bluuebunny/crossref_metadata_2025_split huggingface dataset:
Metabolism of γ‐Glutamyl Amino Acids and Peptides in Mouse Liver and Kidney in vivo
Authors: ORLOWSKI Marian , WILK Sherwin 
Published: March 2005

The metabolism in vivo of γ‐glutamyl amino acids and peptides was studied in the mouse after administration of loading doses of l‐γ‐glutamyl‐2‐aminobutyrate and several other γ‐glutamyl compounds, including glutathione. A great and rapid accumulation of glutamate, glutamine, aspartate and pyrrolidone carboxylate was observed in the kidney. Similarly, after administration of a tracer dose of l‐γ‐[14C]glutamyl‐l‐2‐aminobutyrate a rapid incorporation of label into kidney glutamate, glutamine and aspartate was found. These results suggest that both the hydrolytic and γ‐glutamyl transfer reactions catalyzed by γ‐glutamyl transpeptidase are active in the renal handling of γ‐glutamyl compounds.Indirect evidence was obtained that l‐γ‐glutamyl‐2‐aminobutyrate is partially taken up by the kidney cell in an intact form.In contrast to the kidney, administration of several γ‐glutamyl derivatives did not cause an increase in liver glutamate, glutamine and pyrrolidone carboxylate. After administration of l‐γ‐glutamyl‐2‐aminobutyrate only a slight increase in liver aspartate and pyrrolidone carboxylate was observed. Experiments with l‐γ‐[14C]glutamyl‐l‐2‐aminobutyrate suggest that this derivative is largely first degraded to its component amino acids (probably in the kidney) before entering into the metabolism of the liver cell.γ‐Glutamyl transpeptidase may function in the metabolism and transport of glutathione and other γ‐glutamyl compounds in a manner analogous to the function of dipeptidases and disaccharidases in the metabolism and transport of dipeptides and disaccharides respectively.
Effect of the Immediate Environment on the Reactivity of the Essential—SH Group of Papain
Authors: HALÁSZ Piroska , POLGÁR László 
Published: March 2005

The effect of the microenvironment on the reactivity of the essential—SH group of papain was studied by alkylation with methyl iodide and with the more polar iodoacetamide. Rate and activation parameters for these reactions were determined with two forms of the—SH group: the free mercaptide ion at pH 10.0, and the mercaptide‐imidazolium ion‐pair at pH 5.5.The ion‐pair of papain reacts with methyl iodide at a rate 1470 times less than that of thiolsubtilisin. This suprising difference between the reactivities of the two enzymes suggests that in contrast to thiolsubtilisin, where a non‐polar environment enhances the rate, in the case of papain a more polar environment somewhat inhibits the reaction with the non‐polar methyl iodide. The positive activation entropy for the papain reaction may indicate an ‘ordered’ structure of bound water around the sulfur atom.The high rate and the low activation entropy (organized transition state) of the reaction of papain with iodoacetamide can be explained in terms of hydrogen‐bond formation between the enzyme and the amide group of the alkylating agent.
Proton‐Nuclear‐Magnetic‐Resonance Study of the Conformation of Neurotoxin II from Middle‐Asian Cobra (Naja naja oxiana) Venom
Authors: ARSENIEV Aleksandr S. , BALASHOVA Tamara A. , UTKIN Yurii N. , TSETLIN Viktor I. , BYSTROV Vladimir F. , IVANOV Vadim T. , OVCHINNIKOV Yurii A. 
Published: March 2005

A proton nuclear magnetic resonance (NMR) study at 100 and 300 MHz of neurotoxin II from the venom of Middle‐Asian cobra Naja naja oxiana has been performed in 2H2O and H2O solutions. By means of chemical modification and double resonance all the aromatic residue resonances have been assigned. From the NMR titration curves, pK values of histidine 4 and histidine 31 residues have been determined. For one of the two neighbouring tryptophan residues pH dependence (in the 2–8‐pH range) of the chemical shifts of indole protons has been revealed. According to the different sensitivity of the linewidth of indole NH resonances to pH in H2O solution, the accessibility of each of the tryptophan residues has been estimated. Temperature dependence has been observed for the linewidth of the aromatic resonances of the tyrosine 24 residue. Deuterium exchange rates have been measured for amide protons as well as for C(2)H histidine resonances.The NMR data obtained have allowed the conclusions to be made that the two histidine residues and one of the tryptophan residues should be localized on the surface of the protein globule, that arginine residues should be present in the environment of histidine 4, that histidine 31 and the buried tryptophan are possibly localized in close spatial proximity and that the side chain of tyrosine 24 is buried within the protein globule.
The Effect of Initiation Factor IF‐1 on the Dissociation of 70‐S Ribosomes of Escherichia coli
Authors: NAAKTGEBOREN Nico , ROOBOL Kees , VOORMA Harry O. 
Published: March 2005

By means of exchange studies, in which 3H‐labelled 50‐S subunits and unlabelled 70‐S ribosomes from Escherichia coli MRE 600 were used, it has been demonstrated that the 30‐S subunit is the only target for IF‐3 in the dissociation of 70‐S ribosomes. The interference of IF‐3 with the dynamic equilibrium of 70‐S ⇌ 50‐S + 30‐S occurs by binding of the factor to the 30‐S subunit. The 30‐S. IF‐3 complex in impaired in the association reaction, which implies that IF‐3 is acting as an anti‐association factor.The action of IF‐1 is two‐fold. Firstly IF‐1 increases the rate of exchange of the ribosomal sub‐particles in the 70‐S ribosome without changing the position of the equilibrium. Thus the spontaneous equilibrium is attained more rapidly in the presence of IF‐1. This kinetic effect of IF‐1 is also demonstrated in the IF‐3‐mediated dissociation of 70‐S ribosomes. Secondly IF‐1 is able to increase the IF‐3‐mediated dissociation. It seems likely that the explanation for the latter phenomenon must be sought in the binding of IF‐1 to 70‐S ribosomes, resulting in a loosening of the ribosome structure, as well as to 30‐S. IF‐3 complex, thereby slowing down the association reaction of the subunits.
Kinetics and Regulation of Sarcoplasmic Reticulum ATPase
Authors: DUPONT Yves 
Published: March 2005

The measurement of ATP binding to the sarcoplasmic reticulum membrane reveals that the calcium pump possesses one high affinity (Kd= 2–3 μM) site. Competition with substrate analogs show the high specificity of that site. At high ATP concentration another class of site can be detected with a much higher dissociation constant (Kd∼ 500 μM). This class of sites is of low specificity and ATP is easily displaced by other polyphosphates.The steady state rate of ATP cleavage is measured in the presence of ATP analogs. It is shown that the catalysis is due to the high affinity site. The activation of the hydrolysis rate at high substrate concentration may be related to the effect of binding of ATP to the weak sites. The effect of ATP analogs for various ATP concentration supports this hypothesis.
Ligand‐Dependent Bohr Effect of Chironomus Hemoglobins
Authors: STEFFENS Guy , BUSE Gerhard , WOLLMER Axel 
Published: March 2005

The O2 and CO Bohr effects of monomeric and dimeric hemoglobins of the insect Chironomus thummi thummi were determined as proton releases upon ligation.For the O2 Bohr effect of the monomeric hemoglobin III a maximum value of 0.20 H+/heme was obtained at pH 7.5. Upon ligation with CO, however, only 0.04 H+/heme were released at the same pH. In agreement with this finding isoelectric focusing experiments revealed different isoelectric points for O2‐liganded and CO‐liganded states of hemoglobin III.Analogous results were obtained in the cases of the monomeric hemoglobin IV and the dimeric hemoglobins of Chironomus thummi thummi: here O2 Bohr effects of 0.43 and 0.86 H+/heme were observed. For the corresponding CO Bohr effects values of 0.08 and 0.31 H+/heme were obtained respectively.On the basis of the available structural data the reduced CO Bohr effect in hemoglobin III is discussed as arising from a steric hindrance of the CO ligand by the side chain of isoleucine‐E11, obstructing the movement of the heme‐iron upon reaction with carbon monoxide. It should, however, be noted that ligands, according to their different electron donor and acceptor properties, may generally induce different conformational changes and thus different Bohr effects, in those hemoglobins in which distinct tertiary and/or quaternary constraints have not evolved.The general utilization of CO instead of O2 as allosteric effector is ruled out by the results reported here.
Photooxidation of Human Serum Albumin and Its Complex with Bilirubin
Authors: PEDERSEN Anders Overgaard , SCHØNHEYDER Fritz , BRODERSEN Rolf 
Published: March 2005

Irradiation with visible light of human serum albumin in aqueous solution at pH 8, in the presence of catalytic amounts of rose bengal or methylene blue, resulted in random oxidation of the histidine residues in the protein under consumption of one mole O2, and release of somewhat less than one proton, per histidine residue degraded. An increase of light absorption at 250 nm was proportional to the amount of oxygen consumed. Bilirubin bound to the oxidized protein showed an increased light absorption at its maximum, 460 nm, and a decreased binding affinity, indicating a conformational change of the protein on oxidation of histidine residues. This change also resulted in a slight perturbation of tyrosine light absorption, corresponding to a shift of the chromophore to more polar surroundings. Further, a sensitized oligomerization of albumin was observed, independent of oxidation of the histidine residues, and not consuming oxygen.Irradiation of a complex of human serum albumin with one molecule of bound bilirubin, in the absence of a sensitizing dye, resulted in a fast, non‐oxygen consuming process whereby the light absorption maximum of the pigment was shifted 4 nm towards longer wavelength and part of the bilirubin was converted to a more polar pigment, bound less firmly to the protein. This was followed by a relatively slow oxidation of the pigment under uptake of one mole O2. Parallel photooxidation of the protein carrier could not be detected.It is considered possible that the fast, anaerobic process is operative in phototherapy of hyper‐bilirubinemia in the newborn. Serum albumin is probably not oxidized during this treatment.
Stereospecificity of Phenylyruvate Tautomerase
Authors: RÉTEY Jànos , BARTLV Knut , RIPP Engelbert , HULL William E. 
Published: March 2005

By 1H nuclear magnetic resonance spectroscopy it has been shown that phenylpyruvate tautomerase from beef kidney catalyses the stereospecific exchange of one of the enantiotopic 3‐H atoms in the side‐chain of the substrate with solvent protons, the rate of spontaneous exchange being slow under physiological conditions.
Using monodeuterated phenylpyruvate of known absolute configuration it has been shown that the tautomerase removes specifically the 3‐pro‐R hydrogen atom of the substrate.
The circular dichroism spectra of the two enantiomeric 3‐phenyl‐[3‐2H]pyruvates have been determined.
Some implications of these findings for the investigation of the metabolism of aromatic amino acids are discussed.
Preparation and Coenzymic Activity of Soluble Polyethyleneimine‐Bound NADP+ Derivatives
Authors: ZAPPELLI Piergiorgio , PAPPA Rosario , ROSSODIVITA Antonio , RE Luciano 
Published: March 2005

Alkylation at N‐1 of the NADP+ adenine ring with 3,4‐epoxybutanoic acid gave 1‐(2‐hydroxy‐3‐carboxypropyl)‐NADP+. Enzymic reduction of the latter, followed by alkaline Dimroth rearrangement and enzymic reoxidation, gave N6‐(2‐hydroxy‐3‐carboxypropyl)‐NADP+. On the other hand, bromination at C‐8 of the NADP+ adenine ring, followed by reaction with the disodium salt of 3‐mercaptopropionic acid, gave 8‐(2‐carboxyethylthio)‐NADP+. Carbodiimide coupling of the three carboxylic NADP+ derivatives to polyethyleneimine afforded the corresponding macromolecular NADP+ analogues.The carboxylic and the polyethyleneimine derivatives synthesized have been shown to be co‐enzymically active with yeast glucose‐6‐phosphate dehydrogenase, liver glutamate dehydrogenase and yeast aldehyde dehydrogenase. The degree of efficiency relative to NADP+ with the three enzymes ranged from 17% to 100% for the carboxylic derivatives and from 1% to 36% for the polyethyleneimine analogues. On comparing the efficiencies with the three enzymes of the N‐1 derivatives to the one of the corresponding N6 and C‐8 analogues, the order of activity was N‐1 > N6 > C‐8, except in the case of the carboxylic compounds with glutamate dehydrogenase, where this order was inverted. None of these modified cofactors were active with pig heart isocitrate dehydrogenase.
Purification and Properties of Blood‐Group‐Specific Lectins fromVicia cracca
Authors: RÜDIGER Harold 
Published: March 2005

The lectins from the seeds ofVicia craccareact specifically with human blood group A erythrocytes. They were purified by affinity chromatography on an adsorbent containing matrix‐boundN‐acyl‐d‐galactosamine. By a continuous pH gradient the lectins could be separated into two fractions each of which was shown to consist of several agglutinating species.The behaviour of both fractions in affinity chromatography was paralleled by the pH dependence of the interaction with the hapten sugarN‐acetyl‐d‐galactosamine. Both lectin fractions have the same molecular (125000) and subunit (33000) weights, display the same pH dependence of their titre against A1erythrocytes, and bind toN‐acetyl‐d‐galactosamine at pH 8 with the same constant of about 6 × 103M−1.
RNA Polymerase and RNA Synthesis in the Silk Glands of the Silkworm Bombyx mori
Authors: SRIDHARA S. , PORTIER Marie‐madeleine , DAILLIE Jacques 
Published: March 2005

Total RNA polymerase activity, as well as the proportion of a‐amanitin‐sensitive and resistant during activity, have been measured in the posterior silk glands of the silkworm as a function of growth the fifth larval instar. During the first 5 days, termed the growth phase, the total enzyme activity and particularly the portion that is α‐amanitin‐resistant increases to reach a peak value and thereafter declines during the secretory phase. Much of the enzyme remains firmly bound and insoluble. Heparin not only does not inhibit this insoluble and probably chromatin‐bound activity which would indicate lack of initiation, but it enhances the activity. A large proportion of newly transcribed RNA is released from the transcription complex.The synthesis of RNA has been studied both qualitatively and quantitatively during the same period. RNA synthesis becomes important on the second day of the fifth instar, as does the RNA polymerase, and stays at a high level for several more days. The results from these studies as well as those with incorporation of 32P indicate interference of varying precursor pools in quantitatively measured RNA synthesis. However, RNA content as well as RNA synthesis in vitro show a close correlation with RNA polymerase activity. The labeled RNAs extracted at different days of the fifth instar have been fractionated on sucrose gradients; this demonstrated that the predominant product of RNA synthesis, as followed by [3H]uridine incorporation at short time intervals, is 45‐S preribosomal RNA and 4‐5‐S RNA. This 45‐S RNA is transformed to 19‐S and 30‐S ribosomal RNA as time progresses or after a chase with unlabeled uridine and/or actinomycin D. There also exists a component heavier than 45 S which is fairly rapidly labeled to a small extent.
Terminal Nucleotide Sequences of 17‐S Ribosomal RNA and Its Immediate Precursor 18‐S RNA in Yeast
Authors: DE JONGE Peter , KLOOTWIJK Jacobus , PLANTA Rudi J. 
Published: March 2005

The 5′ and 3′‐terminal nucleotide sequences of 17‐S rRNA and its immediate precursor 18‐S RNA from the yeast Saccharomyces carlsbergensis have been analysed. Identification of the terminal oligonucleotides, as present in Ti ribonuclease digests, was performed by diagonal procedures.The major (molar yield 0.9) 5′‐terminal oligonucleotide of mature 17‐S yeast rRNA proved to be pU‐A‐U‐C‐U‐G. In addition a minor 5′‐terminal oligonucleotide (molar yield 0.15) with the overall composition pU(U2,C2)G was observed. 18‐S precursor RNA was found to contain the same 5′‐terminal sequences as 17‐S rRNA. However, the 3′‐terminal sequences of the two types of RNA appeared to be different. The 17‐S rRNA yields the oligonucleotide A‐U‐C‐A‐U‐U‐AOH while at least half of the 18‐S RNA molecules contain the sequence U‐U‐U‐C‐A‐A‐U‐AOH. In addition 18‐S RNA yields several minor 3′‐terminal oligonucleotides which appear to be structurally related to the major 3′‐terminal sequence. These results demonstrate that the extra nucleotides in 18‐S RNA relative to 17‐S RNA are located exclusively at the 3′‐terminus of the 18‐S RNA molecule.The possibility that the 3′‐terminal nucleotide sequence of 18‐S RNA plays a role in the maturation process is discussed.
2′‐Deoxy‐2′‐azidocytidine, a New Inhibitor of DNA Replication in Mammalian Cells
Authors: SKOOG Lambert , BJURSELL Gunnar , THELANDER Lars , HÄGERSTRÖM Torsten , HOBBS John , ECKSTEIN Fritz 
Published: March 2005

Cell growth is reversibly inhibited by the nucleoside analogue, 2′‐deoxy‐2′‐azidocytidine and the inhibition is a result of interference with DNA replication. The 5′‐diphosphate of the analogue was earlier shown to specifically inactivate the enzyme ribonucleotide reductase in vitro. However, measurements of the pools of deoxyribonucleoside triphosphates in cells incubated in azidocytidine showed only minor changes which appeared to result from and not to be the cause of the inhibition of DNA replication. The DNA synthesized in polyoma‐infected cells after incubation in azidocytidine showed a sedimentation pattern quite different from that seen after inhibition of DNA synthesis with arabinosyl cytosine or hydroxyurea. Experiments with nuclei isolated from azidocytidine‐inhibited, polyoma‐infected cells indicated (a) that the number of replicating molecules is decreased during the inhibition and (b) that upon incubation of the nuclei there is a rapid synthesis of DNA occurring in a new class of DNA molecules which are at a very early state of replication.Neither the 5′‐triphosphate of azidocytidine nor the nucleoside itself inhibit DNA synthesis in vitro in isolated nuclei from polyoma‐infected cells and at present the nature of the DNA‐synthesis‐inhibiting compound acting in the cells after addition of azidocytidine is unknown. Taken together the results suggest that azidocytidine inhibits DNA synthesis at an early stage, possible by blocking the initiation of DNA synthesis at the origin or by interfering with the elongation of newly initiated DNA molecules.
Fate of Histone Messenger RNA in Synchronized HeLa Cells in the Absence of Initiation of Protein Synthesis
Authors: STAHL Hans , GALLWITZ Dieter 
Published: March 2005

The fate of cytoplasmic histone mRNA was studied under conditions in which initiation of protein synthesis in synchronized HeLa cells in S phase was blocked by increasing the osmolarity of the growth medium with NaCl. In contrast to the interruption of DNA replication with hydroxy‐urea, which results in an exponential degradation of translatable histone mRNA with a half‐life of about 10‐13 min, blocking the initiation of protein synthesis leads to only a marginal loss of biologically active histone mRNA in the cytoplasm. When the initiation of protein synthesis was interrupted by treating cells with 150 mM NaCl, 40‐50% of the total cytoplasmic histone mRNA previously translated in polyribosomes appears in the cytoplasm integrated into mRNA. protein particle(s) sedimenting between 15 S and 30 S. On the other hand, in untreated S‐phase cells or in cells blocked with hydroxyurea only 3‐6% of the total translatable histone mRNA is found in the cytoplasm not bound to ribosomes or their subunits. In addition, the degradation of histone mRNA in hydroxyurea‐blocked S‐phase cells is prevented when the initiation of protein synthesis is inhibited with NaCl.These studies clearly indicate that the inhibition of initiation of protein synthesis per se is not the cause for the rapid degradation of cytoplasmic histone mRNA observed when DNA replication is turned off and that the inactivation of these mRNAs is a process dependent on continuous protein synthesis.
Purification of Bovine Adrenal‐Cortex Plasma‐Membrane Vesicles Containing a Highly Corticotropin‐Sensitive Adenylate‐Cyclase System and Angiotensin‐II‐Binding Sites
Authors: SCHLEGEL Werner , SCHWYZER Robert 
Published: March 2005

The purpose of this experimental investigation was to provide a purified plasma membrane fraction containing a highly hormone‐responsive adenylate cyclase system. Bovine adrenal cortex was homogenised and a washed pellet (450000 × g · min) was fractionated by zonal centrifugation in a sucrose and dextran gradient.Adenylate cyclase activity was purified up to 60‐fold to a specific activity of 55, 340 and 210 pmol of adenosine 3′: 5′‐monophosphate (cyclic AMP) produced/minute per mg of protein at 380C for the basal, adrenocorticotrophin and fluoride‐activated states, respectively. The time course of the adenylate cyclase activity is linear. The concentration necessary for half‐maximal stimulation by adrenocorticotrophin‐(1‐24)‐tetracosipeptide is 0.5 μM. The high hormone‐responsiveness of the membrane preparation allows one to demonstrate activation of adenylate cyclase by very weakly agonistic adrenocorticotrophin fragments. The F−‐activated state can be detergent‐dispersed by Lubrol and shows a Km (ATP) different from that of either the basal or adrenocorticotrophin‐stimulated state.Other marker enzymes such as 5′‐nucleotidase, glucose‐6‐phosphatase and cytochrome oxidase were followed during purification. The plasma membrane fraction shows rather homogeneous, relatively large vesicles (mean diameter 0.5 μm). It contains high‐affinity binding sites for angiotensin II (about 2 pmol per mg protein) with an apparent association constant of 2 × 107 (1/mol) at 120C. The yield, 20 mg of membrane protein per preparation, may make it a tool in either affinity‐labelling studies with the peptide hormones mentioned or the starting point for solubilisation and purification of adenylate cyclase.