bluuebunny/crossref_metadata_2025 huggingface dataset:
An Analysis of Fish Genomes by Density Gradient Centrifugation
Authors: HUDSON Alan P. , CUNY Gerard , CORTADAS Jordi , HASCHEMEYER Audrey E. V. , BERNARDI Giorgio
Published: March 2005

DNA was prepared from 33 species of fishes representing 12 of the 31 orders of Teleostei and one order of Chondrichthyes. DNA samples were investigated to determine modal and mean buoyant densities in CsCl (q0 and 〈q〉), main‐band asymmetry, intermolecular compositional heterogeneity and base composition. Modal buoyant densities ranged over 1.697–1.704 g/cm3, generally with identical or close values for DNAs from species belonging to the same order. Main bands were practically symmetrical in most cases; when present, asymmetries were on the heavy side of the band and rather modest. In most cases, compositional heterogeneities were equal to, or lower than, those of bacterial DNAs. Both the CsCl band symmetry and the low compositional heterogeneity strikingly distinguish fish DNAs from the DNAs of warm‐blooded vertebrates and indicate a major discontinuity in genome organization in the vertebrates. The overall properties of the main bands of fish DNAs are very similar to those exhibited by the light components of DNAs from mammals and birds.
Cross‐linking of mRNA to Proteins by Irradiation of Intact Cells with Ultraviolet Light
Authors: WAGENMAKERS Anton J. M. , REINDERS Rita J. , VAN VENROOIJ Walther J.
Published: March 2005

Ehrlich ascites tumor cells were irradiated with ultraviolet light to cross‐link intracellular RNAs to their tightly bound proteins. The efficiency of such cross‐linking in vivo was measured by two independent methods, namely by phenol/chloroform extraction and by isopycnic centrifugation. After short irradiation times more than 70% of the ribosomal RNAs and more than 80% of the mRNA were found to be cross‐linked to protein. The size of the cross‐linked RNAs was essentially unchanged, indicating that the irradiation does not lead to extensive fragmentation of the RNA.

Covalent poly(A)‐containing mRNA‐protein complexes were isolated by oligo(dT)‐cellulose chromatography in the presence of sodium dodecylsulphate. The complexed proteins were freed from RNA by extensive nuclease degradation and analysed by polyacrylamide gel electrophoresis. The results show that some specific proteins are associated with poly(A)‐containing RNA in vivo.
The Core Subunit Structure in RNA Polymerase Holoenzyme Determined by Neutron Small‐Angle Scattering
Authors: STÖCKEL Peter , MAY Roland , STRELL Irmtraud , CEJKA Zdenka , HOPPE Walter , HEUMANN Hermann , ZILLIG Wolfram , CRESPI Henry
Published: March 2005

The core subunit arrangement α2‐β‐β′ within DNA‐dependent RNA polymerase holoenzyme α2ββ′σ from Escherichia coli was investigated by neutron small‐angle scattering using label triangulation. The quaternary structure of multisubunit biomolecules can be studied by this new method if total reconstitution works in a quantitative way and if extensive replacement of C‐bound hydrogen (H) by deuterium (2H) is possible. A substitution of the selected subunits by their fully deuterated analogues was used for the analysis of the overall shapes of the core subunits α2, β and β′in situ and for the determination of the intersubunit centre‐to‐centre distances. The contrast between the buffer and the remaining ‘hydrogenated’ enzyme vanishes if the buffer contains 42%2H2O (matching of scattering length densities). The isotopic hybridization of the enzyme fulfils the conditions of isomorphous replacement as required: molecular functions, like enzyme activity, were completely preserved.

The orientations of the core subunits within the holoenzyme were derived by comparing theoretical and experimental pair distance distribution functions, P(r), obtained from the scattering in tensity differences of the pair‐labelled (e.g. both β and β′ labelled) and both mono‐labelled molecules by direct Fourier transformations. Additional, the subunit shapes were refined by P(r) analyses. The arrangement of the stable core structure within the holoenzyme, which contains σ as a dissociable factor, is presented in a three‐dimensional model.
The Staphylococcal Phosphoenolpyruvate‐Dependent Phosphotransferase System
Authors: SCHÄFER Angela , SCHRECKER Otto , HENGSTENBERG Wolfgang
Published: March 2005

The galactoside‐specific membrane‐bound component of the staphylococcal phosphoenolpyruvate‐dependent phosphotransferase system, enzyme IIlac, was purified to homogeneity. The purification procedure involved several extractions steps at the particulate state, followed by solubilisation with Triton X‐100. Up to this stage the biological activity of enzyme II was preserved. Isolation of the homogeneous protein involved gel filtration of the dodecylsulfate‐denatured material. An apparent molecular weight of the polypeptide chain was estimated by dodecylsulfate gel electrophoresis. The 55000‐Mr protein is visible in dodecylsulfate gels upon induction of the staphylococcal lac operon as a more intensively stained area. Antibodies against the denatured 55000‐Mr protein inhibit the mutant complementation assay of enzyme II offered as membrane fragments. This demonstrates that the 55000‐Mr protein and enzyme IIlac are identical. Polarity and the solubility of the protein in detergents are typical for an integral membrane protein.
Synthesis of Saccharomyces cerevisiae Catalase A in vitro
Authors: AMMERER Gustav , RICHTER Klaus , HARTTER Engelbert , RUIS Helmut
Published: March 2005

mRNA isolated from cells of the yeast Saccharomyces cerevisiae was translated in the cell‐free protein synthesis system from wheat germ. Catalase proteins synthesized were isolated from incubation mixtures by immunoadsorption followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. On dodecyl sulfate gels, catalase A synthesized by the wheat germ system migrates like catalase A protein synthesized in vivo. Evidence is presented that yeast catalase T and A synthesized in vivo are no glycoproteins. Synthesis of the two catalase proteins in the wheat germ system and dissimilarity of proteolytic fingerprints of the two proteins demonstrate conclusively that catalase T and A are biogenetically unrelated.
Inhibition of the Links between Electron Transfer and Proton Translocation in Mitochondria
Authors: TU Shu‐I , LAM Eric , RAMIREZ Fausto , MARECEK James F.
Published: March 2005

The mechanism by which proton extrusion is linked to electron transfer in mitochondria was investigated by means of the primary amine‐specific reagent fluorescamine, and of compounds obtained from the reaction of fluorescamine with simple amines (e.g. benzylamine) and with the mycosamine‐containing antibiotic amphotericin B. The effect of these ‘modifiers’ (i.e. fluorescamine and the acyclic amine‐fluorescamine compounds) on the activities of sites II and III of the electron transfer chain were assayed separately using specific inhibitors to block the action associated with the other site. Both types of modifiers inhibited the proton extrusion across the membrane to a significantly greater extent than the electron transfer process in both sites II and III. In contrast, the lactone derivative (or cyclic form) of the amine‐fluorescamine compounds had no significant inhibitory effect on the proton extrusion and its associated electron transfer. These results are consistent with the hypothesis that the link between proton extrusion and electron transfer in mitochondria is indirect in nature. The results show that: (a) the links involved in sites II and III are identical or very similar in nature; (b) a covalent modification of primary amino groups in the inner membrane is not essential for the expression of these differential inhibitory effects; (c) specific structural features in the amine‐fluorescamine compounds, and in the mitochondria‐fluorescamine derivatives, are crucial for the expression of the inhibitory effects. Our results contradict the ‘redox loop’ model of Mitchell, and are compatible with the proton pump concept for the linked proton translocation in oxidative phosphorylation.
Uniformity of Carbohydrate Chains within Molecular Variants of Rat α1‐Fetoprotein with Distinct Affinity for Concanavalin A
Authors: BAYARD Bernard , KERCKAERT Jean‐Pierre
Published: March 2005

Three rat α1‐fetoprotein fractions were obtained by chromatography on concanavalin‐A‐Sepharose : one non‐reactive, one weakly reactive and one reactive to concanavalin A. The non‐reactive and reactive variants were found to vary in the structure of their carbohydrate chains while the conformation of the weakly reactive form may modulate the accessibility of these chains to the lectin.

N‐Glycosidically linked glycans from unfractionated α1‐fetoprotein were isolated and chemically characterized. Particular attention was paid to develop sensitive methods based upon hydrazinolysis, quantitative re‐N‐acetylation of glycans with [14C]acetic anhydride and thin‐layer chromatography of labeled compounds. With the aid of these methods two main kinds of glycans (1a and 2a) were obtained and fractionated on concanavalin‐A‐Sepharose into non‐reactive (1a) and reactive (2a) molecules. Moreover it was demonstrated that each α1‐fetoprotein variant contained either two glycans 1a or two glycans 2a, not randomly, but a pair of the identical carbohydrate chains at the two glycosylation sites.
Neocarratetraose 4‐O‐Monosulphate β‐Hydrolase from Pseudomonas carrageenovora
Authors: McLEAN Maitland W. , WILLIAMSON Frank B.
Published: March 2005

Neocarratetraose 4‐O‐monosulphate β‐hydrolase has been enriched 73‐fold from the cell‐free extract of Pseudomonas carrageenovora. This enzyme catalyses the hydrolysis of the β‐glycosidic linkage of neocarratetraose 4‐O‐monosulphate [3,6‐anhydro‐α‐d‐galactopyranosyl‐(1–>3)‐1‐d‐galactopyranosyl‐(1–>4)‐3,6‐anhydro‐α‐d‐galactopyranosyl‐(1–>3)‐ d‐galactose 4‐O‐sulphate]. The products of hydrolysis are respectively neocarrabiose [3,6‐anhydro‐α‐d‐galactopyranosyl‐(1–>3)‐ d‐galactose] and neocarrabiose 4‐O‐sulphate [3,6‐anhydro‐α‐d‐galactopyranosyl‐(1–>3)‐ d‐galactose 4‐O‐sulphate]. During purification the χ‐carrageenase and 4‐sulphatase activities present in the bacterial extract were resolved from the title enzyme. From dodecylsalphate/polyacrylamide gel electrophoresis the enzyme has a molecular weight of 94000. Calcium stimulated the activity, Km 0.50 mmol dm−3. Optimal activity was found at pH 7.0 and the Km determined for the nominal substrate was 68 μmol dm−3.

[14C]Neocarratetraose 4‐O‐monosulphate was prepared from [14C]carrageenan (Chondrus crispus) and utilized in a radiochemical assay for neocarratetraose 4‐O‐monosulphate β‐hydrolase. Synthesis of [1‐3H]neocarratetraitol 4‐O‐[35S]monosulphate yielded another substrate. This alditol was hydrolysed to yield a single radioactive species, thus supporting the position of sulphation of neocarratetraose 4‐O‐monosulphate deduced from 13C NMR studies. Neocarratetraose 4‐O‐disulphate [3,6‐anhydro‐α‐d‐galactopyranosyl‐(1–>3)‐4‐O‐sulphato‐β‐d‐galactopyranosyl‐(1–>4)‐3,6‐anhydro‐α‐d‐galactopyranosyl‐(1–>3)‐d‐galactose 4‐O‐sulphate] was not hydrolysed by the purified glycosidase. Tentative evidence for transglycosylation was obtained with both monosulphated substrates. A pathway is described for the sequential degradation of neocarratetraose 4‐O‐disulphate to neocarrabiose.
The Physicochemical Properties of the Galactose‐Specific Lectin from Momordica charantia
Authors: MAZUMDER Tripti , GAUR Namita , SUROLIA Avadhesha
Published: March 2005

A lectin has been isolated from Momordica charantia seeds and purified to homogeneity by affinity adsorption on a cross‐linked arabinogalactan column and subsequent elution with 0.1 M lactose. In sedimentation velocity experiments an s°20,w value of 6.5 S was obtained for the native lectin and of 2.7 S for its subunits obtained by dissociation with 0.1% sodium dodecyl sulphate in the presence of 0.1% 2‐mercaptoethanol. This lectin has a molecular weight of 120 000, is tetrameric in nature with two pairs of non‐identical subunits of molecular weights 28 000 and 30 000. It is a glycoprotein containing 10% hexoses (2% glucose and 8% mannose) and 2.5% hexosamine, all glucosamine. It has two binding sites per molecule and has Ka values of 4.1 × 104 M−1 and 1.8 × 104 M−1 at 5° C and 25° C respectively for p‐nitrophenyl β‐d‐galactopyranoside with ΔH° and ΔS° values of −28.3 kJ mol−1 and −12.3 J mol−p1 K−1 respectively. Hemagglutinating activity of M. charantia lectin is inhibited by galactose and other carbohydrates containing the galactopyranosyl residue. From the chemical modification studies tryptophan and tyrosine residues are found to be important for the carbohydrate binding activity of M. charantia lectin.
The Activation of Ox‐Brain NAD+‐Dependent Isocitrate Dehydrogenase by Magnesium Ions
Authors: WILLSON Vivian J. C. , TIPTON Keith F.
Published: March 2005

Two independent methods were used to assess the dependence of the activity of ox brain NAD+‐dependent isocitrate dehydrogenase on the concentration of magnesium ions. The results indicated the complex between magnesium and isocitrate to be the true substrate for the enzyme. Free isocitrate is neither a substrate nor an inhibitor of the enzyme but free magnesium ions inhibit competitively with respect to the magnesium‐isocitrate complex. The inhibition of the enzyme by ATP and citrate appears to be largely explicable in terms of their effects on the concentration of the complex between Mg2+ and isocitrate. The dependence of the activation of the enzyme by ADP on the concentration of magnesium ions suggests that free ADP, rather than its, complex with Mg2+, is the activator.
The Electrochemical Proton Gradient in Mycoplasma Cells
Authors: BENYOUCEF Mohammed , RIGAUD Jean‐Louis , LEBLANC Gerard
Published: March 2005

The electrochemical proton gradient, generated upon glycolysis by Mycoplasma mycoides var. Capri cells has been determined. The components, the transmembrane pH gradient, ΔpH, and the membrane potential, ΔΨ, were measured using several methods.

The determination of the ΔpH was conducted by measuring the transmembrane distribution of weak acids (acetate and butyrate) and of a weak base (methylamine), using flow dialysis and filtration techniques. The transmembrane electrical potential was determined from the distribution of the lipophilic cation Ph3MeP+ and of Rb+ or K+ in the presence of valinomycin. At extracellular pH 7.2, glycolyzing Mycoplasma cells maintain an internal pH more alkaline (0.5 pH unit) than that of the milieu and an electrical potential of–85 mV, interior negative. The in M. mycoides var. Capri cells is thus about–115 mV.

When the external pH was altered from 7.7 to 5.7, ΔΨ decreased from–90 mV to–60 mV. On other hand although the internal pH decreased, ΔpH was found to increase from 0.2 to 1.0 pH unit. Since the changes in ΔΨ were largely compensated by the changes in ΔpH, remained practically constant at about–115 mV throughout the pH range tested.

Finally, inhibition of ΔpH by N,N′‐dicyclohexylcarbodiimide, carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone or nigericin confirmed that chemiosmotic phenomena contribute to energy transduction across the membranes of M. mycoides var. Capri cells.
Gradation of the Magnitude of the Electrochemical Proton Gradient in Mycoplasma Cells
Authors: BENYOUCEF Mohammed , RIGAUD Jean‐Louis , LEBLANC Gerard
Published: March 2005

The results presented show that in Mycoplasma mycoides var. Capri, regulation of glucose uptake by its non‐metabolizable analogue methyl α‐d‐glucoside, can be used to control intracellular ATP content. This in turn leads to a control of the rate of proton extrusion catalysed by the Mg2+‐dependent ATPase () and the respective amplitudes of the components of . When Mycoplasma cells are incubated with 10 mM methyl α‐d‐glucoside, the amplitude of of the electrical potential ΔΨ and of the chemical gradient ΔpH become continuous functions of external glucose concentration within the limits of the non‐energized and fully energized states.

Analysis of the relationships between graduated amplitudes of ΔΨ, ΔpH and () show that the primary form of energy stored by a generator is the electrical potential.
Interaction of Clonidine and Clonidine Analogues with α‐Adrenergic Receptors of Neuroblastoma × Glioma Hybrid Cells and Rat Brain
Authors: ATLAS Daphne , SABOL Steven L.
Published: March 2005

Clonidine and several analogues of clonidine are shown to be useful probes for α2‐adrenergic receptors in a comparative study of ligand binding and inhibition of adenylate cyclase. The α‐adrenergic properties of a new potential probe, N‐(4‐hydroxyphenacetyl)‐4‐aminoclonidine hydrochloride, are described. [3H]Clonidine binds to α‐receptors of NG108‐15 neuroblastoma × glioma hybrid cell membranes with Kd values of 1.7 and 33 nM for putative high‐affinity and low‐affinity sites, respectively. p‐Aminoclonidine and hydroxyphenacetyl aminoclonidine displace [3H]clonidine from the high‐affinity sites with Kd values of 2.3 and 5.8 nM, respectively. Rat brain α2‐receptors also exhibit high affinity toward clonidine, p‐aminoclonidine, and hydroxyphenacetyl aminoclonidine, as determined by displacement of specifically bound [3H]clonidine. Clonidine, p‐aminoclonidine, and hydroxyphenacetylaminoclonidine elicit modest inhibition (up to 24%) of NG108‐15 adenylate cyclase by interaction with α2‐receptors (Kd,app 300, 50, and 130 nM, respectively); these compounds also partially reverse the inhibition elicited by (–)‐norepinephrine.

Components of the adenylate cyclase assay mixture, particularly ATP, GTP, sodium ions, and a nucleoside‐triphosphate‐regenerating system, decrease the high‐affinity [3H)clonidine binding to NG108‐15 membranes; in the presence of these components, α‐receptors possess only low affinity (Kd 43 nM) for [3H]clonidine. These results are consistent with the concept that certain components required for the receptor‐mediated inhibition of adenylate cyclase convert α2‐receptors from a high‐affinity inactive state to a low‐affinity active state.
Ca2+‐Binding Studies of the Phosphoprotein from Rat‐Incisor Dentine
Authors: ZANETTI Margherita , de BERNARD Benedetto , JONTELL Mats , LINDE Anders
Published: March 2005

Rat incisor dentine was demineralized and extracted with 0.25 M EDTA containing protease inhibitors. The extract was purified by chromatography on DEAE‐cellulose and sulfonated polystyrene. The Ca2+‐binding properties of the phosphoprotein were studied by dynamic dialysis and by using a Ca2+‐selective electrode. Two different binding sites were detected with Kd= 0.9 × 10−7 M and 1.1 × 10−5 M and displaying a Ca2+‐binding capacity of 127 and 176 mol bound Ca2+/mol protein, respectively, assuming a molecular weight of 30 000. Upon enzymatic dephosphorylation of the phosphoprotein, the highest affinity sites disappeared and those with the lowest affinity were reduced The optimum for Ca2+ binding by the phosphoprotein occurred at pH 8.2. The specificity of the Ca2+ ion interaction with the phosphoprotein was investigated by studying the competitive nature of other divalent and monovalent cations. It was found that Ca2+ ions were to a large extent displaced from the phosphoprotein by other cations in physiological concentrations.
Lipoprotein Lipase: Modification of Its Kinetic Properties by Mild Tryptic Digestion
Authors: BENGTSSON Gunilla , OLIVECRONA Thomas
Published: March 2005

Mild tryptic digestion of lipoprotein lipase cleaved its polypeptide chain in the middle, but the pieces were held together by disulphide bonds. The modified enzyme retained its ability to bind to heparin and to anionic detergents and on gel filtration it eluted in a similar position as the native enzyme does. It also retained essentially full activity against soluble substrates. Thus, the overall physico‐chemical properties of the enzyme were not markedly changed and its active site remained intact after treatment with trypsin. The activity of the modified enzyme against long‐chain acylalycerols and phospholipids was, however, much reduced. With some emulsions, the decreased activity could be ascribed in part to a decreased ability of the modified enzyme to bind to the emulsion droplets. Under these conditions apolipoprotein CII partially restored both binding and activity. With a lysophosphatidylcholine‐triacylglycerol emulsion the modified enzyme adsorbed almost completely to the emulsion droplets, but its activity was nonetheless very low. Thus, tryptic cleavage interfered with the ability of the enzyme to become properly orientated at the interface. With this emulsion apoliproprotein CII enhanced the activity of the native enzyme fourfold but the activity of the trypsin‐treated enzyme 30‐fold, so that the activity of the modified enzyme became almost as high as that of the native enzyme. It is concluded that apolipoprotein CII enhances the activity of lipoprotein lipase by stabilizing an effective orientation/conformation of the enzyme at the interface. This effect became more marked when the ability of the enzyme itself to attain this form had been reduced by tryptic cleavage.
Succinic Semialdehyde Dehydrogenases of Escherichia coli
Authors: DONNELLY Mark I. , COOPER Ronald A.
Published: March 2005

Two physically and genetically distinct forms of succinic‐semialdehyde dehydrogenase have been identified in Escherichia coli B. The two enzymes could be separated by filtration on Sephadex G‐150 and their apparent molecular weights were 200000 and 97000. The larger enzyme, which is specific for NADP, is induced by growth on γ‐aminobutyrate. Its induction is highly coordinated with that of γ‐aminobutyrate:2‐oxoglutarate transaminase, the enzyme which initiates degradation of γ‐aminobutyrate. The smaller enzyme, which is induced by growth on p‐hydroxyphenylacetate, has been purified to 98% homogeneity by affinity chromatography in conjunction with conventional methods. Under standard assay conditions this enzyme acts preferentially with NAD but reduces NADP at 15% of the rate observed for NAD, primarily because of a difference in Km. Apparent Km values for succinic semialdehyde and NAD are 13.3 ± 1.3 μM and 33.7 ± 1.4 μM, respectively. The subunit molecular weight was estimated to be 55000, indicating that the native enzyme is dimeric. The NAD‐dependent succinic‐semialdehyde dehydrogenase is aiso induced by exposure of cells to exogenous succinic semialdehyde, a treatment which has no effect on the amount of other enzymes of p‐hydroxyphenylacetate or γ‐aminobutyrate metabolism. Apparently the gene for this enzyme functions independently from the genes encoding the other enzymes of p‐hydroxyphenylacetate degradation. As a consequence of its induction mechanism. this NAD‐dependent dehydrogenase is also present in extracts of E. coli B grown with γ‐aminobutyrate as sole nitrogen source, in addition to the NADP‐specific enzyme involved in γ‐aminobutyrate metabolism. Presumably the NAD‐dependent enzyme is gratuitously induced by succinic semialdehyde formed by transamination of γ‐aminobutyrate.
An Analysis of Activity Determinations in a Series of Coupled Redox Reactions with Special Reference to Hydrogenase
Authors: van DIJK Cees , MAYHEW Stephen G. , VEEGER Cees
Published: March 2005

In this paper a spectrophotometric method is described to study the effects of pH and redox potential on the hydrogen production activity of hydrogenase, which will be applied in the following paper.

The theoretical part of this paper gives the mathematical description of system in which a redox mediator is continuously reduced by a redox system, in a non‐rate‐limiting way and continuously enzymatically oxidized, in a rate‐limiting way. Due to the changes in redox potential during the course of the reaction the ratio reduced oxidized redox mediator declines gradually. It is possible to relate the changes in this ratio to the actual amount of product formed.

The calculation of product formation has been applied to the production of hydrogen by hydrogenase in a system with a redox mediator, whose reduced form functions as the ultimate electron donor for hydrogenate, and the dithionite/(bi)sulphite redox couple as reducing system. The changes in the ratio reduced/oxidized redox mediator as a function of pH and redox potential, followed spectrophotometrically, have been related to the hydrogen production activity of hydrogenase.

The second part of the paper deals with the analysis of the theory and describes the conditions under which the method can be used with methyl viologen as redox mediator.

The conditions for determining the pH dependence of the hydrogen production activity manometrically are also described.
Evidence for the Presence of Two Kinetically Distinct Active Forms of Ribonuclease T2
Authors: YASUDA Toshihiro , INOUE Yasuo
Published: March 2005

A kinetic study has been made of the RNase‐T2‐catalyzed transphosphorylation of two adenine nucleotides, adenylyl(3′‐5′)uridine and adenosine 3′‐(1‐naphthyl)phosphate. Rates were measured at pH values ranging from 2.6 to 8.2. The observed shape of the plot of log kcat against pH for both the natural and the synthetic substrate suggests that there exist two parallel rate‐determining pathways. Two pH‐independent rate constants and three ionization constants of the enzyme‐substrate complexes were obtained by nonlinear iterative least‐squares analysis. Detailed interpretation of the pH profiles was carried out and it is proposed that carboxylate anion is likely to deprotonate O‐2′ at 4 < pH < 6, but at pH > 6 an alternative general base would play this role more effectively than the carboxylate group. Another base in its protonated cationic form is responsible for the general acid catalysis.
Mannosidosis: Isolation and Comparison of Mannose‐Containing Oligosaccharides from Gingiva and Urine
Authors: DANIEL Peter F. , DEFEUDIS Donna F. , LOTT Ira T.
Published: March 2005

Excessive gingival hyperplasia with storage of mannose‐rich oligosaccharides appears to be a unique feature present in a 31‐year‐old mannosidosis patient. Fractionation and analysis of the gingiva established the presence of (Man)2GlcNAc (2.2 μmol/g), (Man)3GlcNAc (3.5 μmol/g), (Man)4GlcNAc (2.8 μmol/g) and higher oligomers (Man)5GlcNAc–(Man)8GlcNAc (0.5 μmol/g); (Man, mannose; GlcNAc, N‐acetylglucosamine). Eight characteristic oligosaccltarides were isolated from the patient's urine by thin‐layer chromatography. The most abundant was (Man)2GlcNAc (161–558 μmol/l); decreasing amounts of higher homologues up to a dekasaccharide. (Man)9GlcNAC (1–4 μmol/1) were also present. In contrast to urine, in which the trisaccharide was predominant, tetrasaccharides and pentasaccharides were more abundant in gingiva.
Dexamethasone‐Binding Proteins in Cytosol and Nucleus of Rat Thymocytes
Authors: TSAWDAROGLOU Nikos G. , GOVINDAN Manjapar V. , SCHMID Wolfgang , SEKERIS Constantine E.
Published: March 2005

Dexamethasone‐binding proteins from the cytosol and the nucleus of rat thymocytes were analyzed by ionexchange chromatography on DEAE‐cellulose. Three dexamethasone‐binding proteins were revealed in cytosol one in the flow‐through (DE‐1) and two (DE‐2 and DE‐3) eluting from the column with 0.13 M and 0.23 M NH4Cl, respectively. In nuclear extracts only one receptor fraction, present in the flow‐through, could be detected.

By a combination of affinity chromatography on Cl‐Sepharose to which dexamethasone 21‐methanesulfonate was linked through a disulfide bond and DEAE‐cellulose chromatography, three receptor proteins were highly purilicd from cytosol, with molecular weights of 45000, 72000 and 90000 and one from nuclear extracts with molecular weight of 72000. Antibodies to the 45000‐Mr and 90000‐Mr proteins were elicited in rabbits. The antibodies to the 45000‐Mr protein cross‐react with the 90000‐Mr protein. Similarly, the antibodies to the 90000‐Mr protein cross‐react with the 45000‐Mr protein. Antibodies to either of the two proteins immunoprecipitate 60–70% of the dexamethasone‐binding activity of rat thymus cytosol. Immunoaffinity chromatography of cytosol and nucleosol on columns of Sepharose linked to the IgG against either the 45000‐Mr or the 90000‐Mr protein leads to binding of these proteins on the columns but not of the 72000‐Mr species. Two nuclear polypeptides with molecular weights of 36000 and 38000 remain attached to the immunoaffinity column; these polypeptides may represent degradation products of the cytoplasmic receptor upon entrance into the nucleus. Antibodies against two dexamethasone‐binding proteins from rat liver cytosol immunoprecipitate the 45000‐Mr and 90000‐Mr cytosol receptors from rat thymus.
Synthesis and Biological Properties of Enzyme‐Resistant Analogues of Substance P
Authors: SANDBERG Bengt E. B. , LEE Chi‐Ming , HANLEY Michael R. , IVERSEN Leslie L.
Published: March 2005

Six analogues of substance P were synthesized with the aim of developing a metabolically stable peptide that would retain the biological activity of substance P. A recently isolated and characterized substance‐P‐degrading enzyme from human brain with a high specificity for substance P described in the preceding paper in this journal was used as a model for the enzymatic inactivation of substance P. The synthetic analogues were designed to protect the peptide bonds on the carboxyl side of residues 6, 7 and 8 of substance P, which represent the sites of cleavage by substance‐P‐degrading enzyme. To test for increased enzymatic resistance, the analogues were incubated with the enzyme, the digests were separated on a high‐performance liquid chromatography reverse‐phase column and the peptide fragments were collected and identified by amino acid analysis. Of the analogue, described, an heptapeptide analogue of residues 5–11. < Glu‐Gln‐Phe‐MePhe‐MeGly‐Leu‐MetNH2, showed almost complete resistance both towards substance‐P‐degrading enzyme and to degradation on exposure to rat hypothalamic slices. This analogue was about a third as potent as substance P in competing for binding to receptor sites for this peptide in rat brain membranes and a tenth as potent in eliciting contractions of the guinea pig ileum. The peptides were synthesized using the solid‐phase technique with polydimethylacrylamide as a solid support and the coupling was achieved with pre‐formed symmetrical anhydrides in dimethylacetamide.

Fluorenylmethyloxycarbonyl was used as an α‐amino protecting group in conjunction with t‐butyloxycarbonyl as an ɛ‐amino protecting group. Ammoniolytic cleavage from the resin was followed by stepwise elution from an SP‐Sephadex column, deprotection with trifluoroacetic acid and chromatography on a Bio‐Rex 70 ionexchanger. The peptides were finally purified on a semi‐preparative reverse‐phase column.
Distinction between Two Subpopulations of β1‐Adrenergic Receptors in Human Adipose Cells
Authors: JACOBSSON Bo , VAUQUELIN Georges , WESSLAU Christian , SMITH Ulf , STROSBERG A. Donny
Published: March 2005

The β‐adrenergic receptors in human adipose membranes were identified by the specific and saturable binding of the β‐adrenergic antagonist (–)‐[3H]dihydroalprenolol. The total number of sites in control membranes was 0.32 ± 0.03 pmol/mg protein and the equilibrium dissociation constant for binding (Kd) was 2.6 nM and 2.5 nM as determined by Scatchard analysis of experiments on equilibrium binding and kinetics respectively. The β1‐adrenergic nature of the receptors was derived from the order of potencies of β‐adrenergic agonists (isoproterenol > norepinephrine > epinephrine) to complete with (–)‐[3H]dihydroalprenolol for binding. Studies of saturation binding, kinetics and competition binding revealed the presence of a single class of β1‐adrenergic receptors.

Prolonged incubation of human adipose cells in the presence of (–)‐norepinephrine decreases the lipolytic response to β‐adrenergic agonists, and reduces by 50% the concentration of β‐adrenergic receptors. The Kd values for (–)‐[3H]dihydroalprenolol and the β‐adrenergic agonists remain unchanged. Catecholamines also produce a rapid conformational change of approximatively 50% of the receptors in control membranes as revealed by their increased sensitivity towards inactivation by the alkylating agent N‐ethylmaleimide. This inactivation process is not observed in desensitized membranes, which indicates that desensitization and inactivation by agonists plus N‐ethylmaleimide affect the same receptor population. The β‐adrenergic receptors in human adipocytes can thus be divided into two subpopulations on the basis of the different consequences of their interaction with agonist molecules.
Dihydrolipoamide Dehydrogenase Component of the Pyruvate Dehydrogenase Complex from Escherichia coli K12
Authors: SCHMINCKE‐OTT Eva , BISSWANGER Hans
Published: March 2005

The regulation of the biosynthesis of dihydrolipoamide dehydrogenase is dependent on the biosynthesis of the pyruvate dehydrogenase complex. The gene coding for the dihydrolipoamide dehydrogenase appears to be included in the regulation of the pyruvate dehydrogenase operon. Possibly a secondary promoter is inserted.

Dihydrolipoamide dehydrogenase was purified in its free form using a dihydrolipoamide‐agarose affinity column and avoiding denaturating conditions.The enzyme shows complete cross‐reactivity with antibodies against the pyruvate dehydrogenase complex and has a higher specific activity than any preparations described thus far. The transition state activation energy of the catalytic activity is smaller for the complex‐bound enzyme than that found for the free enzyme. In its complexed form, the enzyme also proves to be somewhat more stable under alkaline conditions.

The reaction catalyzed by the dihydrolipoamide dehydrogenase shows the behaviour of a ping‐pong mechanism. The Michaelis constants for the substrates NAD and dihydrolipoamide found with the free enzyme are about four times those observed with the enzyme integrated into the native complex. The catalytic reaction of both forms of the dihydrolipoamide dehydrogenase is inhibited by NADH. The mechanism of this inhibition cannot simply be explained by a product inhibition. Rather the further reduction of the catalytically active, half‐reduced enzyme form to the catalytically inactive, fully reduced form has to be considered as causing the inhibition.
Structural Characterization of Polysomal Poly(A)‐Protein Particles in Rat Liver
Authors: TOMCSÁNYI Tihamér , KOMÁROMY László , TIGYI András
Published: March 2005

Poly(A)‐protein particles were prepared from rat liver polyribosomes, washed with 0.5 M KCl or unwashed, after digestion with pancreatic ribonuclease and ribonuclease T1 by two successive rounds of sucrose gradient centrifugation. The particles were sedimented in a range of 5–13 S with a peak at about 9 S. The KCl wash of polysomes had no effect on the sedimentation properties of the particles. The particles isolated in this manner were 99% resistant to further pancreatic ribonuclease treatment and contained about 96% adenylic acid. The length of the poly(A) molecules prepared from the poly(A)‐protein particles showed a broad distribution of about 70–290 nucleotides with a peak around 130 nucleotides, as measured by polyacrylamide gel electrophoresis. In CsCl density gradient the poly(A)‐protein particles banded in a density range of 1.30–1.42 g/cm3 with a peak at 1.36 g/cm3, which amounts to about 80% of the protein content. Sodium dodecyl sulfate/polyacrylamide and urea/sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated six polypeptides with molecular weights of 50000, 54000, 58000, 63000, 76000 and 90000 in the poly(A)‐protein particles, but the main components were dependent on the method. The treatment of polysomes with KCl resulted in a loss of the 90 000‐molecular‐weight component. Amino acid analysis of the polypeptides bound to poly(A) revealed that they contained a relatively large amount of aspartic plus glutamic acid (21.6%) as well as hydrophobic amino acids (41.4%). Digestion of glutaraldehyde‐fixed particles with ribonuclease T2 showed that about 50% of poly(A) was accessible to the enzyme, thus this part of poly(A) was located on the surface of the particles. In the electron micrographs the shadowed poly(A)‐protein particles appeared in a globular, somewhat elongated form and were mostly 14–18 nm in diameter. On the basis of the results a model for the ‘average’ 9‐S particles was constructed.
On the Role of Ribosylthymine in Prokaryotic tRNA Function
Authors: KERSTEN Helga , ALBANI Martin , MÄNNLEIN Elisabeth , PRAISLER Rosemarie , WURMBACH Peter , NIERHAUS Knud H.
Published: March 2005

tRNAPhe and tRNALys were isolated from an Escherichia coli K12 mutant deficient in ribosylthymine (rT) and from the wild‐type strain. The sequence G‐rT‐Ψ‐C which is common to loop IV of practically all tRNAs used in the elongation cycle of protein synthesis reads G‐U‐Ψ‐C in tile tRNAs of the mutant strain. The purified tRNAs were compared in various steps of protein biosynthesis.

The poly(U)‐dependent poly(Phe) synthesis performed with purified Phe‐tRNAPhe and purified elongation factors showed no dependence on the presence or absence of ribosylthymine in the respective tRNAs. In contrast, the corresponding poly(A)‐dependent poly(Lys) synthesis was markedly increased when Lys‐tRNALys lacking rT was used. The analysis of individual functional steps of the poly(A)‐dependent elongation cycle demonstrated that the absence of rT reduced the binding to the A‐site and improved the translocation reaction, whereas the formation of the ternary complex EF‐Tu · GTP · aa‐tRNA as well as both tRNA binding to the P‐site and the peptidyltransferase reaction remained unaffected. The presence of U in place of rT in tRNA increases the misincorporation of leucine in an optimized poly(U)/poly(Phe) system from about 3 in 10000 to 3 in 1000. Our results are in agreement with the view that rT is involved in tRNA binding to the A‐site in contrast to the P‐site, and suggest that the presence of rT in tRNA improves the fidelity of the decoding process at the A‐site of the ribosome.
The Major Components of the Mouse and Human Genomes
Authors: SORIANO Philippe , MACAYA Gabriel , BERNARDI Giorgio
Published: March 2005

The reassociation kinetics of DNA fragments obtained from the major components of the mouse and human genomes (recently isolated in our laboratory) have been investigated. It has been found that the relative amounts of interspersed repeated and unique sequences strikingly differ in the different major components of each genome and in the corresponding major components of the two genomes. Furthermore, within each major component, the interspersed repeated and unique sequences do not differ in dG + dC contents. These findings lead to the general conclusion that the sequence organization of mammalian genomes is not uniform in different chromosomal regions and that it exhibits remarkable variations in different mammals.
Characterization of Hydrolase H, a New Muscle Protease Possessing Aminoendopeptidase Activity
Authors: OKITANI Akihiro , NISHIMURA Toshihide , KATO Hiromichi
Published: March 2005

A newly‐identified protease from rabbit skeletal muscle, named hydrolase H [Okitani, A. et al. (1980) Agric. Biol. Chem. 44, 1705–1708] has been shown to hydrolyze l‐leucine β‐naphthylamide as well as α‐N‐benzoyl‐dl‐arginine β‐naphthylamide. The enzyme hydrolyzes prolamine in the manner of an endopeptidase and hydrolyzes tripeptides and tetrapeptides in the manner of an aminopeptidase. Thus it has been concluded that the enzyme possesses the properties of both endopeptidase and aminopeptidase and that it should be classified as an aminoendopeptidase. Both activities of endopeptidase and aminopeptidase are maximal at pH 7.5–8.0 and enhanced remarkably by thiol compounds. Both activities are stable at pH 7–9 and protected by 2‐mercaptoethanol. They are inhibited by monoiodoacetic acid, leupeptin, Zn2+ and Ni2+, but not affected by EDTA, trypsin inhibitor, pepstatin, antipain and phenylmethylsulfonyl fluoride. These results suggest that the enzyme is a thiol protease. The enzyme is composed of three kinds of subunits, the chain Mr of which are 51000, 72000 and 92000.
Studies on the Mechanism of Action of the Histone Kinase Dependent on Adenosine 3′,5′‐Monophosphate
Authors: GABIBOV Alexander G. , KOCHETKOV Sergei N. , SASHCHENKO Lidia P. , SMIRNOV Ivan V. , SEVERIN Evgenii S.
Published: March 2005

A significant release of inorganic phosphate from ATP in the presence of the cAMP‐dependent pig‐brain histone kinase was detected. The high degree of homogeneity of the enzyme preparations used, identity of Michaelis constants (Km for ATP = 12 μM), the close values of cAMP activation constants (48 nM and 51 nM) for the phosphotransferase and ATPase activities, respectively, are all evidence that ATP decomposition is catalysed by the histone kinase under study. The ATPase activity observed supports the ping‐pong bi‐bi mechanism established earlier for the phosphotransferase reaction and can be regarded as due to decomposition of the phosphoryl enzyme. The transient and steady‐state phases of the ATP hydrolysis were studied. The simplest reaction pathway can be described in terms of a three‐step mechanism. The close values of the rate constant for the elementary stages of the ATPase reaction ebtained in the nucleophile competition study and by computer simulation of the quenched‐flow kinetics give further support for the mechanism proposed. The phosphoryl enzyme decomposition was shown to be a rate‐limiting step under the experimental conditions used (pH 7.8–8.0).
Reconstitution of the Allosteric l‐Lactate Dehydrogenase from Lactobacillus casei Investigated by Hybridization
Authors: MAYR Ulrich , HENSEL Reinhard , KANDLER Otto , PRUSCHA Helmut
Published: March 2005

The reassociation process of the urea‐denatured allosteric l‐lactate dehydrogenase from Lactobacillus casei was investigated by hybridization experiments between the reassociating enzymes from L. casei and Lactobacillus curvatus. The quantitatively evaluated hybridization patterns indicate an assembly pathway from the unfolded subunits to the tetrameric state via dimers. The comparison of the kinetics of reassociation and reactivation of the L. caseil‐lactate dehydrogenase shows that the tetramer is the only active form.
The Complete Amino‐Acid Sequence of Hen Ovalbumin
Authors: NISBET Andrew D. , SAUNDRY Richard H. , MOIR Arthur J. G. , FOTHERGILL Linda A. , FOTHERGILL John E.
Published: March 2005

The complete amino acid sequence of hen ovalbumin, comprising 385 residues, has been determined. The sequence was deduced from the 17 cyanogen bromide fragments and from peptides derived by digestion with a number of proteolytic enzymes. The molecular weight of the polypeptide chain of ovalbumin is 42699.

Ovalbumin has four sites of postsynthetic modification; in addition to the acetylated N terminus, the carbohydrate moiety is located at Asn‐292, and the two phosphorylated serines are at residues 68 and 344. The ‘signal sequence’ of ovalbumin is between residues 234 and 252. The heptapeptide released during the conversion of ovalbumin to plakalbumin by subtilisin digestion corresponds to residues 346–352. The hen ovalbumin polymorphism characterised by an Asn–>Asp replacement results from a mutation at residue 311.

The amino acid sequence of ovalbumin deduced from these amino acid sequence studies is in complete agreement with the sequence of mRNA determined by McReynolds et al. [Nature (Lond.) 273, 723–728 (1978)].
Synthesis and Insertion, bothin vivoandin vitro, of Rat‐Liver Cytochrome P‐450 and Epoxide Hydratase intoXenopus laevisMembranes
Authors: OHLSSON Rolf I. , LANE Charles D. , GUENGERICH F. Peter
Published: March 2005

We described whole cell and cell‐free systems capable of inserting into membranes cytochrome P‐450 and epoxide hydratase made under the direction of rat liver RNA. The systems have been used to study the pathways followed by newly made secretory and integral membrane proteins. The cell‐free system containsXenopus laevisembryo membranes, and demonstrates competition for a common receptor between cytochrome P‐450 and epoxide hydratase, and normal secretory proteins: evidence is provided for differential membrane receptor affinity. Thus, synthesis of secretory and membrane proteins appears to involve a common initial pathway. Microinjection of rat liver RNA into whole oocytes suggests that membrane insertion is neither cell type nor species specific, because functional rat liver enzymes are found inserted in the endoplasmic reticulum of the frog cell. Nonetheless, insertion is highly selective since albumin and several other proteins made under the direction of the injected liver RNA are sequestered within membrane vesicles and are then secreted by the oocyte, whilst epoxide hydratase and cytochrome P‐450 are inserted into membranes but are not secreted.
Processing of Naked 45‐S Ribosomal RNA Precursor in vitro by an RNA‐Associated Endoribonuclease
Authors: DENOYA Claudio , COSTA GIOMI Patricia , SCODELLER Eduardo A. , VÁSQUEZ César , LA TORRE José L.
Published: March 2005

A processing endoribonuclease was isolated from the cytoplasm of chick embryos. The enzyme was easily obtained using an RNA extraction procedure based on a mild deproteinization with Sarkosyl and cold phenol/ chloroform. This technique assured the recovery of several proteins and the endoribonuclease in association with the RNA.

It was demonstrated that this endoribonuclease was capable of promoting, in vitro, a precise processing of naked 45‐S ribosomal RNA precursor to molecules resembling the intermediates as well as the 28‐S and 18‐S cytoplasmic RNAs found in vivo. The presence of magnesium ions was required for the correct processing function of the enzyme. In addition, under the same conditions, the mature ribosomal RNA substrates were degraded at a slower rate by this RNA‐associated RNase.

It was possible to fractionate the enzymatic preparation into two different populations by means of a sucrose gradient: one associated and the other partially free of an RNA component. The effect of the intrinsic RNA associated with the endoribonuclease on the enzymatic activity was tested by analyzing both the enzymatic populations and the total enzymatic preparation treated with pronase or with immobilized pancreatic RNase. In all cases in which the RNA component was present, the enzyme showed processing activity. On the other hand, when the RNA component was absent or at least partially degraded the enzyme proved to be more active in processing precursor molecules and in promoting extensive degradation of mature RNA species.

Although the presence of RNA in association with the enzyme was demonstrated, its role in the regulation of the enzymatic activity is yet not clear.