bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein S1 from Escherichia coli Ribosomes: An Improved Isolation Procedure and Shape Determination by Small‐Angle X‐Ray Scattering
Authors: LABISCHINSKI Harald , SUBRAMANIAN Alap‐Raman 
Published: March 2005

Ribosomal protein S1 from Escherichia coli was studied in solution by small‐angle X‐ray scattering and the following parameters were obtained. The radius of gyration R= 8.0 ± 0.2 nm; largest diameter D= 28 nm; molecular weight = (8–9) × 104. The data also yielded (with the assumption of a rigid particle with almost constant electron density) two radii of gyration of cross‐section Rq1= 2.5 ± 0.1 nm and Rq2= 1.05 ± 0.05 nm and molecular volume = 140 nm3. The experimental scattering curve of S1 was compared with the theoretical scattering curves for several rigid triaxial homogeneous bodies and the closest fit was given by that of a flat elliptical cylinder with the dimensions of 4.5 nm and 0.88 nm for the two semiaxes and 26.5 nm for height. The results from the present X‐ray scattering studies and those from limited proteolytic digestion of protein S1 [J. Mol. Biol. 127, 41–54, (1979)] support the notion that the structure of protein S1 is organized into two distinct subdomains within its elongated overall shape.Protein S1 was purified for this study by an efficient procedure which yielded 12 mg S1/g ribosomes. The isolated protein was fully active in functional tests both before and after X‐ray irradiation.
Effects of Cytosol on Transcription in Isolated Simian‐Virus‐40‐Infected Nuclei
Authors: ICEKSON Isaac , YANIV Moshe 
Published: March 2005

The transcription in vitro of nuclei isolated from monkey kidney cells infected with simian virus 40 was stimulated by a cytosol fraction from the same uninfected cells. Transcription in nuclei was inhibited 60–80% by 0.1 μg/ml of α‐amanitin, in the presence or in the absence of the cytosol preparation.Treatment of nuclei after transcription in vitro with 0.02 M EDTA preferentially solubilized viral‐specific RNA partially bound to the viral chromatin.The increase in incorporation into total RNA seen in the presence of cytosol was not paralleled by any detectable increase in transcription of viral sequences as shown by RNA · DNA hybridizations.
Dissociation and Reassembly of Limulus polyphemus Hemocyanin
Authors: BIJLHOLT Martha M. C. , VAN BRUGGEN Ernst F. J. , BONAVENTURA Joseph 
Published: March 2005

Limulus polyphemus hemocyanin is a 3.3 × 106‐Mr protein containing 48 subunits in an assemblage of eight hexamers. The molecule can be dissociated into monomers and dimers at pH 8.9 in the presence of 0.01 M EDTA. These subunits are heterogeneous and can be separated into five zones (I–V) by DEAE‐Sephadex chromatography.Reassembly experiments were carried out with varied subunit mixtures, based on different combinations of the five chromatographic zones, in order to study the structural role of the diverse subunits in the eight‐hexamer molecule. The reassembly products were analysed by electron microscopy and ultracentrifugation. No structural role for zone I could be found. Zone V and possibly zone II are needed to form structures larger than hexamers. Absence of zone III causes irregular aggregation of hexamers. Zone IV and perhaps zone II are needed to make eight‐hexamer molecules from four‐hexamer molecules. From these results we conclude that there is a high degree of subunit specificity in the inter‐subunit contacts in the native Limulus hemocyanin molecule.
Fatty‐Acid Synthesis in Lactating‐Goat Mammary Gland 2. Medium‐Chain Fatty Acid Synthesis
Authors: GRUNNET Inger , KNUDSEN Jens 
Published: March 2005

Addition of goat, rat, rabbit and cow mammary microsomal fraction to fatty acid synthetase of goat mammary gland induced C10 fatty acid synthesis.When the microsomal fractions from rat, rabbit and cow mammary gland were incubated with their respective fatty acid synthetases only the cow enzyme synthesized significant amounts of C10 compared to the purified fatty acid synthetases alone.The goat mammary microsomal fraction was not able to induce C10 synthesis by rat and rabbit mammary‐gland fatty acid synthetase, but only by the goat and cow enzyme.Both goat and cow mammary‐gland fatty acid synthetase have in contrast to the rabbit and rat enzymes the ability to hydrolyse medium‐chain acyl‐thioesters. We therefore suggest a novel mechanism for medium‐chain fatty acid synthesis in lactating‐goat mammary gland. This mechanism involves an activation of the medium‐chain acyl‐thioester hydrolase within the fatty acid synthetase of goat mammary gland by an unknown microsomal factor.
Actin in Mammalian Lens
Authors: KIBBELAAR Mac A. , SELTEN‐VERSTEEGEN Anne‐Marie E. , DUNIA Irène , BENEDETTI E. Lucio , BLOEMENDAL Hans 
Published: March 2005

In this paper evidence is provided that one of the protein components of the water‐soluble fraction of the calf lens binds specifically to deoxyribonuclease I (DNAse I). On the basis of this property, the polypeptide could be purified by applying DNAse I affinity chromatography. Concomitantly a protein of Mr 55000 and a rather large amount of α‐crystallin copurify with this polypeptide, which has a molecular weight of 42000.Highly purified 42000‐Mr protein was also obtained by extraction of the water‐insoluble fraction of the calf lens with 2‐{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid followed by gel filtration. Amino acid analyses, peptide mapping and electron microscopy show that the protein obtained from both lens fractions is identical to non‐muscle actin. Furthermore the amino acid composition of the 55000‐Mr protein is identical to hog stomach skeletin and very similar to calf brain desmin.
On the Nature of Biochemically Generated Hydroxyl Radicals Studies using the Bleaching of p‐Nitrosodimethylaniline as a Direct Assay Method
Authors: BORS Wolf , MICHEL Christa , SARAN Manfred 
Published: March 2005

An efficient scavenger for radiolytically generated hydroxyl (OH) radicals, p‐nitrosodimethyl‐aniline, was used to try to substantiate the presence of this oxygen radical species in several biochemical systems. Most of these systems which were investigated had previously been assumed to generate OH radicals, e.g. the autoxidation of 6‐hydroxydopamine, the hydroxylating system NADH/phenazine methosulfate, and the oxidation of xanthine or acetaldehyde by xanthine oxidase.We did not observe inhibition of the bleaching of p‐nitrosodimethylaniline in oxygenated solutions by other scavengers of OH radicals nor, in the case of xanthine/xanthine oxidase, by catalase and superoxide dismutase. We therefore conclude that, under biochemical conditions as opposed to radiolysis or photolysis, no freely diffusable OH radicals are formed. Rather, a strongly oxidizing OH‐analogous complex is considered to represent the p‐nitrosodimethylaniline‐detectable species formed under these conditions.
The Dolichol Pathway of Protein Glycosylation in Rat Liver
Authors: GODELAINE Danièle , BEAUFAY Henri , WIBO Maurice , AMAR‐COSTESEC Alain 
Published: March 2005

Incorporation of N‐acetylglucosamine into endogenous lipid and protein acceptors was investigated on heavy microsomes from rat liver, incubated with UDP‐N‐acetyl[14C]glucosamine and GDP‐mannose in the absence of detergent. This subcellular preparation derived for 95% or more from the rough endoplasmic reticulum and was devoid of Golgi components which contain the enzyme that adds the peripheral N‐acetylglucosamine units to glycoproteins.The label was found almost exclusively in dolichyl diphosphate N‐acetylglucosamine, except when the subcellular preparation was treated with pyrophosphate and subsequently incubated with the nucleotide sugars in the presence of GTP. Then, the incorporation of N‐acetylglucosamine was considerably enhanced, and the additional label was associated with dolichyl diphosphate N,N′‐diacetylchitobiose, with dolichyl diphosphate oligosaccharides and with proteins. The time‐course of N‐acetylglucosamine incorporation in these products was compatible with the pathway of dolichyl diphosphate glycoconjugates for the biosynthesis of the core portion of saccharide chains linked to asparagine residues of glycoproteins. The addition of GDP‐mannose to the incubation medium was required to produce labeled dolichyl diphosphate oligosaccharides, but not to incorporate N‐acetylglucosamine in protein.It is concluded that rough microsomes are capable of assembling dolichol‐linked oligosaccharides from exogenous nucleotide precursors and of transferring N,N′‐diacetylchitobiose, or its mannosylated derivatives, from the lipid intermediate to endogenous proteins. However, these metabolic activities are hindered in the original subcellular preparation, and in the absence of GTP. Although the earliest perceptible effect produced jointly by the treatment with pyrophosphate and by GTP was the synthesis of dolichyl diphosphate N,N′‐diacetylchitobiose, the primary action of these factors remains uncertain. They may stimulate directly the reaction forming dolichyl diphosphate N,N′‐diacetylchitobiose from dolichyl diphosphate N‐acetylglucosamine, or activate the synthesis of this latter intermediate from a particular pool of dolichyl monophosphate which is readily converted afterwards into disaccharide and oligosaccharide derivatives and glycosylates protein. The requirement for GTP might have a functional meaning, for GTP acted maximally at a concentration distinctly lower than its actual concentration in liver. The detachment of ribosomes from rough vesicles was the major alteration induced by treatment with pyrophosphate. It is suggested that the removal of ribosomes unmasks the membrane sites where GTP acts.
Methionyl‐tRNA Synthetase from Escherichia coli
Authors: FAYAT Guy , HOUNTONDJI Codjo , BLANQUET Sylvain 
Published: March 2005

Both the aminoacylation and isotopic ATP‐PPi exchange activities of native and trypsin‐modified methionyl–tRNA synthetases from Escherichia coli are specifically inactivated by incubation in the presence of periodate‐treated initiator tRNAMet.The inactivation proceeds through the formation of a reversible Schiff's base between the ɛ‐amino group of a lysine within the catalytic center of the enzyme and the 2′,3′‐aldehyde groups created at the 3′‐terminal ribose of tRNA. The Schiff's base may be stabilized by reduction with sodium borohydride.Intact tRNAfMet competes with the inactivation by its dialdehyde. It has been verified in the case of the modified enzyme that the protection is afforded according to an equilibrium constant identical to that for tRNAfMet binding at the active site of the enzyme.Finally it is shown that the incorporation of one molecule of the dialdehyde of [14C]tRNA completely destroys the activity of the monomeric trypsin‐modified methionyl‐tRNA synthetase.
Kinetics of the Iodination and the Coupling Reaction in Thyroglobulin Catalyzed by Lactoperoxidase and Chloramine T
Authors: LAMAS Luis 
Published: March 2005

The kinetics of the iodination reaction and the coupling reaction in thyroglobulin catalyzed by lactoperoxidase and chloramine T have been compared in order to elucidate whether or not the enzyme is necessary for these reactions once the active iodine has been generated by the peroxidase system.In the presence of added iodide, lactoperoxidase and chloramine T catalyze both reactions. Lactoperoxidase does so in a gradual fashion, while chloramine T acts almost instantaneously. Chloramine T is more rapid but less efficient than lactoperoxidase in both iodotyrosine and iodothyronine formation. At short time intervals the difference is very small, but it becomes larger at longer reaction time intervals.We were able to study the coupling reaction independently of the iodination reaction by using chemically prelabeled thyroglobulin in the absence of added iodide. Lactoperoxidase catalyzes the reaction much more efficiently than chloramine T. Chemically prelabeled thyroglobulin releases iodide during incubation in the presence of the peroxidase system, but not in the presence of chloramine T. Thus iodide might play a role in the coupling reaction as already suggested by Dème et al. [Eur. J. Biochem. 70, 435–440 (1976)] for thyroid peroxidase‐catalyzed iodination of thyroglobulin.From the present results it appears that peroxidase is a more potent catalytic agent than chloramine T for both the iodination and the coupling reactions. This suggests that the enzyme is involved in both reactions beyond simply providing the active form of iodine, though further work is needed to clarify the mechanisms involved.
Calf Spleen NAD Glycohydrolase
Authors: TRAVO Pierre , MULLER Hélène , SCHUBER Francis 
Published: March 2005

The catalytic properties of membrane‐bound calf spleen NAD glycohydrolase were studied in comparison with previous data obtained with a solubilized hydrosoluble form of the enzyme. When the hydrolysis of NAD catalyzed by membrane‐bound NAD glycohydrolase was studied at pH values below 7.5, only insignificant interference by other NAD‐hydrolyzing enzymes was detected, and no proton‐diffusional inhibition was observed. The kinetics could, therefore, be followed using a titrimetric assay for NAD glycohydrolase activity.The effect of pH, ionic strength on the kinetic parameters, and shifts in binding constants for several ligands of the membrane‐bound enzyme indicate that the NAD glycohydrolase activity is influenced by an electrostatic potential due to negative charges (polyelectrolyte effect). No significant changes in kinetic mechanism could be found between both NAD glycohydrolase forms. The association of the enzyme with the membrane results in a remarkably increased thermal stability, in changes in binding properties of the active site and in the emergence of new inhibitor binding sites; e.g. adenosine 3′:5′‐monophosphate (cyclic AMP) and adenosine, which do not inhibit the hydrosoluble form of NAD glycohydrolase, are good inhibitors (respectively competitive and mixed) of the membrane‐bound enzyme. These data (i.e. allotopic changes) probably can be ascribed to enzyme conformational changes induced and stabilized by interaction with membrane constituents.
Analysis of the Sea‐Urchin Genome by Homologous RNA Polymerase II Binding
Authors: Di MAURO Ernesto , PEDONE Francesco , BALLARIO Paola 
Published: March 2005

We observe that Paracentrotus lividus RNA polymerase II can form stable complexes with homologous DNA at sites from which it can start RNA synthesis in vitro. The sea‐urchin genome can therefore be described in terms of the distribution of the DNA sites that form such stable complexes with the homologous RNA polymerase II [class A sites as defined in a prokaryotic homologous system by D. Hinkle and M. J. Chamberlin (1972) J. Mol. Biol. 70, 157‐185].We describe the properties of the complexes that P. lividus RNA polymerase II forms with P. lividus DNA: resistance to heparin, dissociation kinetics, temperature and time dependence of their formation, range of RNA polymerase/DNA weight/weight ratios that give rise to the stable binding events.The amount and the distibution of the sites that form stable complexes with P. lividus RNA polymerase II were determined by the analysis of the dissociation of the complexes formed by the enzyme on DNA fragments of various lengths. P. lividus appears to form 4.5 × 104 stable (t1/2≥ 30 min) complexes/haploid genome. A part of these complexes has a distribution corresponding to values between 2000 and 4500 base pairs; the rest of the complexes is more widely spaced (up to a measured interval of 30000 base pairs).
Purification of Two DD‐Carboxypeptidases/Transpeptidases with Different Penicillin Sensitivities from Proteus mirabilis
Authors: SCHILF Wolfgang , MARTIN Hans Herbert 
Published: March 2005

Two membrane‐bound enzymes of Proteus mirabilis with the dual functions of peptidoglycan DD‐carboxypeptidase and transpeptidase (named DD‐carboxypeptidase/transpeptidase H and L) were isolated and purified by selective solubilization with the nonionic detergent Genapol X‐100, affinity chromatography on matrix‐bound ampicillin, and preparative isoelectric focusing in the presence of detergent.Purified enzymes H and L were, respectively, penicillin‐binding proteins 4 and 5 among seven major penicillin‐binding proteins present in P. mirabilis.The enzymes differed in the following properties. Enzyme H had an Mr of 49000; isoelectrie point at pH 8.1; high sensitivity to benzylpenicillin and permanent inactivation because of high stability of the enzyme‐antibiotic complex El* (half‐life 300 min); fragmentation of benzylpenicillin with formation of phenylacetylglycine during the slow decay of El*; it functioned as an endopeptidase on peptide‐crosslinked side chains of peptidoglycan. Enzyme L had an Mr of 43000; isoelectrie point at pH 5.9; low sensitivity to benzylpenicillin and low stability of El* (half life 7.2 min) with rapid recovery of enzyme activity; no function as an endopeptidase.The properties of enzyme L were identical with those of the single active DD‐carboxypeptidase found previously in the spheroplast L‐form of P. mirabilis grown in the presence of benzylpenicillin. We conclude that the partial penicillin resistance of P. mirabilis, with growth as L‐form and synthesis of peptide‐crosslinked peptidoglycan, depends on the continuing function of enzyme L as a DD‐carboxypeptidase and transpeptidase in the presence of the antibiotic.
Bovine Kidney Pyruvate Dehydrogenase Complex
Authors: KRESZE Georg‐B. , RONFT Hedwig , DIETL Brigitte 
Published: March 2005

Bovine kidney pyruvate dehydrogenase multienzyme complex is inactivated rapidly by papain. However, none of the component activities of the complex is destroyed during inactivation of the overall reaction.
The core component, lipoate acetyltransferase, is cleaved by papain to give principal fragments with Mr 26500 and 26000 (as determined by dodecylsulfate gel electrophoresis). Much more slowly, the α chain of the pyruvate dehydrogenase component is attacked.
Fragmented lipoate acetyltransferase retains its complete enzymatic activity and remains of high molecular weight. It is unable, however, to bind the other component enzymes, pyruvate dehydrogenase and lipoamide dehydrogenase. Therefore, the multienzyme complex is disassembled when treated with papain.
A method is described which allows the rapid and convenient isolation of nicked lipoate acetyltransferase as well as unfragmented pyruvate dehydrogenase and lipoamide dehydrogenase from papain‐treated complex under very mild conditions. The two uncleaved component enzymes have identical properties and similar specific activities as enzyme preparations obtained by other, more laborious procedures.
Degradation of Basement‐Membrane Collagen by Neutral Proteases from Human Leukocytes
Authors: UITTO Veli‐Jukka , SCHWARTZ David , VEIS Arthur 
Published: March 2005

A neutral extract from human leukocytes was shown to have proteolytic activity which could degrade triple‐helical basement membrane collagen from bovine lens capsules into specific triple‐helical sub‐fragments. The enzyme responsible is not identical to the leukocyte collagenase active against interstitial collagen types I, II and III.After denaturation, the neutral protease reaction products showed three major peptides with molecular weights of 70000, 50000 and 30000, by dodecylsulphate gel electrophoresis analysis. Electron microscopic observation and viscosity measurements showed the initial twice‐pepsinized collagen to be comprised of intact rod‐like molecules about 300 nm long. The fragments produced by the protease were also triple‐helical and were resistant to the action of trypsin at 20°C. The fragments were completely degraded by purified bacterial collagenase or by raising the reaction temperature above the melting temperature of the basement membrane collagen during incubation with leukocyte extract.Enzyme activity has its pH optimum between 7 and 9, EDTA, EGTA and 1, 10‐phenanthroline (metal‐chelating agents) completely inhibited enzyme activity, as did serum. Partial inhibition of the activity was obtained with phenylmethylsulfonyl fluoride and soybean trypsin inhibitor.
On the Number of Steroid‐Binding Sites of Δ5‐3‐Oxosteroid Isomerase
Authors: PENNING Trevor M. , WESTBROOK Edwin M. , TALALAY Paul 
Published: March 2005

The number of steroid‐binding sites in Δ5‐3‐oxosteroid isomerase of Pseudomonas testosteroni (EC 5.3.3.1) has been determined from measurements of the red shift of the ultraviolet chromo‐phore of 19‐nortestosterone upon binding to the enzyme. The experiments include spectroscopic measurements when limiting concentrations of either 19‐nortestosterone or isomerase are titrated with varying concentrations of the complementary ligand. Analysis of the results indicates one binding site per subunit (Mr 13394). Scatchard plots indicate a single family of equivalent binding sites. 5,10‐Secoestr‐5‐yne‐3,10,17‐trione is a suicide substrate of isomerase [Batzold & Robinson (1975) J. Am. Chem. Soc. 97, 2576]. The time course for inactivation of isomerase with an excess of 5,10‐seco[7‐3H]estr‐5‐yne‐3,10,17‐trione was parallel to the covalent incorporation of steroid and gave a final stoichiometry of nearly one steroid molecule per subunit of enzyme. Alkylation of [14C]iso‐merase with excess of this 3H‐labeled steroid followed by gel‐filtration and dialysis gave an inactivated enzyme with a 3H/14C ratio that corresponds to one molecule of steroid bound per subunit; this stoichiometry was constant over a wide range of protein concentrations (0.1 − 10 mg/ml).Diffusion of [3H]progesterone into hexagonal crystals of isomerase showed that at saturation one steroid molecule was bound per protomer. Taken together these findings strongly support the conclusion that one molecule of steroid is bound per subunit of isomerase both in solution and in the crystal state.
The Enhancement of Cytotoxicity of N‐Methyl‐N‐nitrosourea and of y‐Radiation by Inhibitors of Poly(ADP‐ribose) Polymerase
Authors: NDUKA Nsirim , SKIDMORE Christopher J. , SHALL Sydney 
Published: March 2005

Inhibitors of poly(ADP‐ribose) polymerase show a synergistic potentiation of cytotoxicity with certain DNA‐damaging agents. Non‐toxic concentrations of 5‐methylnicotinamide dramatically potentiate the cytotoxicity of N‐methyl‐N‐nitrosourea as tested by the cloning ability of mouse leukaemia (LI210) cells. A dose‐enhancement factor of about 10 is observed. This potentiation is dependent on the concentration of 5‐methylnicotinamide. The methylxanthines theobromine, theophlline and caffeine also increase the cytotoxicity of methylnitrosourea. Thymidine, in the presence of sufficient deoxycytidine to overcome the perturbation of deoxynucleotide metabolism, also potentiates the cytotoxicity of methylnitrosourea. Nicotinate, which is not an inhibitor of poly‐(ADP‐ribose) polymerase, has no effect on methylnitrosourea toxicity.A very small, but consistent, enhancement of the toxicity of y‐radiation by the same inhibitors has been observed.We suggest that this potentiation of cytotoxicity is mediated by inhibition of (ADP‐ribose)n biosynthesis; and that the biosynthesis is stimulated by DNA damage. We therefore propose that (ADP‐ribose)n takes part in cellular repair mechanisms, either by modifying chromatin structure or by a specific participation in DNA repair.