,N‐[3H] ‐trimethylamino‐β‐alalyl‐N‐hydroxy‐succinimide ester ([3H]Me3N‐βAla‐NSuc) or with 125I/lactoperoxidase. The cells were labeled in the logarithmic and confluent growth phases as well as in a highly differentiaed state following treatment with 2% dimethylsulfoxide. The labeled exterior membrane proteins were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Major changes in the exterior membrane proteins were observed during maturation and differentiation of the cells. Most of these changes were clonal‐specific, while others were common to several clones. Two proteins of Mr 55000 and 65000 were labeled by both 125I/lactoperoxidase and Me3N‐[3H]‐βAla‐NSuc. The level of labeling was dependent on the clonal lines used and the state of the cell maturation. A group of proteins displaying a molecular weight between 150000 and 200000 was found to be related to the transition of a culture from logarithmic to confluent growth phases. An additional protein, with an apparent molecular weight of 95000, was common to differentiated cells of the two inducible clones used. In general the maturation of logarithmic phase cells into confluent cells resulted in a less complex electrophoretic distribution of the pattern of labeling. After dimethylsulfoxide treatment, further reduction in the complexity of the externally labeled proteins was observed.
On the Secondary and Tertiary Structure of Murein
Authors: LABISCHINSKI Harald , BARNICKEL Gerhard , BRADACZEK Hans , GIESBRECHT Peter
Published: March 2005

X‐ray diffraction, density measurements, and stereochemical data were used in order to disclose the architecture of murein, the rigid component of almost all bacterial cell walls. Dry densities of 1.38–1.39 g/cm3 were observed for Micrococcus luteus and Staphylococcus aureus. The X‐ray data for gram‐positive (S. aureus, M. luteus) and gram‐negative (Escherichia coli) strains were almost identical, showing, in addition to some diffuse scattering with broad maxima corresponding to Bragg values of 0.22 and 0.45 nm, two sharp peaks indicating periodicities of about 0.7 and 0.94 nm. These latter periodicties were not found in whole bacteria but emerge immediately after breakage of the whole cells. In gram‐positive species a weak oriented reflex at 4.2 nm appeared, while all other reflexes remain ring‐shaped. Diffraction patterns obtained under the influence of humidity and certain metal ions showed strong variations in the position of the 0.45‐nm halo.

These data, together with stereochemical considerations, invalidate the hiterto advanced models of a chitin‐like structure of the glycan chains in murein. Instead, the following model proposed and discussed here is consistent with the experimental data. The structure is made up of layers, which in S. aureus and M. luteus are separated by about 4.2 nm. The layers are more or less randomly rotated against each other. The sugar chains run parallel to these layers and, hence, to the surface of the cell wall. They do not possess a twofold screw axis as in chitin or cellulose. Thus, the peptide strands protude from the glycan chain axis in different directions. The peptide strands seem to assume fairly rigid conformations and to be mainly responsible for the regular layer‐like arrangement of murein.
Conformational Energy Calculation on the Peptide Part of Murein
Authors: BARNICKEL Gerhard , BRADACZEK Hans , LABISCHINSKI Harald , GIESBRECHT Peter
Published: March 2005

Conformational energy calculations have been carried out on N‐acetyl‐l‐alanyl‐d‐γ‐glutamyl‐l‐lysyl‐d‐alanyl‐d‐alanine as a model of the peptide moiety of peptidoglycan. Although many conformations were of comparable energy, particular favoured conformations were selected by assuming conformational similarity between the pentapeptide and the tetrapeptide found during biosynthesis subsequent to the cross‐linking of the peptide chains in murein. The common feature of these conformations, which include the global minimum of the pentapeptide, is a ring‐shaped backbone. The global minimum is stabilised by a hydrogen bond between the ‐NH group of l‐alanine and the ‐CO group of the penultimate d‐alanine. The distance between the d‐lactyl group and the side‐chain of the diamino acid is about 1.5 nm. The ring‐like strctures will accomodate chemical modifications that have been observed in peptidoglycan. The present ring‐like structure differs considerably from the models proposed as yet. Energetically β‐pleated sheet conformations and a flat 2.27 helical structure are not favoured. Furthermore, an α helix cannot occur. The suggested new model exhibits no significant relationship to the solid state conformation of β‐lactam antibiotics.
5‐Thia‐5‐Deazaflavin, a 1e−‐Transferring Flavin Analog
Authors: FENNER Helmut , GRAUERT Rolf , HEMMERICH Peter , MICHEL Heinrich , MASSEY Vincent
Published: March 2005

By sulfur substitution of the N‐5 atom in flavins and flavocoenzymes a flavin analog is obtained, 5‐thiaflavin, which is found to be isoelectronic and isosteric with natural flavin in the fully reduced and half‐reduced states, but not in the oxidized state. Among the three ‘redox shuttles’ characterizing the flavin system, viz. upper 1e−, lower 1e− and 2e− shuttle, only the second one is retained in thiaflavin, which limits the redox activity of this system to 1e− transfer.

The structure and properties of the molecular species participating in the thiaflavin redox system are discussed in comparison with the flavin system. The corresponding chemistry of a ‘2e− flavin’, 5‐deazaflavin, has been treated in the preceding paper.

5‐Thiaflavin is found to exhibit a stable neutral radical, which is analogous to the ‘blue’ flavosemiquinone. Unlike normal flavin, where the radical is in a dismutation equilibrium, thiaflavin radical shows reversible formation of a covalent dimer, which is stable in aprotic solution and disproportionates only in water, with irreversible formation of a sulfoxide. The ultraviolet and infrared spectra of the dimer are in agreement with the structure of two 5‐thiaflavin molecules linked covalently at their 4a carbons. This corroborates the earlier hypothesis that the essential intermediate in the dismutation of normal flavin is likewise a covalent dimer.

Thiaflavin is tightly bound by apoflavodoxin. The protein catalyses the autoxidation to the radical state. Thiaflavodoxin radical is even more stable (towards further oxidation) than is the free thiaflavin radical.

The redox potential of the couple reduced thiaflavin/thiaflavin radical (sFlred/sFl) is surprisingly high. From the reversible equilibrium established with ferricyanide, sFlred+ Fe(CN)63−⇄ sFl·+ Fe(CN)64−, the standard potential of the sFlred/sFl· couple, Em at pH 7, has been estimated as + 0.38 V.
The Quaternary Structure of Bovine α‐Crystallin
Authors: SIEZEN Roland J. , HOENDERS Herman J.
Published: March 2005

Limited proteolysis of α‐crystallin with 10 different endopeptidases in vitro shows that only 5–20 residues of the carboxy termini of A and B chains are accessible to these enzymes, without disruption of the quaternary structure. The A chains are more susceptible to proteolysis in vitro than B chains, which corresponds with the degradation in vivo upon aging of α‐crystallin in the lens.

Six proteases specifically cleave off the carboxyl‐terminal pentnpcptide of all B chains, which indicates that the B (Thr170 ‐Ala171) bond, the only observed degradation site of B chains in vivo, is surface‐exposed and proteolytically very labile. None of the endopeptidases split the A (Asp151 ‐Ala152) bond, the main degradation site of A chains in vivo, but a nearby segment A156–163 iS very accessible to several enzymes. We postulate that this A151–152 bond is close to the surface of α‐crystallin and, although proteolytically stable in the native protein, it could become exposed as a result of age‐related changes in secondary and/or tertiary structure. Two minor tryptic cleavage sites in A chains of native α‐crystallin become more accessible in reassociated a‐crystallin, and particularly in pure A‐chain reaggregates, which indicates a change in secondary/tertiary structure upon dissociation‐reassociation.

Although the accessibility of B chains suggest that they are in equivalent positions in the α‐crystallin quaternary structure, the A chains exhibit various rates of proteolysis and therefore occur in more than one orientation.

The anomalously slow migration of B chain in dodecylsulfate gel electrophoresis is abolished after removal of its carboxyl‐terminal pentapeptide, Ala‐Ala‐Pro‐Lys‐Lys.
Conformational Changes in Bovine‐Liver Glutamate Dehydrogenase: a Spin‐Label Study
Authors: ZANTEMA Alt , VOGEL Hans J. , ROBILLARD George T.
Published: March 2005

A spin‐labelled analogue of p‐chloromercuribenzoate reacts specifically with glutamate dehydro‐ genase. The most marked change in the properties of the spin‐labelled enzyme is a fivefold decrease in the rate of reduction of the coenzyme by l‐glutamate and no change in the rate of oxidation by 2‐oxoglutarate. The electron spin resonance spectrum is a sensitive probe for the conformational state of the enzyme. Spin‐labelled glutamate dehydrogenase in tlie presence of saturating concentrations of NADPH and 2‐oxoglutarate or l‐glutamate shows a complete conformational change while in the presence of NADP+ and 2‐oxoglutarate only half of the protomers have changed conformation. The conformational change upon addition of NADPH to the spin‐labelled glutamate dehydrogenase in the presence of 2‐oxoglutarate happens in a concerted way between 20 and 80% saturation with NADPH. One of the conformations is favoured by the activator ADP while the other is favoured by the inhibitor GTP.
Magnetic‐Resonance Studies of the Geometry of Bound Substrate, Coenzyme and Activator on Bovine‐Liver Glutamate Dehydrogenase
Authors: ZANTEMA Alt , DE SMET Marie‐José , ROBILLARD George T.
Published: March 2005

ADP and ATP with a spin‐label linked to the terminal phosphate are activators of glutamate dehydrogenase and bind to the same site as the activator ADP. There is hardly any interaction with the coenzyme site. Glutamate dehydrogenase can be modified with a ketone spin‐label at a site in the active centre [Andree and Zantema, (1978) Biochemistry, 17, 778 – 783]. The spin‐labelled activators interact with ketone spin‐labelled glutamate dehydrogenase in the same way as with native glutamate dehydrogenase relative to the activator site, but show a stronger binding to the coenzyme site. Upon binding to the coenzyme site a spin‐spin interaction between the ketone spin‐label and the spin‐labelled activators is observed.

Nuclear magnetic resonance studies of the linewidth of 2‐oxoglutarate and NADP+ bound to their functional sites on glutamate dehydrogenase without and with spin‐labels result in distances between the ligand nuclei and the spin‐labels. The results show that NADP+ binds in an open conformation consistent with the conformation in other dehydrogenases. The activator ADP binds in the neighbourhood of the active centre, but with very little or no overlap with tlje coenzyme site.
The Lipoxygenase of Reticulocytes
Authors: RAPOPORT Samuel M. , SCHEWE Tankred , WIESNER Rainer , HALANGK Walther , LUDWIG Peter , JANICKE‐HÖHNE Marina , TANNERT Christof , HIEBSCH Christa , KLATT Dietmar
Published: March 2005

A lipoxygenase has been purified from rabbit reticulocyte‐rich anaemic blood cells. It possesses a molecular weight of 78000 and an isoelectric point of 5.5 and contains 5% neutral sugars and two iron atoms per enzyme molecule. The lipoxygenase has proved to be identical with the inhibitors of respiratory proteins described formerly.

The actions of the lipoxygenase on linoleic acid, phospholipids, mitochondrial and erythrocyte membranes and electron transfer particles were studied. A special feature of the reticulocyte lipoxygenase is the suicidal character of its action on lipids. With electron transfer particles the reticulocyte lipoxygenase causes a loss of acid‐labile sulfur which accompanies respiratory inhibition; the strong respiratory inhibition is not exerted by soybean lipoxygenase. The reticulocyte lipoxygenase acts preferably on mitochondrial membranes as compared with cell membranes of the erythrocyte; erythrocyte cytosol moderates the action on mitochondrial membranes. Furthermore, the lipoxygenase reaction can concomitantly and irreversibly inactivate sulfhydryl enzymes as demonstrated with muscle glyceraldehyde‐3‐phosphate dehydrogenase. The occurrence of the lipoxygenase here described is restricted to reticulocytes; very low amounts were observed in bone marrow and no lipoxygenase was detectable in normal blood. During the course of an experimental anaemia the lipoxygenase is produced owing to superinduction in large amounts, which may persist for a long time since they escape inactivation.

Preliminary evidence was obtained for the occurrence of other lipoxygenases in tissues of lung, spleen, kidney and also epithelial tumours
DNA‐Dependent RNA Polymerase from the Archaebacterium Sulfolobus acidocaldarius
Authors: ZILLIG Wolfram , STETTER Karl O. , JANEKOVIĆ Davorin
Published: March 2005

Purified DNA‐dependent RNA polymerase from Sulfolobus acidocaldarius is composed of 10 different subunits, one of which is present as four copies. Their molecular weights are 122000, 101000, 44000, 32000, 24000, 17500, 13800, 11800 (four copies), 11200, 10800, summing up to a total Mr of 423500. The sedimentation velocity is 13.5 S, indicating that at 0.5 M NH4Cl the enzyme exists in the monomeric form. At pH 9.2 in cellogel electrophoresis two of the subunits migrate towards the cathode. The composition is quite different from that of a typical eubacterial RNA polymerase. Its complexity reminds one of eucaryotic RNA polymerase.

Maximal transcription of DNA from a Halobacterium halobium phage φH (φH DNA) proceeds at pH 8.5 and 75 °C. The enzyme is stable up to 75 °C and strictly requires a DNA template. φH DNA and poly[d(A‐T) · d(A‐T)] are the most efficient. The temperature dependence of the transcription rate is characteristic for the template. Actinomycin D and heparin prevent transcription, while rifampicin, streptolydigin and α‐amanitin have no effect.

During storage, even at −70°C, the enzyme loses its activity to transcribe φH DNA, whereas transcription of poly[d(A‐T) · d(A‐T)] remains unaffected.
Some Comments on the Article:
Authors: TARDIEU Annette
Published: March 2005

In their paper, K. Muller, P. Laggner, O. Glatter and G. Kostner report X‐ray scattering and density experiments on human plasma low‐density lipoprotein B, performed at different solvent densities (i.e. contrast variation method). The interpretation of the experimental data lead those authors to question the use of that method and particularly the use of the characteristic scattering functions in the study of serum lipoproteins. In the present paper it is shown that several aspects of their analysis are in fact highly questionable and that the objections raised are therefore unfounded.
Purification, Characterization and Localization of Serine Protease of Morris Hepatoma 8999
Authors: BANNO Yoshiko , MORRIS Harold P. , KATUNUMA Nobuhiko
Published: March 2005

A serine protease of hepatoma 8999, isolated in the mitochondrial fraction, was purified and crystallized. The purified enzyme was apparently homogeneous on ultracentrifugal analysis and polyacrylamide disc gel electrophoresis. The ratio of absorbance at 280 nm and 260 nm, A280/A260, was 1.90 and its absorption coefficient, A1%280 was 10.5 cm−1 estimated from dry weight measurements. Its s20, w, value was 2.23 S and its molecular weight was estimated to be 24000 ± 1000. The enzyme contained twice as much lysine, arginine and histidine as chymotrypsinogen did, but had a very similar amino acid composition to serine protease from skeletal muscle. Its isoelectric point was pH 10.6.

The substrate specificity of the enzyme was the same as that of chymotrypsin A. Its Km, and kcat, values for N‐acetyl‐l‐tyrosine ethyl ester, N‐acetyl‐l‐phenylalanine ethyl ester and N‐acetyl‐l‐tryptophan ethyl ester were 0.35 mM and 10.69 s−1, 0.38 mM and 10.7 s−1, and 0.11 mM and 11.8 s−1, respectively. Its activity was completely inhibited by phenylmethylsulfonyl fluoride and partially inhibited with tosylphenylalanine chloromethyl ketone.

The enzyme was shown to be located in different granules from the intracellular particules (light and heavy mitochondrial fraction) by sucrose density gradient centrifugation, and it was stained in mast cells of the hepatoma 8999 by the immunofluorescent technique.

Serine protease is present in different amounts in various organs of rat and the enzyme from hepatoma 8999 gave a single band that fused completely with those of the enzymes from skeletal muscle, heart, liver and kidney, respectively, on Ouchterlony double‐diffusion analysis using antiserum to the crystalline enzyme of hepatoma 8999, but the enzyme from small intestine did not react with the antiserum.
Nuclear‐Magnetic‐Resonance Study on Met‐enkephalin and Met‐enkephalin
Authors: HIGASHIJIMA Tsutomu , KOBAYASHI Junichi , NAGAI Ukon , MIYAZAWA Tatsuo
Published: March 2005

The 270‐MHz 1H and 68‐MHz 13C nuclear magnetic resonance spectra of Met‐enkephalin (Tyr‐Gly‐Gly‐Phe‐Met) and Met‐enkephalinamide are analyzed in a variety of solvents. For the dipolar form of Met‐enkephalin in (C2H3)2SO solution, significant concentration dependences are found of C‐α proton chemical shifts, indicating an aromatic ring‐current effect in molecular aggregates. An anomalous temperature dependence is observed of the amide proton chemical shift of the Met‐5 residue. Furthermore, the chemical shifts of C‐α protons of the dipolar form are found to depend appreciably on temperature. From the analyses of the temperature dependences together with concentration dependences of C‐α proton resonances, the dipolar form of Met‐enkephalin is found to be in an equilibrium of folded and extended conformations at low concentration in (C2H3)2SO solution. Solvent‐composition dependences of the amide and C‐α proton resonances and carbonyl and α‐carbon resonances of the dipolar form in 2H2O/(C2H3)2SO solution are consistent with the conformation equilibrium and the association equilibrium. The folded conformation of the dipolar form in (C2H3)2SO solution is stabilized by the intramolecular attraction between the positively charged N‐terminal group and negatively charged C‐terminal group. The presence of the folded conformation is confirmed by the measurements of Gd(III)‐induced relaxation enhancements of C‐α protons. Nuclear Overhauser effects on the dipolar form are not consistent with the predominant formation of the β‐turn structure with the intramolecular hydrogen bond (Gly‐2) C ═ O · H ─ N (Met‐5). For the dipolar form of Met‐enkephalin in 2H2O solution and for the cationic form of Met‐enkephalinamide in (C2H3)2SO solution and in 2H2O solution there is no evidence for the formation of folded conformations.
Characterization of an Adenosine Triphosphatase from Myeloblasts Infected with the Avian Myeloblastosis Virus
Authors: BANERJEE Ranajit K.
Published: March 2005

An adenosine triphosphatase (ATPase EC 3.6.1.3) was partially purified from myeloblasts of chicken infected with the avian myeloblastosis virus and some of its molecular, catalytic and immunological properties were compared with that of the ATPase purified from the virus. Both the enzymes possessed almost same electrophoretic mobility, molecular weight, s20,w value, substrate specificity, metal‐ion requirement, apparent K value and sensitivity to inhibitors and activator. Evidence also indicated immunological identity of the two enzymes. The insensitivity of this enzyme to rutamycin or ouabain and extreme sensitivity to most of the detergents, trypsin and mercurials are the remarkable properties of this enzyme.
Purification, Some Properties and the Complete Primary Structures of Two Protease Inhibitors (DE‐3 and DE‐4) from Macrotyloma axillare Seed
Authors: JOUBERT Francois J. , KRUGER Helene , TOWNSHEND Grace S. , BOTES Dawie P.
Published: March 2005

The Macrotyloma axillare plant, belonging to the Leguminosae family, is a perennial climbing or trailing herb 0.2 – 3.5 m long. The plant is indigenous to South Africa and it occurs in the warm dry northern parts of the Transvaal. It has been introduced into Australia, where the seed are used as animal food. Two protease inhibitors, DE‐3 and DE‐4, were purified from Macrotyloma axillare seed by gel filtration on Sephadex G‐50 followed by ion‐exchange chromatography on DEAE‐cellulose. They each comprise 76 amino acid residues including 14 half‐cystine residues. The complete primary structures of the two protease inhibitors have been elucidated and their sequences are 67% identical. The inhibitor specificities, the sequences, the invariant amino acid residues and the reactive inhibitor sites of protease inhibitors DE‐3 and DE‐4 resemble the corresponding properties of the Bowman‐Birk double‐headed protease inhibitor group. The cysteine residues are in similar locations to those in protease inhibitors of known structure so they are presumed to link similarly.
Nuclear‐Magnetic‐Resonance Study of the Conformation of a Dinucleotide in Solution Using the Lanthanide Probe Method
Authors: GERALDES Carlos F. G. C. , WILLIAMS Robert J. P.
Published: March 2005

The conformation of the dinucleotide adenylyl(3′‐5′)adenosine 2′‐phosphate (ApA2′p) in aqueous solution at different pH values and temperatures has been studied using the lanthanide(III) ethylenodiaminetetraacetate(EDTA) 1:1 complexes as shift and relaxation probes. The conformational analysis, based on mixing different conformations in fast exchange within the nuclear magnetic resonance time scale, agrees well with the results from vicinal coupling constants and dimerization shifts obtained for the metal‐free system. The dinucleotide exists in a temperature‐dependent and pH‐dependent conformational equilibrium between unstacked and base‐stacked forms. At neutral pH and low temperature, the stacked form predominates, and it is a predominantly right helical structure, characterized as an anti,3E, g−, g−g−, g′g′, gg, 3E, anti conformation. This situation contrasts with adenylyl(3′‐5′)adenosine (ApA), where both right and left helices contribute to the stacked form. The nucleotidyl units of the unstacked form of ApA2′p have average conformations which are very similar to those of the corresponding mononucleotides in similar conditions.
Oligosaccharide and Ganglioside Neuraminidase Activities of Mucolipidosis I (Sialidosis) and Mucolipidosis II (I‐Cell Disease) Fibroblasts
Authors: CANTZ Michael , MESSER Helgdrd
Published: March 2005

Fibroblasts cultured from the skin of patients with the genetic diseases mucolipidosis I and mucolipidosis II were found deficient in a neuraminidase specific for both an α2→3 and and α2→ 6 type of neuraminosyl linkage of sialyl oligosaccharides. Obligate heterozygotes (parents) showed an intermediate activity in mucolipidosis I, but a normal one in mucolipidosis II.

The neuraminidase activity of mucolipidosis I fibroblasts towards gangliosides, measured at pH 4.5 in the presence of Triton X‐100, was within the range of normal controls with gangliosides Gm3 and GD3, but somewhat diminished with a bovine brain ganglioside preparation. In mucolipidosis II, neuraminidase activity was markedly decreased towards the bovine brain and Gm3 ganglioside substrates, and partially deficient with ganglioside GD3.

The results suggest that in mucolipidosis I the degradation of glycoprotein‐derived sialyl oligosaccharides is impaired due to the genetic deficiency of an oligosaccharide neuraminidase, whereas the ganglioside catabolism is unaffected.

The simultaneous decrease of both oligosaccharide and ganglioside neuraminidase activities in mucolipidosis II fibroblasts is considered to be another example of the multiple lysosomal hydrolase deficiency observed by others in these cells.

Our data also suggest that the α2→3 and α2→6 oligosaccharide neuraminidase activities are genetically related, yet that they are distinct from the activities towards gangliosides possessing an α2→3 (GM3) or an α2→8 (GD3) terminal neuraminosyl linkage.
Two Chromatin Fractions with Different Metabolic Properties of Non‐histone Proteins and of Newly Synthesized RNA
Authors: DJONDJUROV Lalju , IVANOVA Emilia , TSANEV Roumen
Published: March 2005

Digestion of chromatin with micrococcal nuclease under mild conditions results in the release of a minor chromatin fraction showing an increased RNA and non‐histone protein content, a fast turnover of the non‐histone proteins and the presence of rapidly labelled heterogeneous nuclear RNA (hnRNA) with half‐life of about 20 min. Further digestion of the chromatin leads to the elimination of about 19% of the initial chromosomal DNA, thus leaving a second chromatin fraction relatively resistant to nuclease attack. This fraction has a low protein and RNA content and contains only metabolically stable non‐histone proteins. No differences in the histone complement of the two fractions was found except for a 40% deficiency of H1 in the minor fraction.
The Kinetics of ATP‐Dependent Exonuclease V from Micrococcus lysodeikticus
Authors: PALITTI Franco , VELLANTE Antonio , CERIO‐VENTURA Gabriella , FASELLA Paolo , SALERNO Costantino , WHITEHEAD Edward
Published: March 2005

The ATP‐dependent exonuclease V from Micrococcus lysodekticus shows a Michaelian relation between steady‐state velocity and the concentration of T7 DNA substrate. The Km, (expressed as a mass concentration) does not change when the T7 DNA is broken into smaller fragments by a restriction enzyme. This is interpreted to mean that the predominant process by which the exonuclease‐V–DNA complex breaks down is digestion of the entire DNA molecule rather than physical dissociation, in accord with the already known processive nature of degradation by this enzyme. The way that the V and Km, towards DNA vary with ATP and ADP concentration suggests that enzyme‐DNA complex is predominantly formed by reaction of DNA with an enzyme‐ATP complex rather than with bare enzyme.
Metabolic Carbohydrate‐Labelling of Glycolipids from Mouse Splenocytes
Authors: ROSENFELDER Günter , VAN EIJK Ronald V. W. , MÜHLRADT Peter F.
Published: March 2005

Splenic lymphocytes from CBA/J, AKR/A/J, BALB/c/A, C57/BL/6J, C3H/HeJ and C3H/Tif nu/nu mice and B lymphocyte or T lymphocyte preparations derived from CBA/J mouse spleen were cultivated in the presence of either concanavalin A, phytohemagglutinin, Salmonella minnesotaR595 lipopolysaccharide or Proteus mirabilis soluble lipoprotein. The mitogens stimulated the incorporation of [14C]galactose into acid‐insoluble cell material with the same specificity for B or T cells as that known for thymidine incorporation.

The glycolipids extracted from mitogen‐activated, carbohydrate‐labelled B or T cells were compared by thin‐layer chromatography and characteristic differences between B and T cells were noted in the ganglioside as well as in the neutral glycolipid fractions. In addition, subsets of B or T cells, namely lipopolysaccharide‐responsive or lipoprotein‐responsive B‐cell populations or nylon‐purified T cells may be recognized by characteristic neutral glycolipid bands.
The Hormonal Control of Glycogen Metabolism
Authors: FOULKES J. Gordon , COHEN Philip
Published: March 2005

Inhibitor‐1 has been shown to be phosphorylated in skeletal muscle in vivo. In normal fed animals the degree of phosphorylation was 31 ± 7% and this value increases to 70 ± 12% following an intravenous injection of adrenaline.

The results imply that the phosphorylation of inhibitor‐1 may be equally as important as the phosphorylation of phosphorylase kinase in elevating the levels of phosphorylase a. The role of inhibitor‐1 in metabolism is discussed.
DNA Polymerase β from Brain Neurons Is a Repair Enzyme
Authors: WASER Jürg , HÜBSCHER Ulrich , KUENZLE Clive C. , SPADARI Silvio
Published: March 2005

DNA polymerase β was isolated from rat cortex neurons and characterised. Its properties were strikingly similar to those of other mammalian β‐polymerases. In adult rats, this was the major DNA polymerase occurring in neuronal nuclei, which contained no α‐polymerase, 99.2%β‐polymerase and only 0.8%γ‐polymerase. Isolated neuronal nuclei of this developmental stage were shown to perform ultraviolet‐induced repair DNA synthesis in vitro. Since β‐polymerase was virtually the exclusive DNA polymerase in these nuclei it was concluded that the β enzyme was responsible for the observed DNA repair. This was further substantiated by demonstrating a virtually complete suppression of DNA repair in irradiated nuclei by 2′,3′‐dideoxyribosylthymine 5′‐triphosphate (d2TTP), a potent β‐polymerase inhibitor. However, the presence of minute amounts of γ‐polymerase in neuronal nuclei and its susceptibility to d2TTP did not allow one to rule out an ancillary role of DNA polymerase γ in DNA repair. In view of the similarity of the neuronal DNA polymerase β with all other mammalian β‐polymerases it may be speculated that the ability to perform repair DNA synthesis is not unique to the neuronal enzyme but is a general function of all β‐polymerases.
Porcine Pancreatic Lipase
Authors: BIANCHETTA Josiane D. , BIDAUD Jacqueline , GUIDONI Andréa A. , BONICEL Jacques J. , ROVERY Mireille
Published: March 2005

The single polypeptide chain of about 460 amino acids of porcine pancreatic lipase (EC 3.1.1.3) has been fragmented into five peptides by cyanogen bromide cleavage [Rovery, M., Bianchetta, J. & Guidoni, A. (1973) Biochim. Biophys. Acta, 328, 391–395]. The sequence of the first three cyanogen bromide peptides (CNI, CNII, CNIII), including a total of 234 amino acids, was fully elucidated. Automatic or manual Edman degradation was performed on the different peptides. Fragmentations of the CN peptides were accomplished by digestions with trypsin (after citraconylation or 1,2‐cyclohexanedione treatment), chymotrypsin and Staphylococcus aureus external protease. Hydrolysis of unreduced material by pepsin and thermolysin, performed in order to determine the S‐S bridge positions, provided useful overlapping peptides.

The glycan moiety of lipase is bound to Asn‐166. The non‐essential tyrosine specifically blocked by diisopropylphosphorofluoridate is Tyr‐49 in a cluster of asparagine and glutamine residues. The existence of a highly hydrophobic sequence (206–217) at the C terminus of the CNII fragment is noteworthy.
Dissociation and Aggregation of Lactic Dehydrogenase by High Hydrostatic Pressure
Authors: SCHMID Gerhard , LÜDEMANN Hans‐Dietrich , JAENICKE Rainer
Published: March 2005

As shown by earlier experiments high hydrostatic pressure affects the catalytic function of lactic dehydrogenase from rabbit muscle. In the presence of substrates denaturation occurs, whereas in the absence of substrates and −SH‐protecting reagents oxidation of sulfhydryl groups takes place [Schmid, G., Lüdemann, H.‐D. & Jaenicke, R. (1975) Biophys. Chem. 3, 90–98; (1978) Eur. J. Biochem. 86, 219–224]. Avoiding oxidation effects by reducing conditions in the solvent medium and by chelation of heavy metal ions, the remaining high‐pressure effects consist of dissociation of the native quaternary structure into subunits followed by aggregation. Both reactions are influenced by temperature and enzyme concentration. Short incubation (≤ 10 min) at pH 6.0–8.5 and pressures of 0.3–1.0 kbar causes dissociation which is reversed at normal pressure. At 5°C the activation volume is found to be ΔV≠=–62 ± 3 cm3· mol−1. Above 1.2 kbar irreversible aggregation takes place; the reaction is favoured by low temperature and decreased pH. The activation volume for the aggregation process at 5°C is ΔV≠=–97 ± 3 cm3· mol−1.

The results may be described by a reaction sequence comprising pressure‐induced dissociation of the native enzyme into its subunits followed by subunit aggregation to form inactive high‐molecular‐weight particles.
Purification of Albumin mRNA from Rat Liver
Authors: HOFER Erhard , ALONSO Angel , KRIEG Liselotte , SCHÄTZLE Ursula , SEKERIS Constantin E.
Published: March 2005

Albumin mRNA was purified from rat liver by indirect immunoprecipitation of albumin‐synthesizing polysomes. Monospecific antibodies against albumin, raised in rabbits, were added to rat liver polysomes and the antibody‐polysome complex formed was adsorbed to goat antibodies against rabbit immunoglobulins, coupled to p‐aminobenzyl‐cellulose according to Schütz et al. [Nucleic Acid Res. 4, 71–84 (1979)]. Approximately 8–10% of the polysomes were bound to the matrix. They were then eluted from the column, RNA was isolated and mRNA recovered by chromatography on oligo(dT)‐cellulose. By translation of the mRNA in vitro in a wheat germ system, it was estimated that approximately 90% of the albumin‐synthesizing mRNA was precipitated and that the albumin mRNA fraction obtained was enriched 8–10‐fold for albumin‐coding sequences. Complementary DNA was transcribed from this mRNA. Albumin‐specific cDNA was purified by hybridization of the cDNA to polysomal rat liver mRNA up to a limited rot value of 2 × 10−2 mol · 1−1· s, isolation of the double strands by hydroxyapatite chromatography and recovery of the hybridized cDNA after alkali digestion of RNA. Kinetic data obtained by hybridization of this cDNA against total cytoplasmic and purified mRNA demonstrate that more than 90% of this cDNA comprises only one sequence component. An r0t1/2 value of 2.2 × 10−3 mol · l−1· s was estimated for the immunoprecipitated mRNA fraction. This indicates already a high degree of purity of the mRNA component of this fraction, as this RNA still contains approximately 50% ribosomal RNA. By electrophoresis of glyoxal‐denatured RNA in agarose gels, a molecular weight of 780000 was found for albumin mRNA, a value which corresponds well with a sequence complexity of 880000, estimated from the kinetics of the mRNA‐cDNA hybridization. Titrations of albumin cDNA with total polysomal poly(A)‐containing RNA indicated a content of approximately 10% for albumin mRNA in this fraction. Total nuclear RNA was shown to contain approximately 0.1% albumin‐specific sequences, whereas poly(A)‐containing nuclear RNA was about 4‐fold enriched for albumin sequences.
Polymerization of the Bacterial Elongation Factor for Protein Synthesis, EF‐Tu
Authors: BECK Barbara D.
Published: March 2005

The bacterial elongation factor for protein synthesis, EF‐Tu, polymerizes into fibrils at pH 6.0. These fibrils are 0.7 μM in diameter, at least 200 μm in length, and are positively birefringent. Electron microscopic observations of negatively stained images demonstrates that the EF‐Tu fibrils consist of bundles of individual filaments, approximately 5 nm in diameter, aligned parallel to the long axis of the fibril. Polymerized EF‐Tu exchanges nucleotide rapidly and interacts with the other elongation factor, EF‐Ts. The antibiotic kirromycin induces the polymerization of EF‐Tu into fibrils and even larger structures under nonyolymerizing conditions.
Higher‐Order Structures of Chromatin in Solution
Authors: SUAU Pedro , BRADBURY E. Morton , BALDWIN John P.
Published: March 2005

Neutron scatter studies have been made on gently prepared chicken erythrocyte chromatin over a range of ionic strength. At low ionic strength the mass per unit length of the ‘10‐nm’ nucleofilament corresponds to one nucleosome per 8–12 nm and a DNA packing ratio of between 6 and 9. From the contrast dependence of the cross‐section radius of gyration of the nucleofilament the following parameters have been obtained; RgDNA, the cross‐section radius of gyration (Rg) when DNA dominates the scatter; RgP, the cross‐section Rg when protein dominates the scatter; Rc, the cross‐section Rg, at infinite contrast and α, the constant which describes the dependence of the cross‐section Rg, on contrast variation. From our understanding of the structure of the core particle, various arrangement of core particles in the nucleofilament have been tested. In models consistent with the above parameters the core particles are arranged edge‐to‐edge or with the faces of the core particles inclined to within 20° to the axis of the nucleofilament. With increase of ionic strength the transition to the second‐order chromatin structure has been followed. This gave the interesting result that above 20 mM NaCl or 0.4 mM MgCl2 the cross‐section Rg increases abruptly to about 9 nm with a packing ratio of 0.2 nucleosome/nm and with further increase of ionic strength the Rg increases to 9.5 nm while the packing ratio increases threefold to 0.6 nucleosome/nm. This suggests a family of supercoils of nucleosomes which contract with increasing ionic strength. In its most contracted form the diameter of the hydrated supercoil has been found from the radial distribution function to be 34 nm. Models for the arrangements of core particles in the 34‐nm supercoil are discussed.
The Mode of Inhibitory Action by Aphidicolin on Eukaryotic DNA Polymerase α
Authors: OGURO Mieko , SUZUKI‐HORI Chiyo , NAGANO Hiroshi , MANO Yoshitake , IKEGAMI Susumu
Published: March 2005

The mode of action by aphidicolin on DNA polymerase α from the nuclear fraction of seaurchin blastulae was studied. The inhibition of DNA polymerase α by aphidicolin was uncompetitive with activated DNA and competitive with the four deoxynucleoside triphosphates using activated DNA as a template‐primer. For truncated (residual or limited) DNA synthesis with only three deoxynucleoside triphosphates, aphidicolin inhibited the residual synthesis more strongly in the absence of dCTP than in the absence of each of the other three deoxynucleoside triphosphates. The inhibition was reversed with excess dCTP but not with the other three deoxynucleoside triphosphates. That is, aphidicolin inhibited DNA polymerase α by competing with dCTP with a Ki value of 0.5 μg/ml and by not competing with the other three deoxynucleoside triphosphates. dTMP incorporation with the activated DNA was more sensitive to aphidicolin than dGMP or dTMP incorporation with poly(dC) · (dG)12–18 or poly(dA) · (dT)12–18. Similar results were obtained for DNA polymerase α (B form) from mouse myeloma MOPC 104E.
Purification of Rat‐Liver Microsomal UDP‐glucuronyltransferase
Authors: BOCK Karl Walter , JOSTING Dorothee , LILIENBLUM Werner , PFEIL Hugo
Published: March 2005

Glucuronidation reactions catalysed by rat liver microsomal UDP‐glucuronyltransferase are differentially inducible by 3‐methylcholanthrene and phenobarbital. To elucidate the molecular basis of this functional heterogeneity the enzyme was purified from livers of rats pretreated with the inducing agents.

Using cholate solubilization, chromatography on Bio‐Gel A‐1.5m and on DEAE‐cellulose in the presence of the nonionic detergent Brij 58, two enzyme forms could be separated. Both forms were subsequently purified to apparent homogeneity by affinity chromatography on UDP‐hexanolamine Sepharose 4B. 3‐Methylcholanthrene‐inducible enzyme activity towards 1‐naphthol, 4‐nitrophenol, 3‐hydroxybenzo(a)pyrene and N‐hydroxy‐2‐naphthylamine copurified with one enzyme form (enzyme 1). In contrast phenobarbital‐inducible enzyme activity towards morphine, chloramphenicol and 4‐hydroxybiphenyl was associated with the other enzyme fraction (enzyme 2). Sodium dodecylsulfate/polyacrylamide gels showed similar molecular weights of 54000 for enzyme 1 and 56000 for enzyme 2.

The results suggest the presence of at least two forms of UDP‐glucuronyltransferase in rat liver. Factors affecting enzyme activity in purified and membrane‐bound states are discussed.
The Use of Equilibrium Density‐Gradient Ultracentrifugation in the Isolation and Characterisation of Glycoproteins with Blood Group P1 Activity from Sheep Hydatid‐Cyst Fluid
Authors: COSSEY Ailsa , DIMICHIEL Anthony , DUNSTONE John
Published: March 2005

Equilibrium density‐gradient ultracentrifugation in caesium choride and caesium sulphate has been used in the isolation and fractionation of the glycoproteins specific for blood‐group P1 from hydatid cyst fluids. The fractions obtained have distinct and systematic differences specifically related to their buoyant densities, chemical compositions and specific‐activities for group P1.

High levels of specific‐activity were maintained over a large range of chemical compositions. The peptide content varied systematically from 2.5% for the densest fraction to 37% for the least dense fraction. The amino acid composition was essentially constant over all fractions. The proportion of glucosamine decreased and the proportions of galactosamine, mannose and glucose increased with increasing peptide content of the fractions. The data presented suggest the presence of oligosaccharide side‐chains of various lengths and compositions and/or the presence of oligosaccharide side‐chains with very different chemical compositions, of which only some are associated with the specificity for group P1.

The properties of the glycoproteins from hydatid cyst fluids have been compared with those of the glycoproteins from human ovarian cysts. Although some similarities have been demonstrated there are significant differences.