bluuebunny/crossref_metadata_2025_split huggingface dataset:
Carbohydrate and Amino‐Acid Composition of Human Glomerular‐Basement‐Membrane Fractions Purified by Affinity Chromatography
Authors: Mahieu Philippe M. , Winand Roger J. 
Published: March 2005

Human glomerular basement membranes can be substantially solubilized by autoclaving in 0.1 M Tris‐acetate buffer pH 7.4, at 110°C, during 3 h or by treatment with 8 M urea containing mercaptoethanol. The best solubilization (80% of soluble material) was obtained by autoclaving. Therefore it was this material which has been purified from tritiated glycoproteins, by affinity chromatography, using Sepharose cyanogen‐bromide coupled with rabbit anti‐human glomerular‐basement‐membrane γ‐G globulin; at least 80% of solubilized basement membranes are adsorbed onto this γ‐globulin.Unadsorbed material contains the same carbohydrate units as the whole membranes but are richer in heteropolysaccharide units. Its molecular weight is lower than 69000. Adsorbed glyco‐protein components, eluted from the Sepharose by acidic medium at 4°C, are further purified by preparative polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate. Four major fractions exhibiting molecular weights from 80000 to 300000 are obtained; they have the same carbohydrate units as the whole membranes, but have more disaccharide units. Similarly, amino acid composition of these purified fractions exhibits more 3‐hydroxyproline and hydroxy‐lysine residues than the whole membranes. This procedure appears therefore to be a valuable technique for preparing large quantities of labelled glomerular‐basement‐membrane antigens containing both types of oligosaccharidic chains.
Isolation and Characterization of Rat‐Liver Mitochondrial Ribosomes Highly Active in Poly(U)‐Directed Polyphenylalanine Synthesis
Authors: Greco Margherita , Cantatore Palmiro , Pepe Gabriella , Saccone Cecilia 
Published: March 2005

The procedure used to obtain mitochondrial ribosomes from rat liver highly active in catalyzing a poly(U)‐dependent polyphenylalanine synthesis is described.Two kinds of particles with sedimentation coefficients of 80‐S and 55‐S, present in the mitochondrial lysate, are able to catalyze protein synthesis in vitro.Sensitivity to specific inhibitors of protein synthesis demonstrates that the activity of 55‐S particles is only of mitochondrial origin whereas that of 80‐S particles is partly due to mitochondrial ribosomes and partly to contaminating cytoplasmic ribosomes.Characterization of the two kinds of mitochondrial particles has been carried out by studying their sedimentation pattern in different experimental conditions and by analyzing the RNA extracted by polyacrylamide gel electrophoresis. 16‐S and 12‐S RNA seem to be specific mitochondrial ribosomal species.The results suggest that the 55‐S particle represents the basic unit for protein synthesis in rat liver mitochondria. Particles with higher sedimentation value probably represent aggregates of mitochondrial monosomes.
Role of Manganous Ion in the Kinetics of Pig‐Heart NAD‐Specific Isocitrate Dehydrogenase
Authors: COHEN Phyllis F. , COLMAN Roberta F. 
Published: March 2005

The NAD‐dependent isocitrate dehydrogenases require the presence of divalent metal cation for activity. In order to elucidate the role of metal in this reaction, Michaelis constants for manganese were determined as a function of pH and isocitrate concentration. At constant pH, the Km values for total manganese as well as free manganous ion decreased as the concentration of isocitrate in the reaction mixture was increased; in contrast, the Michaelis constant in terms of the metal · isocitrate complexes was invariant, suggesting that manganese binds as a metal chelate of isocitrate. A previous analysis of the Michaelis constants for isocitrate [Cohen, P. F., and Colman, R. F., Biochemistry (1972) 11, 1501] indicated that in the presence of 1–10 mM manganese, the substrate binds as free dibasic isocitrate. A model is proposed which allows for the formation of the active enzyme · manganese · isocitrate complex from either of two pathways: in the presence of low concentrations of manganese, the enzyme may combine directly with the metal chelate of dibasic isocitrate; alternatively, in the presence of high concentrations of manganese, the enzyme first binds metal ion to yield an enzyme · manganese complex which subsequently reacts with free dibasic isocitrate.The affinity of the enzyme for the manganese · dibasic‐isocitrate complex varies with pH, exhibiting a lower Michaelis constant at pH 8 than 6. It is shown that the catalytic function of the free enzyme is dependent on the ionization of an amino acid residue with pKe= 7.26. The measurable PK of this ionizable group is decreased to 6.39 in the enzyme · manganese and enzyme · manganese isocitrate complexes.The Km for manganese was determined in the presence of various chelating agents. Although citrate and GDP increase the Michaelis constant in terms of total manganese, the Km for the actual substrate (manganese · dibasic‐isocitrate) is not changed. This result can be attributed to the competition between these chelators and isocitrate for manganous ion. In contrast, ADP lowers the Km values for the metal · isocitrate complex, as well as that for total manganese, suggesting that a direct interaction with the enzyme is involved.
The Synthesis of Three AMP‐Analogues: N6‐(6‐Aminohexyl)‐adenosine 5′‐Monophosphate, N6‐(6‐Aminohexyl)‐adenosine 2′,5′‐Bisphosphate, and N6‐(6‐Aminohexyl)‐adenosine 3′,5′‐Bisphosphate and Their Application as General Ligands in Biospecific Affinity Chromatography
Authors: BRODELIUS Peter , LARSSON Per‐Olof , MOSBACH Klaus 
Published: March 2005

Phosphorylation of 6‐chloropurine riboside with phosphorus oxychloride and phosphorus trichloride gave a mixture of the two isomers, 6‐chloropurine‐riboside 2′,5′‐bisphosphate and 6‐chloropurine‐riboside 3′,5′‐bisphosphate. Reaction with 1,6‐diaminohexane followed by resolution of the isomeric mixture on a Dowex 1‐X2 column yielded N6‐(6‐aminohexyl)‐adenosine 2′,5′‐bisphosphate and N6‐(6‐aminohexyl)‐adenosine 3′,5′‐bisphosphate.The inhibition of several NADP+‐dependent and NAD+‐dependent dehydrogenases by N6‐(6‐aminohexyl)‐adenosine 2′,5′‐bisphosphate, N6‐(6‐aminohexyl)‐adenosine 3′,5′‐bisphosphate and N6‐(6‐aminohexyl)‐adenosine 5′‐monophosphate was examined.These three AMP‐analogues were attached to Sepharose 4B by the cyanogen bromide method and the binding of several NAD(P)+‐dependent enzymes were investigated. NADP+‐dependent enzymes were bound to Sepharose substituted with N6‐(6‐aminohexyl)‐adenosine 2′,5′‐bisphosphate, whereas NAD+‐dependent enzymes were not bound under the same conditions. Conversely, when N6‐(6‐aminohexyl)‐adenosine 5′‐monophosphate was used as the immobilised ligand only the NAD+‐dependent enzymes were bound, as well as glucose‐6‐phosphate dehydrogenase showing weak affinity. These observations strongly suggest that these two immobilised analogues represent true biospecific and group‐specific adsorbents. The enzymes were eluted with their complementary nucleotides, NAD(H) and NADP(H). These techniques were utilised to purify several NADP+‐dependent enzymes from a crude Candida utilis extract by chromatography on the new biospecific adsorbent.
Translation Products in vitro of Mitochondrial Messenger RNA from Neurospora crassa
Authors: KÜNTZEL Hans , BLOSSEY Hans‐Christian 
Published: March 2005

A submitochondrial polypeptide‐synthesizing system prepared from Neurospora crassa actively incorporates radioactive amino acids in vitro in the absence of added messenger RNA. Translation products were fractionated into what are most probably single polypeptide chains by filtration through Sephadex G‐100 in the presence of dodecylsulfate. The majority of incorporated amino acids is present in two partially resolved fractions of apparent molecular weights 12000 (P1) and 10000 (P2). P2 (but not P1) forms large‐molecular‐weight aggregates during concentration in the presence of 0.5% dodecylsulfate. Both P1 and P2 aggregate in the presence of 0.01% dodecylsulfate. P1 can be separated from P2 by selective extraction with 90% methanol.Purified mitochondrial DNA from Neurospora crassa was transcribed in vitro by Escherichia coli RNA polymerase. The resulting transcript was translated in a cell‐free system from E. coli into two polypeptide fractions having apparent molecular weights identical to P1 and P2.These data suggest that in Neurospora crassa the major proteins coded by mitochondrial cistrons and synthesized by endogenous mitochondrial polyribosomes are polypeptides of apparent molecular weights 12000 and 10000.
Control of the Rate of Hepatic Uptake and Catabolism of Liposome‐Entrapped Proteins Injected into Rats. Possible Therapeutic Applications
Authors: GREGORIADIS Gregory , NEERUNJUN Diane E. 
Published: March 2005

131I‐ or 125I‐labelled albumin and β‐fructofuranosidase were entrapped in liposomes composed of phosphatidylcholine, cholesterol and a charged lipid (molar ratio 7:2:1). Investigations on the fate of such liposomes injected into rats revealed the following.1. Initially there is an increased rate of removal from plasma (rapid phase) followed by a decreased one (slow phase). The rate of removal during the rapid phase was highest for negatively charged liposomes and lowest for positively charged liposomes with neutral liposomes in between. Uniform rates of removal, regardless of charge, were obtained during the slow phase.2. The rate of hepatic uptake of such liposomes paralleled that observed for their removal from plasma during the rapid phase.3. Concurrent administration of a large quantity of positively charged liposomes and of a smaller quantity of negatively charged liposomes led to a diminution of the rapid phase of the latter suggesting a common site of uptake for liposomes of either charge.4. Blockade of the reticuloendothelial system with carbon led to an increased hepatic localization of liposomes during the rapid phase suggesting a bimodal uptake of liposomes with both the Kupffer and the liver parenchymal cells involved.5. Cross‐linking of liposome‐entrapped albumin and β‐fructofuranosidase increased the stability of the latter and following injection into rats rendered both proteins less vulnerable to degradation by the liver lysosomal enzymes.6. The possibility of controlling both the rate of removal from plasma and catabolism of liposome‐entrapped proteins adds more scope to the use of liposomes in therapy.
d‐Galactose Dehydrogenase from Pseudomonas fluorescens
Authors: BLACHNITZKY Ernst‐Otto , WENGENMAYER Friedrich , KURZ Gerhart 
Published: March 2005

1. A procedure is described for the isolation of d‐galactose dehydrogenase from Pseudomonas fluorescens, grown on d‐galactose as carbon source. A 750‐fold purification was achieved with an overall yield of about 40%. The final preparation had a specific activity of about 850 μmol NADH formed per min per mg protein.2. The enzyme has been shown to be homogeneous by ultracentrifugation, by disc gel electrophoresis, by gel isoelectric focusing and by dodecylsulfate gel electrophoresis.3. The molecular weight of the native enzyme has been determined to be 64000 by sedimentation equilibrium studies and by gel permeation chromatography.4. The enzyme dissociates in the presence of 0.1% sodium dodecylsulfate to polypeptide chains, whose molecular weight was determined as 32000 by electrophoresis, indicating that the native enzyme is composed of two subunits.5. The amino acid composition of d‐galactose dehydrogenase has been determined.6. The pI of the enzyme has been shown to be 4.28. The pH optimum is between 9.1 and 9.5. At pH 9.1 and 30 °C the limiting Michaelis constant for NAD+ is Ka= 0.24 mM, for NADP+Ka= 2.3 mM and for d‐galactose with NAD+ and with NADP+ as cosubstrate Kb= 0.7 mM. The dissociat on constant for NAD+ is Kia= 0.54 mM and for NADP+Kia= 6.2 mM.7. d‐Galactose dehydrogenase from Ps. fluorescens catalyzes the dehydrogenation of d‐galactose by either NAD+ or NADP+. The enzyme oxidizes the following sugars arranged according to relative rates in decreasing order: d‐fucose > d‐galactose > l‐arabinose > 2‐deoxy‐d‐galactose ≫ 4‐deoxy‐d‐galactose > 2‐deoxy‐2‐amino‐d‐galactose.8. The correspondence between amino acid composition, subunit size and structure and between specificity of d‐galactose dehydrogenase from Ps. fluorescens and from Ps. saccharophila is discussed.
Metabolism of Palmotoxins B0 and G0in vitro
Authors: BASSIR Olumbe , EMEROLE Godwin O. 
Published: March 2005

The metabolism of the palmotoxins is yet unknown. Our preliminary report (1973) shows that they are probably metabolized in vitro in a similar manner to the aflatoxins. This involves demethylation and a possible hydroxylation.The rate of demethylation of 50 nmol toxin attains a maximum in about 75 min. Optimum pH was between 7.5 and 7.6 using 0.3 M phosphate buffer, In comparison with aflatoxin B1, palmotoxins B0 and G0 were found to be demethylated and hydroxylated at a slower rate. Two fluorescent metabolites were identified for palmotoxin B0 on thin‐layer chromatography and only one for palmotoxin G0. Fluorescence spectra of the isolates from the toxins showed excitation and fluorescence maxima similar to those of the parent compounds. Ultraviolet spectra of the isolates showed a shift to higher wavelength when compared with the parent compound. Glucuronide conjugates were identified with both the palmotoxins and aflatoxin B1. Phenobarbitone was found to induce the palmotoxins B0 and G0 as well as the aflatoxin‐B1‐metabolizing enzymes. The activity of these enzymes were inhibited by carbon monoxide aeration.
Replication of the Double‐Stranded Replicative‐Form DNA of Bacteriophage M‐13 in Plasmolysed Escherichia coli Cells
Authors: STAUDENBAUER Walter L. 
Published: March 2005

Escherichia coli cells, infected with bacteriophage M‐13 am‐5, when plasmolysed by treatment with 2 M sucrose incorporate radioactivity from deoxyribonucleoside triphosphates into phage‐specific DNA. After suppressing host DNA synthesis by treatment with mitomycin C prior to infection, more than 80% of the incorporated label is found in superhelical replicative form I molecules. If the incorporation is carried out in the presence of bromodeoxyuridine triphosphate, the radioactive label is found in equal amounts in two classes of hybrid replicative form molecules in which either the viral or the complementary strand is labeled.DNA synthesis depends on a functional gene‐2 product and shows a specific requirement for ATP and magnesium as divalent cation. Incorporation is inhibited by mitomycin C, nalidixic acid, phenyl ethanol, and sulfhydryl‐blocking agents. It is concluded that the observed DNA synthesis occurs by a semi‐conservative replication process resembling replicative form replication in vivo.
The Stimulus‐Secretion Coupling of Glucose‐Induced Insulin Release
Authors: MALAISSE Willy J. , SENER Abdullah , MAHY Marianne 
Published: March 2005

1. The possible significance of the insular sorbitol pathway in glucose‐induced insulin release was investigated.2. Glucose, but neither fructose nor galactose, stimulated sorbitol synthesis by isolated islets. Aldose reductase inhibitors such as 2,4‐dimethylglutaric and 3,3‐tetramethylene glutaric acid abolished the glucose‐induced sorbitol formation, but failed to affect glucose‐induced insulin release. About 75% of the sorbitol formed by the islets was recovered in the incubation medium, the efflux of sorbitol occurring independently of insulin release. Exogenous fructose and sorbitol exerted a modest insulinotropic action characterized by a shift to the left of the sigmoidal curve relating insulin release to glucose concentration. The relative insulinotropic potency of glucose (100%), fructose (16%) and sorbitol (7%) paralleled their rate of oxidation by the isolated islets.3. These data suggest that metabolism through the sorbitol pathway plays little role in the process of glucose‐induced insulin release, although exogenous sorbitol exerts a modest insulinotropic action.
The Peptide Moiety of Blood‐Group‐Specific Glycoproteins
Authors: GOODWIN Shirley D. , WATKINS Winifred M. 
Published: March 2005

A purified glycoprotein with blood‐group‐A specificity, was digested with insoluble pronase to give a serologically active product enriched in peptide‐bonded serine and threonine residues; this was degraded with 0.25 M sulphuric in glacial acetic acid for 24 h. The resultant N‐acetylgalactosaminyl‐peptides were hydrolysed with 0.2 M hydrochloric acid for 24 h at 110°C. Fractionation of the products on Sephadex G‐10, followed by preparative separation on an amino‐acid analyser and paper electrophoresis at pH 1.9 yielded the following homogeneous oligopeptides; one tripeptide with the N‐terminal sequence Thr‐Thr‐Ser, three tetrapeptides with the sequences, Thr‐Ser‐Thr‐Ser, Thr‐Pro‐Thr‐Ser and Pro‐Thr‐Thr‐Ser, one pentapeptide with the sequence Thr‐Pro‐Thr‐Ser‐Ser, one hexapeptide with the sequence Pro‐Thr‐Thr‐Thr‐Pro‐Ser and one heptapeptide with the sequence Ala‐Pro‐Thr‐Thr‐Ser‐Gly‐Ser. Galactosamine was present in several of these fragments and the hexapeptide and heptapeptide both contained sufficient of this sugar for all the serine and threonine residues to be glycosylated.The following five tetrapeptides sequences were deduced from mixtures of two peptides which differed by the amino‐acid substituent at only one position in the chain; Pro‐Thr‐Ala‐Ser, Thr‐Thr‐Pro‐Ser, Thr‐Thr‐Ala‐Ser, Ser‐Pro‐Thr‐Ser, and Ser‐Ala‐Thr‐Ser. A tripeptide mixture which differed only in the nature of the third amino acid yielded the following three tripeptide sequences: Pro‐Thr‐Ser, Pro‐Thr‐Gly and Pro‐Thr‐Ala.These peptides do not indicate a clearly defined amino‐acid sequence surrounding the glycosylated hydroxy amino acids but establish that in the human blood‐group‐specific glycoproteins there is close packing of the serine and threonine residues in the region of the peptide moiety carrying the carbohydrate chains.
A Ribonuclease H from Ustilago maydis
Authors: BANKS Geoffrey R. 
Published: March 2005

A ribonuclease, which can degrade about 90% of the RNA strand of an RNA · DNA hybrid, has been extensively purified from Ustilago maydis. It is highly specific for a calf thymus RNA · DNA hybrid, little or no degradation of the RNA strands occurring after heat denaturation of the hybrid, or of single and double‐stranded DNA. With the exception of poly(rU) · poly(dA), all the ribonucleotide strands in hybrid homopolymers were susceptible to degradation, the double‐stranded homo‐polyribonucleotides being resistant. Both poly(rU) and poly(rI), however, were degraded.The enzyme possesses a molecular weight of 100000–130000 and requires magnesium ions, ammonium sulphate and a sulphydryl reagent for maximum activity. It is an endoribonuclease, releasing oligoribonucleotides terminated by 5′‐phosphate and 3′‐hydroxyl groups, but is unable to remove a ribonucleotide covalently joined to the 3′‐terminus of DNA. Two mutant strains of U. maydis, which are temperature‐sensitive for DNA synthesis, possess elevated levels of the enzyme. This organism contains at least two other enzymes which can degrade the hybrid substrates.
Le centre actif de la 3‐oxostéroide Δ1‐déshydrogénase d'Arthrobacter simplex
Authors: PÉNASSE Lucien , NOMINÉ Gérard 
Published: March 2005

The Active Center of the 3‐Oxosteroid Δ1‐dehydrogenase from Arthrobacter simplexThe kinetic parameters of the semi‐purified, hydrocortisone induced 3‐oxosteroid Δ1‐dehydrogenase from Arthrobacter simplex have been determined with twenty different steroids. The values thus obtained with nortestosterone and six of its stereoisomers and with derivatives bearing substituents at positions 10, 13 and 17, show that for the reaction to take place, first the presence of a 3‐oxo group, a Δ4‐double bond and axial hydrogens at 1α and 2β positions, are necessary, secondly no bulky substituents must be present in the middle part of the α‐face and at position 4; thirdly at positions 10β and 13β no substituents with more than two carbon atoms must be present. With steroids bearing a methyl group in position 10, the chirality of this center has to be natural, and with 19‐norsteroids, the conformation of the whole molecule has to be almost flat.The active center could be represented as a cleft in the protein molecule one side of which is in contact with the α‐face of the steroid, the other side being located a few tenths of nanometer above the β‐face, The Δ4‐double bond is required for the reaction but not for binding. There is no contact point in the area of carbon atom 17.
Studies on Polysome‐Membrane Interactions in Mouse Myeloma Cells
Authors: HARRISON Timothy M. , BROWNLEE George G. , MILSTEIN Cesar 
Published: March 2005

The nature of the interaction of membrane‐bound polysomes with the endoplasmic reticulum has been studied in MOPC 21 mouse plasmocytoma tissue‐culture cells which secrete an immunoglobulin G. Treatment of microsomes at high salt concentrations (500 mM KCl) results in the detachment of 40–45% of the bound ribosomes. This released (“loose”) fraction is predominantly monosomal in contrast to the remaining “tight” polysomes. It is shown that both ribosomal fractions contain immunoglobulin light‐chain mRNA which argues against a functional distinction between them in the cell.Polysomes have been dissociated to apparently undegraded ribosomal subunits at 0°C by incubation with puromycin in the presence of a magnesium ion buffer. Application of this procedure to membrane‐bound polysomes results in partial release of ribosomal subunits from the membrane. The ribosomal material which is not so released sediments predominantly at 60 S and this fraction can be detached by subsequent washing at 500 mM KCl. Thus, by use of the polysome‐dissociating procedure together with high salt concentrations, essentially total release of the ribosomal components can be obtained.The results indicate that the salt‐labile interactions are mediated via the large ribosomal subunits and are the only anchoring forces to the membrane at early stages of protein synthesis. The tight membrane‐bound polysomes result from the additional anchoring effect of the polypeptide chains as they grow through the membrane.