bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determination of ADP‐ribose and Poly(ADP‐ribose) by a New Radioimmunoassay
Authors: BREDEHORST Reinhard , FERRO Ari M. , HILZ Helmuth 
Published: March 2005

A specific and sensitive radioimmunoassay for ADP‐ribose has been developed on the basis of the selective conversion of ADP‐ribose to 5′‐AMP by alkaline treatment. Antibodies highly specific against 5′‐AMP allowed quantification of ADP‐ribose converted to 5′‐AMP in the range of 1–40 pmol, and in the presence of large quantities of nucleic acids or 3′‐AMP.Poly(ADP‐ribose) could also be determined when degraded to ADP‐ribose by poly(ADP‐ribose) glycohydrolase. Determination of the chain length of purified polymer was possible by a parallel determination of ADP‐ribose residues after glycohydrolase treatment and of 5′‐AMP from the nonreducing end obtained by phosphodiesterase catalyzed hydrolysis.The high specificities of the alkaline conversion of ADP‐ribose to 5′‐AMP and of the radioimmunoassay for 5′‐AMP allowed quantification of protein‐bound ADP‐ribose residues in crude tissue extracts as verified by comparison with chromatographically purified samples.
Characteristics of a Coupled Cell‐Free Transcription and Translation System Directed by Vaccinia Cores
Authors: PELHAM Hugh R. B. , SYKES Jane M. M. , HUNT Tim 
Published: March 2005

A coupled transcription and translation system is described in which protein synthesis is directed by mRNA synthesised in situ by vaccinia virus cores. The cell‐free system is based on a micrococcal‐nuclease‐treated reticulocyte lysate.
The polypeptides made in vitro include many authentic early vaccinia proteins, but also other proteins which were not detected in infected cells.
Concentrations of cores which inhibit host cell protein synthesis in vivo caused a delayed inhibition of translation in vitro; this was partly, but not entirely, due to dsRNA associated with the cores.
The mRNA made was methylated by core enzymes. Inhibition of methylation reduced the rate of translation tenfold; unmethylated RNA bound ribosomes poorly, but was nevertheless translated faithfully.
The Primary Structure of Bovine Pancreatic Phospholipase A2
Authors: FLEER Eduard A. M. , VERHEIJ Hubertus M. , DE HAAS Gerard H. 
Published: March 2005

The complete amino acid sequence of bovine phospholipase A2 (EC 3.1.1.4) was determined. This enzyme has a molecular weight of 13 782 and consists of a single polypeptide chain of 123 amino acids cross‐linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digesting the reduced and thialaminated derivative of the protein with trypsin, staphylococcal protease and cyanogen bromide. A number of chymotryptic peptides were used for alignment and to obtain overlaps of at least two residues.The sequence of the peptides was determined by Edman degradation by means of direct phenylthiohydantoin identification in combination with identification as dansyl amino acids.Although 71% of all residues of phospholipase A2 from bovine, porcine and equine sources are conserved, bovine phospholipase A2 differs from the others by the total number of residues and by substitutions at 20 (porcine) and 33 (equine) positions.
A Universal Lipid Exchange Protein from Rat Hepatoma
Authors: DYATLOVITSKAYA Ella V. , TIMOFEEVA Natalya G. , BERGELSON Lev D. 
Published: March 2005

The postmicrosomal protein fraction from rat hepatoma 27 adjusted to pH 5.1 stimulates phospholipid exchange between rat liver microsomes and mitochondria with higher rates and in a less specific way than the corresponding fraction from rat liver. A phospholipid exchange protein has been purified to homogeneity from the hepatoma pH‐5.1 supernatant by gel filtration on Sephadex G‐75 and ion‐exchange chromatography on carboxymethylcellulose. The isolated protein had a molecular weight of 11 200 as determined by electrophoresis on polyacrylamide in the presence of dodecyl sulfate and of 11 168 as calculated from the amino acid composition. Isoelectric focusing showed a single band at pH 5.2.In the assay system rat liver microsomes → mitochondria the protein exhibits a complete lack of substrate specificity transferring all the major microsomal phospholipids to about the same extent. The possible role of the isolated phospholipid exchange protein in the chemical dedifferentiation of hepatoma cell membranes is discussed.
A Hydrogen‐Deuterium Exchange Study of the Amide Protons of Polymyxin B by Nuclear‐Magnetic‐Resonance Spectroscopy
Authors: PERKINS Stephen J. , RADDA George K. , RICHARDS Rex E. 
Published: March 2005

Proton magnetic resonance spectra at 270 MHz of polymyxin B, a cationic oligopeptide antibiotic, show the influence of the inorganic counteranion present in solution.
Hydrogen‐deuterium exchange rates for the amide protons are of two types, depending on whether the anion is monovalent or polyvalent. Polyvalent anions catalyse the acid‐catalysed reaction more than the monovalent anions.
The structure in solution was monitored using the proton signals of the amides, the phenylalanine aromatic protons, and the leucine methyl and γ‐CH protons in several polymyxin salts. The temperature coefficients of the chemical shifts of the N‐H protons are used to identify two β turns in the cyclic ring of polymyxin B. The variation in chemical shift of the N‐H protons, the aromatic protons and the leucine protons are correlated with anionic size and electronegativity.
Do Mitochondria Produce Oxygen Radicals in vivo?
Authors: NOHL Hans , HEGNER Dietmar 
Published: March 2005

Heart mitochondria from rats at 3 months and 23 months of age were investigated for their capacity to generate oxygen radicals and hydrogen peroxide.
Highest values for O2+‐formation were obtained with submitochondrial particles freed from superoxide dismutase after the addition of succinate and antimycin A to start the reaction. Under these conditions superoxide‐radical formation with mitochondria from old rats (2.54 nmol × min−1× mg−1) exceeded formation rates in the young controls (1.9 nmol × min−1× mg−1) by 25%.
A constant fraction of 20% of the radicals produced escaped quenching by intramitochondrial superoxide dismutase. This fraction was independent of the rate of radical formation; its magnitude was deduced from generation rates of hydrogen peroxide in the presence and absence of exogenous superoxide dismutase.
Free oxygen radicals could be obtained with intact rat‐heart mitochondria as well following the addition of ATP, a system which is closer to physiological states. Formation rates of oxygen radicals observed under these conditions were similar to those seen in the particulate fractions which escaped quenching by mitochondrial superoxide dismutase.
Peroxidative degradation of membrane lipids was found to parallel steady‐state concentrations of free oxygen radicals.
The results are discusssed in terms of a radical‐generating mechanism which functions in vivo at the level of the mitochondrial electron‐transferring system.
Evidence for the Involvement of a Glucose‐6‐Phosphate Carrier in Microsomal Glucose‐6‐Phosphatase Activity
Authors: NILSSON Olle S. , ARION William J. , DEPIERRE Joseph W. , DALLNER Gustav , ERNSTER Lars 
Published: March 2005

Protease and diazobenzene sulfonate have been used to probe the transverse topology of the microsomal glucose‐6‐phosphatase system.
Treatment of intact microsomes with these reagents under the conditions used here did not affect the permeability of the membrane to mannose 6‐phosphate, nucleoside diphosphatase, or dextran of 70 000 molecular weight. Nor did these treatments inactivate the hydrolytic site of glucose‐6‐phosphatase, a finding in agreement with earlier conclusions that this site is on the inside of the membrane.
On the other hand, treatment of intact microsomes with diazobenzene sulfonate or proteases does inactivate (or increase the apparent Km of) some other component which is rate‐limiting for glucose‐6‐phosphatase activity in intact but not in disrupted microsomes. The simplest explanation for this phenomenon is that there is a protein carrier in the microsomal membrane which transports glucose 6‐phosphate from the medium to the lumen, where it is hydrolyzed, and that diazobenzene sulfonate and proteases attack this carrier.
The lack of effect of these reagents on microsomal inorganic pyrophosphatase activity suggests that the glucose‐6‐phosphate carrier cannot transport pyrophosphate.
Finally, it was found that treatment of microsomes with ammonia breaks down their permeability barrier but also removes significant amounts of microsomal phospholipid and inactivates a number of microsomal enzymes. We do not recommend it as a general approach to altering microsomal permeability.
Nature of the Collagenous Protein in a Tumor Basement Membrane
Authors: TIMPL Rupert , MARTIN George R. , BRUCKNER Peter , WICK Georg , WIEDEMANN Hanna 
Published: March 2005

The EHS sarcoma (named after its discoverer, Engelbreth‐Holm, and its primary characterizer, R. Swarm) contains an extracellular matrix of basement membrane. The collagenous component of the membrane was extracted with dilute acetic acid from tumors grown in lathyritic mice and separated from other proteins by chromatographic methods. This collagen has a composition resembling that reported for other basement membrane collagens. The polypeptide chains of the tumor collagen are linked by disulfide bonds and migrate after reduction intermediate between the α chains und β components of type‐I collagen. The circular dichroism spectra obtained from it resemble those obtained from other collagens.The intact protein contained about 280 residues of glycine per 1000 residues. After reduction of disulfide bonds in the basement membrane collagen, treatment with either pepsin or trypsin gave rise to resistant fragments containing one‐third glycine. These fragments formed segment‐long‐spacing crystallites 210 nm long in the case of those treated with pepsin and 350 nm long in the case of those treated with trypsin. The band pattern differs from that found with other collagens.Antibodies prepared against the tumor collagen were found to localize to the tumor matrix and to known basement membranes occurring in mouse tissues. The data suggest that these basement membranes may contain a collagenous protein similar to the tumor protein.
Synthesis of an Oligonucleotide Inhibitor of Protein Synthesis in Rabbit Reticulocyte Lysates Analogous to that Formed in Extracts from Interferon‐Treated Cells
Authors: HOVANESSIAN Ara G. , KERR Ian M. 
Published: March 2005

A heat‐stable, low‐molecular‐weight inhibitor of protein synthesis is formed on incubation of haemin‐supplemented rabbit reticulocyte lysates with ATP and double‐stranded RNA (dsRNA). It inhibits the translation of both added encephalomyocarditis virus RNA (EMC RNA) and endogeneous messenger RNA in reticulocyte lysates and mouse L‐cell extracts.The enzyme responsible for the synthesis of the inhibitor binds to dsRNA and can be purified on a column of poly(I) · poly(C) bound to an inert support. The highly purified enzyme in its stable column‐bound state can be conveniently employed to synthesise the inhibitor and to label it with [3H]ATP, or [α‐32P]ATP or [γ‐32P]ATP as substrate. The radioactive inhibitor synthesised in this way with material from rabbit reticulocyte lysates shows the same spectrum of resistance and sensitivity to alkali and a variety of enzymes as corresponding material similarly synthesised with extracts from interferon‐treated mouse L‐cells. The inhibitors from the two systems have comparable absorbance spectra, are chromatographically and electrophoretically indistinguishable and are apparently identical in specific activity in the inhibition of protein synthesis in the cell‐free system.The inhibitor is also formed on inhibition of protein synthesis by dsRNA in reticulocyte lysates. On comparison of the spectrum of polypeptide products synthesised in response to EMC RNA in the reticulocyte lysate, the effects of the inhibitor or dsRNA were similar: a distinctly different effect was obtained with the haemin‐controlled repressor, a known inhibitor of initiation. The significance of these results with respect to the mechanism of action of the inhibitor and its role in the inhibition observed in response to dsRNA is discussed.
Yeast DNA Polymerases: Antigenic Relationship, Use of RNA Primer and Associated Exonuclease Activity
Authors: WINTERSBERGER Erhard 
Published: March 2005

Highly purified preparations of DNA polymerases A and B from yeast were compared with respect to antigenic relationship, ability to use ribonucleotide primers and associated nuclease activity. The following results were obtained.

Antiserum directed against DNA polymerase A inhibits this enzyme but does not interfere with the activity of DNA polymerase B or of mitochondrial DNA polymerase, nor does it precipitate the latter two enzymes.
DNA polymerase A is capable of using oligo(ribouridylic acid) as a primer for the polymerization of dTMP. This reaction is not catalysed by polymerase B to any significant extent.
Whereas DNA polymerase A is devoid of nuclease activity, DNA polymerase B catalyses an exonucleolytic release of mononucleotide units from the 3′ end of polynucleotides. The results of several experiments suggest that this nuclease activity is associated with the DNA polymerase B molecule.
N‐Acetylglutamate Synthetase from Rat‐Liver Mitochondria
Authors: SHIGESADA Katsuya , TATIBANA Masamiti 
Published: March 2005

N‐Acetyl‐l‐glutamate synthetase was purified approximately 300‐fold from the extract of rat liver mitochondria. The enzyme shows a high substrate specificity for l‐glutamate and acetyl‐CoA. No or very low activity is observed with other amino acids and acyl compounds. The reaction velocity fits normal Michaelis‐Menten kinetics with respect to both l‐glutamate (Km, 3.0 mM) and acetyl‐CoA (Km, 0.7 mM).Acetylglutamate, the reaction product, and some of its structural analogues, such as N‐acetylaminoadipate, N‐propionylglutamate, and N‐acetylglutamine, significantly inhibited the enzyme reaction. The Ki for acetylglutamate was 0.07 mM. The enzyme activity is sensitive to thiol‐blocking agents and is inhibited by divalent cations.The enzyme activity is markedly stimulated by arginine, presumably by an allosteric mechanism. Other intermediates of the urea cycle and various structural analogues of arginine were practically not effective. Arginine increases the maximal velocity with no influence on Km values for the substrates. The activation shows a sigmoidal dependence on arginine concentrations and reaches a half‐maximal level at 5–10 μM. The enzyme behaves as a mixture of multiple forms upon gel filtration (the range of molecular weight, 30000 to 300000) and its mean value is considerably increased in the presence of arginine.The specific activity of the enzyme increases with higher enzyme concentrations in the assay. The effect is more marked in the presence of arginine than in its absence. These observations suggest that the enzyme undergoes association and dissociation with concomitant changes in catalytic properties.
A Study of Unwinding of DNA and Shielding of the DNA Grooves by RNA Polymerase by Using Methylation with Dimethylsulphate
Authors: MELNIKOVA Anna F. , BEABEALASHVILLI Robert , MIRZABEKOV Andrei D. 
Published: March 2005

The dimethylsulphate method has been used to study the complexes of RNA polymerase (Escherichia coli) with DNA of T7 phage, poly[d(A–T)] and fragments of calf thymus DNA protected against DNase digestion by RNA polymerase. The binding of RNA polymerase to DNA significantly increases the formation of 1‐methyl‐adenine produced by methylation of the single‐stranded DNA region, diminishes by about 10% the formation of 3‐methyl‐adenine by methylation within the minor groove and does not affect the formation of 7‐methyl‐guanine by methylation within the major DNA groove. The presence of nascent RNA decreases the formation of 1‐methyl‐adenine in DNA of the complex by about 30%. The initiation of RNA synthesis or RNA synthesis itself does not influence the methylation of the major groove but shielding of the minor groove increases by about twice as much.These results suggest that RNA polymerase, upon binding, breaks Watson‐Crick base‐pairing in a DNA region of about 15‐base‐pairs long, that nascent RNA forms a duplex with DNA of about 10‐base‐pairs long; and that the enzyme weakly interacts with DNA along its grooves and preferentially makes contacts with the minor groove.
Interaction of the Acetylcholine (Nicotinic) Receptor Protein from Torpedo marmorata Electric Organ with Monolayers of Pure Lipids
Authors: POPOT Jean‐Luc , DEMEL Rudy A. , SOBEL André , VAN DEENEN Laurens L. M. , CHANGEUX Jean‐Pierre 
Published: March 2005

Membrane fragments rich in cholinergic (nicotinic) receptor protein were purified from the electric organ of Torpedo marmorata. Their lipid composition is essentially characterized by the prominence of cholesterol, phosphatidylethanolamine and phosphatidylcholine, long‐chain fatty acyl constituents, and the absence of sphingomyelin. Solubilised receptor was purified from these fragments and the concentrations of sodium cholate lowered by dialysis to 0.01% (w/v). When this preparation was injected under a lipid monolayer, an increase of surface pressure developed, which was not observed with the detergent alone nor in the absence of lipid film. When covalently radiolabelled receptor preparations were injected at a constant surface pressure the radioactivity recovered with the film was proportional to the increase in area. It is concluded that the pressure or area increases are due to the penetration of the cholinergic receptor protein into the lipid film.Incorporation experiments into films formed from various pure lipids showed that the protein interacts more readily with cholesterol than with ergosterol, phosphatidylcholine, or other phospholipids. Its affinity is also higher for long‐chain phosphatidylcholines than for short‐chain ones. The degree of unsaturation and fluidity of the 3‐sn‐phosphatidylcholine (lecithin) films are of secondary importance. Parallel experiments with covalently and non‐covalently labelled receptor preparations showed that part of the protein recovered with the film lost its α‐toxin binding ability during the penetration.Similar data were obtained with the receptor purified from Electrophorus electricus electric organ.
Phosphoglycerate Kinase B from Ram Testis
Authors: STEWART Andrew A. , SCOPES Robert K. 
Published: March 2005

The testis‐specific isoenzyme of phosphoglycerate kinase (phosphoglycerate kinase B) has been isolated from ram testes using a procedure which separates it from ‘normal’ phosphoglycerate kinase which is also present in testis tissue. The purification procedure is described.
The best preparations had no detectable impurity on electrophoresis, and had specific activities comparable with the same enzyme from other sources.
Kinetic studies indicated that the two isoenzymes have identical properties, within experimental error, for substrate affinity (for MgATP, 3‐phosphoglycerate and MgADP), energy of activation and thermal denaturation.
The molecular weights of both isoenzymes were not distinguishably different from those previously reported, as measured by polyacrylamide/dodecylsulphate electrophoresis. The amino acid compositions showed only slight differences, and tryptic peptide maps showed that there was close homology of sequence. Starch gel electrophoresis at pH 6.5 indicates that the B isoenzyme has 1–2 more positive charges than the A.
Phosphoglycerate kinase A isolated from sheep muscle was shown, within experimental error, to be identical to the phosphoglycerate kinase A isolated from testis.
The results further substantiate the suggestion that the B isoenzyme is coded by a gene which was duplicated from the phosphoglycerate kinase A gene.
Effect of Methylamine on Trypsin Catalysis
Authors: VAJDA Tamás , SZABÓ Tibor 
Published: March 2005

The effect of the basic group of specific substrates of trypsin on the kinetics of the enzyme was studied. The basic group was substituted by methylamine added to a non‐specific substrate. The kinetic constants of tryptic hydrolysis of N‐acetyl‐l‐alanine ethyl ester involving methylamine, at pH 6.3–8.5 were determined. For comparison, the constants of trypsin and α‐chymotrypsin were also determined.It can be concluded that methylamine increases the rate of hydrolysis catalyzed by trypsin. The relative increase of the pH‐independent limiting value of (kc/Km)max is shown to be fivefold and the pKa decreases by 0.4 unit. The increase of kc is nearly fourfold. The rate constants of the hydrolysis steps catalyzed by trypsin and trypsin · methylamine, respectively, were assayed by means of the nucleophile 1,4‐butanediol at pH 6.6. There is a sixfold increase of the original rates caused by methylamine for acylation (k2) and a threefold increase for deacylation (k3) but the substrate dissociation constant (Ks) was almost unchanged.A comparison of the individual rates of the methylamine‐activated tryptic hydrolysis of nonspecific substrates with the appropriate data of the trypsin‐catalyzed hydrolysis of specific substrates indicates that there is an approximately 1000‐fold difference between the rate constants for the acylation step, while in deacylation the difference is insignificant.The methylammonium ion bound to trypsin is supposed to keep the enzyme through the catalysis in a more active conformation than the original one. This indirect effect might bear only a small part of the trypsin‐substrate interaction.
Particulate Tyrosinase of Human Malignant Melanoma
Authors: NISHIOKA Kenji 
Published: March 2005

Studies of mammalian tyrosinase have been conducted principally with soluble forms (T1 faster, T2 slower migrating by gel electrophoresis). However, a majority of tyrosinase from mouse and human melanoma tissue is of a particulate (T3) nature, being bound to melanosomes. In addition, T1 and T2 enzymes have been considered isozymes with T3 being a separate enzyme.Using human malignant melanoma tissue, solubilization and purification of particulate tyrosinase have been attempted. Melanosome‐bound tyrosinase was solubilized with sodium cholate and purified 1500‐fold by ammonium sulfate fractionation, DEAE‐cellulose column chromatography, concanavalin A affinity chromatography, and Sephadex G‐150 gel filtration. Tryptic cleavage of this solubilized tyrosinase yielded a fast migrating tyrosinase by gel electrophoresis. This trypsin‐cleaved tyrosinase was purified by Sephadex G‐150 and DEAE‐cellulose columns with an apparent final yield of 56% and an approximate 10000‐fold purification. This preparation gave a single band by 7% gel electrophoresis and 5% sodium dodecyl sulfate gel electrophoresis. In addition, electrophoresis indicated that this tyrosinase was a glycoprotein showing positive staining by periodic acid/Schiff stain. This enzyme oxidized both l‐tyrosine and l‐3,4‐dihydroxyphenylalanine at a similar rate. The molecular weight was approximated at 66 700 by sodium dodecyl sulfate gel electrophoresis. This enzyme migrated to the T1 position by gel electrophoresis; however, upon treatment with neuraminidase, it shifted to the T2 position. Sialic acid content was determined as 4.8%. Copper content was estimated as 2 atoms/molecule. These results tend to support the dynamic conversion of T3→ T1→ T2 rather than their existence as separate enzymes.