bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Properties of RNA‐Dependent DNA Polymerase from Cytoplasmic A‐Type Particles of Murine Mammary Tumor Virus
Authors: KOHNO Michiaki , ISHIHAMA Akira 
Published: March 2005

The RNA‐dependent DNA polymerase associated with cytoplasmic A‐type particles of murine mammary tumor virus was isolated to near homogeneity by a procedure which includes dissociation of proteins from RNA by centrifugation in a step gradient of cesium chloride, followed by an affinity chromatography on poly(rC)‐agarose column. Two species of DNA polymerase were separated by the chromatography: enzyme I in 0.55 M NaCl and enzyme II in 0.80 M NaCl eluate, respectively.
The purified DNA polymerases consist of two major polypeptides, with molecular weights of 94000 and 42000, as the intrinsic subunits. Both enzyme protomers with a sedimentation coefficient of 6.3–6.4 S and a molecular weight of 115000–120000 associate to form active oligomers in low‐ionic‐strength buffer.
Both enzymes catalyzed the hydrolysis of RNA in RNA. DNA hybrids as well as the RNA‐dependent synthesis of DNA; these are the intrinsic activities of the reverse transcriptase from B‐type particles of murine mammary tumor virus as well as from avian and mammalian C‐type oncornaviruses. The general catalytic properties are similar to those of the enzyme from B‐type particles. Compared with DNA polymerases I, DPU'A polymerase II exhibited a high affinity for all the template‐primers tested and, in addition, a high preference for (rC)n· (dG)12–18.
Activities of NAD‐Specific and NADP‐Specific Isocitrate Dehydrogenases in Rat‐Liver Mitochondria
Authors: SMITH Colleen M. , PLAUT Gerhard W. E. 
Published: March 2005

The contributions of NAD‐specific and NADP‐specific isocitrate dehydrogenases to isocitrate oxidation in isolated intact rat liver mitochondria were examined using dl‐threo‐α‐methylisocitrate (3‐hydroxy‐1,2,3–butanetricarboxylate) to specifically inhibit flux through NADP‐specific isocitrate dehydrogenase.Under a range of conditions tested with respiring mitochondria, the rate of isocitrate oxidation was decreased by about 20–40% by inhibition of NADP‐isocitrate dehydrogenase, and matrix NADP became more oxidized. (a) For mitochondria incubated with externally added dl‐isocitrate and citrate, the rate of isocitrate oxidation obtained by extrapolation to infinite α‐methylisocitrate concentration was approximately 70% of the uninhibited rate in both state 3 and state 4. (b) With pyruvate plus malate added as substrates of citric acid cycle oxidation and isocitrate generated intramitochondrially, a concentration of α‐methylisocitrate (400 μM) sufficient for 99.99%, inhibition of NADP‐isocitrate dehydrogenase inhibited isocitrate oxidation in states 4 and 3 by 21 ±6% and 19 ± 11% (mean ± SEM), respectively. (c) With externally added isocitrate and citrate, the addition of NH4Cl increased isocitrate oxidation by 3–4‐fold, decreased NADPH levels by 30–40% and 2‐oxoglutarate accumulation by about 40%. The further addition of 600 μM α‐methylisocitrate decreased the NH4Cl‐stimulated isocitrate oxidation by about 40% and decreased NADPH to about 30% of the level prevailing in the absence of NH4Cl; nevertheless, the rate of isocitrate oxidation was still twice as large in the presence of NH4Cl and α‐methylisocitrate as in their absence.Experiments were also performed with intact mitochondria incubated with respiratory inhibitors to determine additional factors which might affect the flux through the two isocitrate dehydrogenases. (a) In the coupled reduction of acetoacetate by isocitrate, where the rate of reoxidation of reduced pyridine nucleotides is limited by NAD‐specific 3–hydroxybutyrate dehydrogenase, 85–100% of the rate of 3‐hydroxybutyrate formation was retained in the presence of 400–900 μM α‐methylisocitrate. (b) In a system where the rate of isocitrate oxidation is limited by the rate of NADPH reoxidation by glutathione reductase, the rate of glutathione reduction extrapolated to infinite α‐methylisocitrate concentration was from 20–40% of the uninhibited rate. (c) In the coupled synthesis of glutamate from isocitrate and NH4Cl, where the reoxidation of NADPH and NADH can occur via glutamate dehydrogenase, the rate of glutamate production extrapolated to infinite α‐methylisocitrate concentration was about 60% of the uninhibited rate.
Intracellular Enzymes of Collagen Biosynthesis in 3T6 Fibroblasts and Chick‐Embryo Tendon and Cartilage Cells
Authors: RISTELI Leila , RISTELI Juha , SALO Lüsa , KIVIRIKKO Kari I. 
Published: March 2005

An increase of about 4–9‐fold was found in prolyl and lysyl hydroxylase activities per cell when 3T6 fibroblasts were grown from the early‐log to the stationary phase, whereas essentially no changes were found in hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyl‐transferase activities. A control experiment using a different assay procedure indicated that the increase in prolyl hydroxylase activity was not due to any methodological artefact. The results demonstrate for the first time that these four intracellular enzymes of collagen biosynthesis are not regulated in an identical manner within one cell type.Freshly isolated chick embryo cartilage cells, which synthesize type I1 collagen, had higher lysyl hydroxylase and hydroxylysyl galactosyltransferase activities than freshly isolated chick embryo tendon cells, whereas no difference was found between them in prolyl hydroxylase and galactosylhydroxylysyl glucosyltransferase activities. The greater extent of the hydroxylation of lysyl residues and galactosylation of hydroxylysyl residues in type I1 than in type I collagen is thus probably due in part to a higher activity of the corresponding enzymes in the cartilage cells, but the greater extent of the glucosylation of galactosylhydroxylysyl residues cannot be explained by any higher activity of the glucosyltransferase.Prolyl and lysyl hydroxylase activities were markedly lower in the cultured 3T6 cells than in freshly isolated chick embryo tendon cells, particularly when expressed per unit solubilized cell protein. These data agree with the lower rate of collagen production by the 3T6 cells. Unexpectedly, the two hydroxylysyl glycosyltaransferase activities were somewhat higher in the 3T6 cells than in the tendon cells when expressed per cell, a finding which may explain the pronounced glycosylation of the collagen synthesized by the 3T6 cells.On incubation of the 3T6 cells with 5 μg/ml puromycin for 10 h the intracellular enzyme activities of collagen biosynthesis decreased by about 15–30% in the early‐log phase and by about 10–20% in the late‐log phase. The results suggest that the half‐lives of all four enzyme activities are at least about 24 h in the early‐log phase and possibly even longer in the late‐log phase.
On the Efficiency of Oxidative Phosphorylation in Membrane Vesicles of Azotobacter vinelandii and of Rhizobium leguminosarum Bacteroids
Authors: LAANE Colja , HAAKER Huub , VEEGER Cees 
Published: March 2005

The effect of O2 on the rate of ATP synthesis and the P/O ratio has been studied in membrane vesicles of Azotobacter vinelandii and Rhizobium leguminosarum bacteroids (strain PRE). It was observed that the P/O ratio rises to a maximum value when the O2 input is increased, but declines when O2 becomes detectable in the medium.
Evidence is presented, that in A. vinelandii H2 donates its electrons at a fourth proton‐translocating site. The presence and the efficiency of this site is independent of the growth conditions and it appeared that no flavoproteins are directly involved in the oxidation of H2. The unidirectional hydrogenase in membrane vesicles of A. vinelandii is not inhibited by acetylene. The function of hydrogenase in the process of nitrogen fixation by A. vinelandii is discussed.
Two types of membrane vesicles of R. leguminosarum bacteroids were prepared. Membranes isolated in a low salt medium lose the ability to couple succinate oxidation to oxidative phosphorylation; upon isolation in a high salt medium this ability is preserved. In contrast the efficiency of oxidative phosphorylation with NADH is not affected by the isolation procedure. The efficiency of energy coupling in membranes vesicles of R. leguminosarum bacteroids can be enhanced by the addition of fatty‐acid‐free bovine serum albumin. No active membrane‐bound hydrogenase could be detected in membrane vesicles of R. leguminosarum bacteroids.
Crosslinking Studies with the Pyruvate Dehydrogenase Complexes from Azotobacter vinelandii and Escherichia coli
Authors: DE ABREU Ronney A. , DE VRIES Joop , DE KOK Arie , VEEGER Cees 
Published: March 2005

The spatial arrangements of the individual components of the pyruvate dehydrogenase complexes from Azotobacter vinelandii and Escherichia coli have been studied by crosslinking the complex with bis(imido esters) of different chain lengths and with dimethyl‐3,3′‐dithiobis(propionimidate), a bis(imido ester) containing a cleavable disulfide linkage between the two reactive groups. The three‐component complexes of A. vinelandii and E. coli show almost identical crosslink patterns. These indicate that each component exists in at least a dimeric structure and that all three components contact each other. In contrast, in the four‐component complex from A. vinelandii heterologous dimer formation was only found between lipoamide dehydrogenase and high‐molecular‐weight trans‐acetylase and between pyruvate dehydrogenase and low‐molecular‐weight transacetylase. A model is presented to account for these observations.In the presence of pyruvate, Mg2+ and thiamin diphosphate extensive crosslinking with bis‐(imido esters) takes place between the transacetylase chains of the E. coli complex and also between the high‐molecular‐weight transacetylase chains of the A. vinelandii complex. This indicates a conformational change of the transacetylase core induced by acetylation of this component, exposing lysyl residues previously hidden.Crosslinking with N,N′‐o‐phenylene‐bismaleimide of the four‐component A. vinelandii complex, pretreated with N‐ethylmaleimide, in the presence of pyruvate, Mg2+ and thiamin diphosphate shows a close positioning of the −SH groups generated by reductive acetylation of the transacetylase isoenzymes (less than 0.6 nm). With the E. coli complex most of the material thus cross‐linked consists of higher polymers with a concomittant decrease of transacetylase monomers. No homologous dimer formation was observed.
The Effect of Octanoate and Palmitate on the Metabolism of Valine in Perfused Hindquarter of Rat
Authors: SPYDEVOLD Øystein 
Published: March 2005

The effect of octanoate and palmitate on the oxidation of 14C‐labelled valine has been studied in perfused hindquarter of rat.

The oxidation rate of valine increases 30 times when the concentration of valine is increased from 0.1 mM to 5 mM.
Octanoate at a 5 mM concentration effected a 10‐fold increase in the flux through the α‐keto‐isovalerate dehydrogenase step and a 5‐fold increase at 0.5 mM concentration.
Palmitate (1 mM) effects only a moderate increase in the valine oxidation.
With no octanoate there was a great accumulation of α‐ketoisovalerate in both the muscle and the perfusion medium.
With octanoate little α‐ketoisovalerate accumulated whereas 3‐hydroxyisobutyrate was found in high concentration both in the muscle and in the medium.
Octanoate stimulated the production of citric‐acid‐cycle intermediates and lactate.
The results are discussed in relation to valine metabolism in the body.
3′ Esters of ADP as Energy‐Transfer Inhibitors and Probes of the Catalytic Site of Oxidative Phosphorylation
Authors: SCHÄFER Günter , ONUR Günes 
Published: March 2005

A large series of 3′ esters of ADP has been synthesized. Several of these can serve as photoaffinity labels; others exhibit fluorescent properties. The corresponding AMP and ATP derivatives have also been synthesized in some cases.
The influence of the 3′‐O‐acyl nucleotides on energy‐linked functions of beef‐heart submitochondrial particles has been investigated. The following results were obtained.a) 3′ Esters of ADP are powerful and highly specific inhibitors of oxidative phosphorylation. The inhibition is competitive to ADP and Ki values as low as 0.05 μM, for the 3′‐O‐(1)naphthoyl ester of ADP, could be observed.b) The inhibition of oxidative phosphorylation by 3′ esters of ADP appears to be non‐competitive versus inorganic phosphate.c) The nucleotide analogs are not phosphorylated themselves. The corresponding ATP analogs can not drive energy‐linked processes.d) The 3′ esters of AMP are ineffective as inhibitors, whereas the ATP derivatives are only comparatively weak inhibitors.e) Uncoupled or solubilized ATPase is almost two orders of magnitude less sensitive against inhibition by 3′ esters than coupled systems. The analogs exert maximal inhibition specifically in systems involving an ‘energized’ state of the coupling device.f) Azido‐group‐bearing analogs can be used for irreversible photoinactivation of the coupling ATPase. Photoinactivation also is most efficient when carried out with ‘energized’ particles.g) The inhibitory properties are similar also in ATP‐driven NAD+ reduction by succinate, and in the uncoupler‐sensitive ATP ⇌ Pi exchange. The required concentrations for half‐maximal inhibition are somewhat higher than in oxidative phosphorylation, but lower than with uncoupled ATPase.
From molecular models, from substituent properties, and from the conditions required for inhibition it is concluded that these highly effective analogs of ADP may act as conformation‐specific probes at the catalytic site of oxidative phosphorylation. The results are interpreted in terms of a model suggesting that, in the process of ATP synthesis, a hydrophobic cavity on the enzyme is exposed only in the energized state, accepting the large 3′ substituent. The substituent is assumed to inhibit phosphoryl transfer and/or conformational transitions inherent in the process of ADP phosphorylation by steric hinderance.
Yeast Phenylalanyl‐tRNA Synthetase
Authors: BALTZINGER Mireille , FASIOLO Franco , REMY Pierre 
Published: March 2005

The localization of the binding sites of the different ligands on the constitutive subunits of yeast phenylalanyl‐tRNA synthetase was undertaken using a large variety of affinity and photoaffinity labelling techniques. The tRNAPhe was cross‐linked to the enzyme by non‐specific ultraviolet irradiation at 248 nm, specific irradiation in the wye base absorption band (315 nm), irradiation at 335 nm, in the absorption band of 4‐thiouridine (s4U) residues introduced in the tRNA molecule, or by Schiff's base formation between periodate‐oxidized tRNAPhe (tRNAPheox) and the protein. ATP was specifically incorporated in its binding site upon photosensitized irradiation.The amino acid could be linked to the enzyme upon ultraviolet irradiation, either in the free state, engaged in the adenylate or bound to the tRNA.The tRNA, the ATP molecule and the amino acid linked to the tRNA were found to interact exclusively with the β subunit (Mr 63000). The Phenylalanine residue, either free or joined to the adenylate, could be cross‐linked with equal efficiency to either type of subunit, suggesting that the amino acid binding site is located in a contact area between the two subunits.The Schiff's base formation between tRNAPheox and the enzyme shows the existence of a lysyl group close to the binding site for the 3′‐terminal adenosine of tRNA. This result was confirmed by the study of the inhibition of yeast Phenylalanyl‐tRNA synthetase with pyridoxal phosphate and the 2′,3′‐dialdehyde derivative of ATP, oATP.
Immunochemical Studies of Beef Pancreas Tryptophanyl‐tRNA Synthetase and Its Fragments
Authors: SCHEINKER Vladimir Sh. , BERESTEN Sergey F. , MAZO Alexander M. , AMBARTSUMYAN Noona S. , ROKHLIN Oskar V. , FAVOROVA Ol'ga O. , KISSELEV Lev L. 
Published: March 2005

The immunoglobulin G (IgG) fraction of the antiserum from rabbits immunized with homogeneous beef pancreas tryptophanyl‐tRNA synthetase inhibits the enzyme activity in the reactions of both tRNATrp aminoacylation and tryptophan activation. Fab fragments of IgG act in a similar way. Common antigenic determinants have been detected in tryptophanyl‐tRNA synthetases from beef, pig, chicken and rat livers using pure antibodies against beef pancreas tryptophanyl‐tRNA synthetase. This observation indicates the evolutional stability of certain structural features of tryptophanyl‐tRNA synthetases.The interaction of antibodies with the fragments of beef tryptophanyl‐tRNA synthetase produced by endogenous and tryptic proteolysis of the enzyme has been studied. One third of the antiserum antibodies interacting with the C‐terminal fragment of the enzyme (Mr∼ 40000) inhibits its activity whereas the antibodies to the N‐terminal fragment (Mr∼ 20000) have no effect on the enzyme activity.The immunochemical identity of the two synthetase fragments differing in their enzymatic activity supports the assumption that the loss of enzymatic activity of the tryptic fragment is caused by lack of a small peptide which is retained in case of endogenous proteolysis; probably the amino acid residues of this peptide participate in formation of active centre of tryptophanyl‐tRNA synthetase.A radioimmunochemical method is described for determining the number of antigenic determinants. One molecule of tryptophanyl‐tRNA synthetase was found to bind 9(± 1) molecules of Fab fragments.Antibodies against tryptophanyl‐tRNA synthetase from beef pancreas do not inhibit noticeably the activity of reverse transcriptase from avian myeloblastosis virus. No antigenic determinants in common have been detected in reverse transcriptase and tryptophanyl‐tRNA synthetase by radio‐immunochemical assays.
Chemical Evidence for a Codon‐Induced Allosteric Change in tRNALys Involving the 7‐Methylguanosine Residue 46
Authors: WAGNER Rolf , GARRETT Roger A. 
Published: March 2005

[32P]tRNALyS, from Escherichia coli, was modified with kethoxal, in the presence and absence of the oligonucleotide codon (A)4. The presence of the codon resulted in a faster modification rate of the tRNA at three guanine sites which were identified by a diagonal fingerprint method. A large increase in the modification rate occurred at the 7‐methylguanosine residue 46 (m7G‐46) in the presence of the codon; weakly enhanced modification was observed at G‐15 and G‐57.It is concluded that the formation of a codon‐anticodon complex induces, primarily, a confor‐mational change involving disruption of the m7G‐46 from the m7G‐46 · G‐22 · C‐13 base triple. Subsequently, the guanines of G‐15 and G‐57, in the D and T loops, respectively, become slightly more reactive, suggesting a weak tendency for these two interacting arms to unfold.The results are interpreted in terms of an equilibrium between two main conformers and a third minor one; the possible significance of these conformers, in protein biosynthesis, is considered.
A Lipopolysaccharide‐Binding Cell‐Surface Protein from Salmonella minnesota
Authors: GEYER Rudolf , GALANOS Chris , WESTPHAL Otto , GOLECKI Jochen R. 
Published: March 2005

Protein extracts obtained from Salmonella minnesota Re mutant cells by treatment with EDTA/NaCl solution contain a protein which exhibits high affinity to bacterial lipopolysaccharides. The isolation and partial characterization of this lipopolysaccharide‐binding protein is described.
The protein was purified from EDTA extracts by a two‐step procedure consisting of ion‐exchange chromatography on CM‐Sephadex and preparative polyacrylamide gel electrophoresis at pH 9.5. The yield of the total purification procedure was around 16%.
The resulting protein preparation was homogeneous on the basis of disc gel electrophoresis, dodecylsulfate gel electrophoresis, isoelectric focusing in polyacrylamide gel and immunoelectrophoresis.
The isoelectric point of the protein was found to be 10.3 at 4°C. Its molecular weight determined by dodecylsulfate gel electrophoresis is 15000. Its amino acid composition is characterized by the absence of histidine and proline, a low content in tyrosine and high amounts of alanine, lysine, aspartic and glutamic acid residues, or their respective amides.
The lipopolysaccharide‐protein association was shown to be mainly due to ionic interactions of the basic protein with negatively charged groups (probably phosphate and pyrophosphate groups) of the lipid A moiety.
Purified lipopolysaccharide‐binding protein is immunogenic in rabbits, thus enabling the preparation of specific antiserum.
The protein is located at the surface of Salmonella minnesota Re mutant cells as revealed by antiserum absorption with total bacteria. Ferritin‐labelling studies further demonstrated that it is evenly spread over the entire cell surface.
Comparative antiserum absorption studies using smooth and rough strains of Salmonella minnesota, Salmonella typhimurium, Escherichia coli, Klebsiella and Shigella revealed the presence of lipopolysaccharide‐binding protein (or a serologically cross‐reacting antigen) in most of the strains tested. From these results the protein can be considered as a common antigen of Enterobacteriaceae.
Formylmethionyl‐tRNA Binding Properties of Escherichia coli Ribosomal Protein S1
Authors: LI Percy T. , SHEA Timothy , ELLIS Steven , CONWAY Thomas W. 
Published: March 2005

Formylmethionyl‐tRNA has been found to bind to Escherichia coli ribosomal protein S1. Complexes of S1 and methionine‐labeled fMet‐tRNAMetf were detected by binding to cellulose nitrate membranes. Complex formation was stimulated by incubation at low ionic strength, at 37°C and in the absence of Mg2+. Protein S1 also bound [3H]poly(rC) under the same conditions or in buffers of higher ionic strength, 10 mM in Mg2+, and during incubation at 0°C. Unlabeled poly(rC) was an effective inhibitor of the binding of labeled fMet‐tRNA when both nucleotides were added simultaneously to the reaction mixtures; however, approximately 80% of previously bound f[35S]Met‐tRNA remained bound after the subsequent addition of unlabeled poly(rC). Binding of f[35S]Met‐tRNA, but not the binding of [3H]poly(rC), was inhibited by polyamines or aminoglycoside antibiotics. Protein S1 that had been reacted with N‐ethylmaleimide failed to bind f[35S]Met‐tRNA but was still capable of binding [3H]poly(rC).The data support the concept of two distinct RNA‐binding sites on the S1 molecule. Initiator tRNA appears to occupy a site concerned with the RNA unwinding property of S1, whereas poly(rC) binds primarily at a separate site. During initiation on the ribosome, the poly(rC) site may interact with the 3′ end of 16‐S ribosomal RNA, while the other site may interact with either mRNA or fMet‐tRNA.
Mitochondrial Protein‐Synthesizing Machinery in Saccharomyces cerevisiae Grown in Different Metabolic Conditions
Authors: BALDACCI Giuseppe , FALCONE Claudio , FRANCISCI Silvia , FRONTALI Laura , PALLESCHI Claudio 
Published: March 2005

The isoacceptor patterns of mitochondrial seryl and alanyl‐tRNAs from Saccharomyces cerevisiae grown under different conditions and committed to fermentative or respiratory metabolism have been studied by reversed‐phase chromatography.
An extensive variability of the chromatographic patterns of the four isoacceptors for serine and of the three isoacceptors for alanine has been observed as a function of carbon source and stage of growth, but the main differences were observed in the different stages of growth on glucose.
In order to distinguish the effects due to stage of growth from those due to relief from glucose repression, the isoacceptor patterns of mitochondrial tRNA were compared in repressed and derepressed resting cells; results show that some of the mitochondrial species are almost undetectable in resting repressed cells, but are the major ones after recovery of respiration.
Two of the serine isoacceptors (species 1 and 2), one of which is absent or not acylable in repressed resting cells, are different gene products.