bluuebunny/crossref_metadata_2025_split huggingface dataset:
Studies on the Mechanism of Action of the ATP‐Dependent DNAase from Alcaligenes faecalis
Authors: YOUNG Douglas B. , ROSAMOND John 
Published: March 2005

An ATP‐dependent DNAase has been purified to homogeneity from extracts of Alcaligenes faecalis, and has been shown to couple the degradation of DNA to the hydrolysis of ATP. Enzyme activity also requires divalent ions, with Mn2+, Mg2+ and Co2+ being effective cofactors for both DNAase and ATPase activities. We have studied the intermediates formed by the enzyme during the degradation of duplex DNA with each of these cofactors using sedimentation velocity, binding to nitrocellulose filters and sensitivity to a nuclease specific for single‐stranded DNA.With Mn2+ or Co2+, the enzyme acts processively to produce mostly acid‐soluble material and acid‐insoluble single‐strand fragments up to 400‐nucleotides long. However, with Mg2+ present, the enzyme produces intermediates comprising a duplex region with one or more single‐strand tails, while little acid‐soluble oligonucleotide is formed. From these results, we propose a model to describe the mechanism by which the ATP‐dependent DNAase from A. faecalis degrades duplex DNA.
Pyruvate Dehydrogenase Component of the Pyruvate Dehydrogenase Complex from Escherichia coli K12
Authors: SAUMWEBER Harald , BINDER Renate , BISSWANGER Hans 
Published: March 2005

Free pyruvate dehydrogenase component of the Escherichia coli K12 pyruvate dehydrogenase complex was isolated from a mutant lacking the dihydrolipoamide transacetylase component. The procedure, employing three chromatographic steps, yields a product that is electrophoretically pure. The purified enzyme reassociates with the residual complex lacking this component to a fully active enzyme complex.The kinetic characteristics of the free component were compared to that of the enzyme integrated into the native complex molecule. No essential differences could be detected regarding the behaviour of the catalytic reaction with variations in temperature, pH and substrate concentration. An inhibition, competitive to pyruvate, of the pyruvate dehydrogenase component (Ki= 18 μM) by fluoropyruvate was observed with both enzyme forms.Pyruvate exerts a cooperative effect both upon the partial enzyme reaction of the pyruvate dehydrogenase component as well as upon the overall reaction of the native enzyme complex. However, there is a clear difference in the shape of the saturation curves of both types of reaction. Experiments with the free enzyme, with the component integrated into the native complex molecule and with enzyme complexes which are partially deficient in the pyruvate dehydrogenase component demonstrate that the type of saturation curves obtained were characteristic for the reaction observed rather than for the interaction of a high number of subunits of the pyruvate dehydrogenase complex.
The Major Components of the Mouse and Human Genomes
Authors: CUNY Gérard , SORIANO Philippe , MACAYA Gabriel , BERNARDI Giorgio 
Published: March 2005

Main‐band DNA from mammals and birds can be resolved by density gradient centrifugation techniques into three or four families of fragments of different dG + dC contents. These major DNA components are similar in their buoyant densities and relative amounts in all species tested and are observed in DNA preparations ranging in Mr from 2 × 106 to over 200 × 106.In the present work, the four major components of mouse and human DNAs were prepared and characterized in several basic properties: relative amounts, dG + dC contents, buoyant densities and compositional heterogeneity. The results obtained lead to the following conclusions: (a) the major DNA components of mouse and man form at least 85% and possibly the totality of the main bands of these DNAs; (b) they have very low compositional heterogeneities over a wide molecular weight range; (c) they derive from very large chromosomal DNA segments of fairly homogeneous base composition, for which the name ‘isochores’ is proposed.A comparison of the compositional heterogeneity of main‐band DNAs from warm‐blooded and cold‐blooded vertebrates confirms our previous conclusion that these DNAs are characterized by a different sequence organization.
Activation of Lymphocyte Populations with Concanavalin A or with Lipoprotein and Lipopeptide from the Outer Cell Wall of Escherichia coli Correlation of Early Membrane Changes with Induction of Macromolecular Synthesis
Authors: RESCH Klaus , BESSLER Wolfgang 
Published: March 2005

The occurrence of early membrane changes following the interaction of mouse lymphocytes with the B‐lymphocyte mitogens, lipoprotein or lipopeptide, and with the T‐lymphocyte mitogen concanavalin A was compared to the induction of RNA and DNA synthesis induced by the mitogens in several lymphocyte populations. Membrane alterations were determined by measuring the incorporation of phospholipid precursors into phosphatidylcholine after the addition of the mitogens. It was shown that both B‐cell and T‐cell mitogens caused an increase in the incorporation of [14C]oleate and [14C]acetate in T B‐cell mixtures after 4 h of stimulation. The B‐cell mitogens, lipoprotein and lipopeptide, induced a marked incorporation of phospholipid precursors in spleen and lymph node cells of athymic mice. Corresponding to their cell specificity for the induction of macromolecular synthesis, no membrane changes were induced by the bacterial mitogens in thymocytes. On the other hand, the T‐cell mitogen concanavalin A was active in causing marked membrane changes in thymocytes. Remarkably, however, concanavalin A also induced the incorporation of phospholipid precursors in spleen and lymph node cells of congenitally athymic mice. Since membrane events in response to concanavalin A were not found after treating normal spleen and lymph node cells with anti‐Thyl serum and complement, the positive response observed in athymic mice probably resulted from the presence of pre‐T‐lymphocytes.Our results show that early alterations in membrane phospholipid metabolism in mature B or T lymphocytes are correlated with the subsequent induction of macromolecular synthesis, suggesting that these membrane changes are causally linked to cell activation. In addition the data suggest a similar molecular mechanism for the activation of lymphocytes by both T‐cell or B‐cell mitogens.
Multiple Forms of Rat‐Serum α1‐Protease Inhibitor
Authors: IKEHARA Yukio , MIYASATO Mihoko , OGATA Shigenori , ODA Kimimitsu 
Published: March 2005

α1‐Protease inhibitor was purified from rat serum by two different methods, of which an immunoaffinity method should be preferentially used to obtain all of the multiple forms. The protein thus obtained showed a single protein band in dodecylsulphate/polyacrylamide gel electrophoresis corresponding to a molecular weight of 54000, and contained 13.2% carbohydrate by weight. By column isoelectric focusing in a pH 3.5–5.0 gradient the purified α1‐protease inhibitor was separated into five forms with pl values from 4.3–4.7. The amino acid composition of each form was identical, while sialic acid content was significantly different from each other. The most acidic form contained 6.7 residues/molecule, the most basic form, 5.1 residues/molecule, and three forms between them showed proportionally intermediate values between the two.When α1‐protease inhibitor was treated with neuraminidase, the five forms were converted finally into three major forms with pl values of 5.3–5.7. In addition, the major form (band 3) of the inhibitor was also converted into three forms after complete removal of sialic acid. These results suggest that α1‐protease inhibitor originally exists as three forms with different charges, possibly due to modification of amino acids which might not be detectable by the amino acid composition analysis in the present study. A possible explanation was presented for involvement of sialic acid in appearance of multiple forms originating from three parental forms.
Photochemical Cross‐Linking of Elongation Factor G to 70‐S Ribosomes from Escherichia coli by 4‐(6‐Formyl‐3‐azidophenoxy)butyrimidate
Authors: MAASSEN J. Anthonie , MÖLLER Wim 
Published: March 2005

Ribosomal proteins situated at or near the binding site of elongation factor G (EF‐G) on the Escherichia coli ribosome have been identified by use of the heterobifunctional cross‐linker 4‐(6‐formyl‐3‐azidophenoxy)butyrimidate. Four different preparations of EF‐G, in which the number of cross‐linker molecules coupled to EF‐G ranged from four to seven, all cross‐linked to 50‐S subunit proteins L1, L3 and L11 as well as to 30‐S subunit proteins S3 and S4.Cross‐linking of EF‐G to ribosomal proteins was tested electrophoretically. In the case of L7/L12 and L11 immunological methods were also used.Cross‐linking of EF‐G to L1, L3, L11, S3 and S4 is specific as judged from the fact that addition of unmodified EF‐G and of thiostrepton results in less cross‐linking. The cross‐linking data suggests that the binding site for EF‐G includes several proteins which are located at the interface between the 30‐S and 50‐S subunits.
The ATP Substrate Site of a Cyclic‐Nucleotide‐Independent Protein Kinase from Porcine Liver Nuclei
Authors: BAYDOUN Hassan , HOPPE Jürgen , FREIST Wolfgang , WAGNER Karl G. 
Published: March 2005

The ATP substrate site of a second messenger‐independent protein kinase of the type NIT from porcine liver nuclei was mapped using a series of 30 ATP derivatives with modifications at the base, ribose or triphosphate moiety. Ki values for these derivatives were determined by competition with [γ‐32P]ATP; they range from 4 μM to 1.5 mM. For a comparison with data previously reported for the catalytic subunit of cAMP‐dependent protein kinase I from rabbit skeletal muscle, the Ki values were transformed into δΔG values. These values are related to the Ki value of unsubstituted ATP and indicate the decrease of affinity caused by the different substitutions.With both enzymes the major binding affinity is derived from the interaction of the adenine base. The contributions of the two ribosyl OH groups are marginal and the triphosphate moiety interacts most strongly with its β‐phosphoryl group. Between the two enzymes the most striking differences, however, were observed for the specificity of the nucleobase interaction. While an unmodified N‐6 amino group is required in the case of the cAMP‐dependent protein kinase, the nuclear enzyme seems to tolerate extensive modification at this position, such as the introduction of a keto group or a bulky benzyl residue. Obviously, the ATP site of the nuclear kinase has an open cleft next to the N‐6 of the adenine and binding of the adenine occurs by hydrophobic interaction without the formation of hydrogen bonds to any of the adenine nitrogens.
Glycosaminoglycan Biosynthesis in Arterial Wall
Authors: LEVY Peggy , PICARD Jacques , BRUEL Arlette 
Published: March 2005

Incubation of microsomal fractions with labelled 3′‐phosphoadenylyl sulfate results in incorporation of [35S]sulfate into endogenous glycosaminoglycans. Specific radioactivity observed incorporated into heparan sulfate chains is 10‐fold greater than that incorporated into chondroitin sulfate chains. This is in agreement with the results obtained for glycosylation of glycosaminoglycans in arterial wall membrane fractions. Sulfation of heparan sulfate was studied since it contains N‐ and O‐sulfate groups in contrast with the other sulfated glycosaminoglycans which contain only O‐sulfate groups.Sulfation of heparan sulfate occurs rapidly, since sulfate incorporation is detected after exposure for only 0.5 min.Heparan sulfate was identified on the basis of its resistance to hyaluronidase and chondroitin ABC lyase, its susceptibility to heparitinase, its sensitivity to nitrous acid and the presence of glucosamine as the only hexosamine.The chemical composition of the purified heparan sulfate fractions provides evidence for the high degree of sulfation of its chains.Studies into the distribution of sulfate residues on heparan sulfate at different times of sulfation indicate that N‐sulfate groups are not randomly introduced into the polymer.The relationship between the processes of N‐ and O‐sulfation was studied. The present results demonstrate that preferential N‐sulfation is obtained for incorporation of labelled precursor over a short period, the O‐sulfation occurring on previously N‐sulfated heparan sulfate.
Changes in Amount of Hypo‐Modified tRNA Having Guanine in Place of Queuine during Erythroid Differentiation of Murine Erythroleukemia Cells
Authors: SHINDO‐OKADA Nobuko , TERADA Masaaki , NISHIMURA Susumu 
Published: March 2005

The amounts of hypo‐modified tRNAs having guanine in place of queuine in murine erythroleukemic cells decreased markedly when the cells differentiated into mature erythroid cells. The amounts of these hypo‐modified tRNAs can be determined easily by measuring incorporation of labeled guanine into tRNA with Escherichia coli tRNA‐guanine transglycosylase. The decrease was detected at on early stage of erythroid differentiation: namely, before any detectable increase in the percentage of cells containing hemoglobin. The amount of guanine‐accepting tRNA species was nearly proportional to the percentage of undifferentiated cells in the population, regardless of the type of inducer used. Decrease in the amounts of hypo‐modified tRNAs in the cells was effectively blocked by 12‐O‐tetradecartoylphorbol 13‐acetate, which inhibits differentiation of these cells.8‐Azaguanine, which is known to be substrate of tRNA—guanine transglycosylase. was incorporated almost exclusively into the first position of hypo‐modified tRNA in murine erythroleukemic cells when they were pulselabeled in culture with 8‐azaguanine, suggesting strongly that tRNA‐guanine transglycosylase in the cells is actually involved in incorporation of 8‐azaguanine into tRNA in vivo. The amount of 8‐azaguanine incorporated into tRNA in differentiated cells was one third of that in undifferentiated cells, the decrease being parallel with that in the amount of guanine‐accepting tRNA in these cells. The results suggest that the appearance of hypomodified tRNAs in the transformed cells was due to lack of substrate for queuosine biosynthesis in tRNA.
Different Transport Pathways of Individual Precursor Proteins in Mitochondria
Authors: ZIMMERMANN Richard , HENNIG Bernd , NEUPERT Walter 
Published: March 2005

Transport of mitochondrial precursor proteins into mitochondria of Neurospora crassa was studied in a cellfree reconstituted system. Precursors were synthesized in a reticulocyte lysate programmed with Neurospora mRNA and transported into isolated mitochondria in the absence of protein synthesis. Uptake of the following precursors was investigated: apocytochrome c, ADP/ATP carrier and subunit 9 of the oligomycin‐sensitive ATPase.Addition of high concentrations of unlabelled chemically prepared apocytochrome c (1–10 μM) inhibited the appearance in the mitochondrial of labelled cytochrome c synthesized in vitro because the unlabelled protein dilutes the labelled one and because the translocation system has a limited capacity [apparent V is 1–3 pmol × min−1× (mg mitochondrial protein)−1]. Concentrations of added apocytochrome c exceeding the concentrations of precursor proteins synthesized in vitro by a factor of about 104 did not inhibit the transfer of ADP/ATP carrier or ATPase subunit 9 into mitochondria. Carbonylcyanide m‐chlorophenylhydrazone, an uncoupler of oxidative phosphorylation, inhibited transfer in vitro of ADP/ATP carrier and of ATPase subunit 9, but not of cytochrome c.These findings suggest that cytochrome c and the other two proteins have different import pathways into mitochondria. It can be inferred from the data presented that different ‘receptors’ on the mitochondrial surface mediate the specific recognition of precursor proteins by mitochondria as a first step in the transport process.
Biosynthesis of the Light‐Harvesting Chlorophyll a/b Protein
Authors: CUMING Andrew C. , BENNETT John 
Published: March 2005

Antibodies raised against the 26000‐Mr polypeptides of the light‐harvesting chlorophyll a/b proteins of pea leaves specifically immunoprecipitated two 32000 ‐Mr polypeptides synthesized when pea leaf poly(A)‐containing RNA was translated in vitro. On the basis of immunochemical relatedness and by comparison of their partial tryptic digestion products, the 32000 ‐Mr products formed in vitro are identified as precursors to the authentic polypeptides of the light‐harvesting chlorophyll a/b complex.
The specificity of the immunoprecipitation permitted the development of an assay for the cellular levels of translationally active light‐harvesting protein mRNA in plants exposed to different light regimes. Low levels of the mRNAs were detectable in dark‐grown plants. Exposure to continuous illumination caused these levels to increase by at least ten‐fold and led to the appearance of large quantities of the light‐harvesting chlorophyll a/b complex. In plants exposed to intermittent illumination (2 min of white light every 2 h for 2 days), the light‐harvesting complex did not accumulate, although levels of mRNA specifying the polypeptides of the complex were high (50% of those in continuously illuminated plants).
Messenger RNAs encoding the light‐harvesting proteins were detected in polysomes of intermittently illuminated leaves. These polysomes were active in a wheat‐germ 100000 ×g supernatant ‘run‐off’ system, to form light‐harvesting protein precursors, under conditions when only nascent polypeptide chains initiated in vivo were elongated and terminated. These results demonstrate that the inability of intermittently illuminated leaves to accumulate the light‐harvesting proteins is not due to a selective inhibition of the translation of the corresponding mRNAs.
Intermittently illuminated leaves were labelled with [35S]methionine in darkness, and incorporation of radioisotope into the light‐harvesting proteins and their precursors was assayed immunologically. No pool of untransported or unprocessed 32000 ‐Mr precursor polypeptides could be detected in the soluble fraction (cytoplasm and stroma). However, low levels of the mature 26000 ‐Mr polypeptides were detected in the membrane fraction. It is concluded that the newly synthesized light‐harvesting chlorophyll a/b proteins fail to accumulate in intermittently illuminated leaves because they undergo rapid turnover. The site of light‐harvesting protein breakdown is probably the thylakoid membrane, and the cause of breakdown is probably the absence of chlorophyll a and chlorophyll b molecules that are required for eventual stabilization of the proteins within the photosynthetic membrane.
Synergism Between Coenzyme and Carboxylate Binding to Liver Alcohol Dehydrogenase
Authors: ANDERSSON Pia , KVASSMAN Jan , OLDÉN Bertil , PEJTERSSON Göata 
Published: March 2005

The interaction of decanoate and benzoate with the substrate‐binding site in liver alcohol dehydrogenase has been characterized by fluonmetric equilibrium binding studies, using auraimne O as a reporter ligand.
The affinity of the enzyme for the carboxylates examined decreases about 50‐fold on complex formation with NADH. The coenzyme‐competitive inhibitors ADP‐ribose and Pt(CN)2−4 have a similar destabilizing effect on decanoate binding, indicating that the effect of NADH derives from its negatively charged pyrophosphate group.
Carboxylate binding to free enzyme requires the protonated form of an ionizing enzymic group with pKa 9.2, while there is no corresponding effect of pH on binary complex formation with auramine O. Carboxylate binding to the enzyme · NADH+ complex exhibits no significant dependence on pH over the pH range 7–10.
These results, combined with previously reported data for decanoate binding to the enzyme · NAD+ complex, provide clear evidence that carboxylates (in contrast to auramine O) are bound at the active‐site zinc ion of the enzyme in a process regulated by the ionization state of zinc‐bound water. The synergistic effect of NADH and NAD+ on carboxylate binding can be qualitatively and quantitatively explained in terms of electrostatic interactions analogous to those proposed to account for the effect of coenzymes on the pKa of zinc‐bound water.
The Synthesis of Ricinus communis Agglutin
Authors: ROBERS Lynne M. , LORD J. Michael 
Published: March 2005

Polyadenylated RNA isolated from the endosperm tissue of maturing castor been seeds was translated in a call‐free rabbit reticulocyte lysate syste. Rabbit antibodies raised against Ricinus communis agglutinin were used to identify nascent agglutinin chains. In contrast to the authentic agglutinin polypeptides with molecular weights of 31000 (A chains) and 37000 (glycosylated B chains), immunoreactive translational products of Mr 33500 and 59000 were observed. The inclusion of canine pancreatic microsomes in the translational system resulted in the contranslational segregation of these immunoreactive products into the lumen of the vesicles and their modification, to molecular weights of 32000 and 66000–69000 respectively. These contranslational size modifications resulted from the cleavage of leader sequences and, in the case of the larger product, concomittant core glycosylation. 32000‐Mr and 66000–69000‐Mr proteins were also observed amonst the immunoreactive products initially formed during the labeling of intact endosperm tissue in vivo, together with 37000‐Mr and 39000‐Mr proteins. Pulse‐chase experiments showed that 66000–69000‐Mr proteins slowly disappeared while the smaller proteins were further cleaved to chains of Mr 31000 (authentic A chain), 34000 and 37000 (authentic glycosylated B chains).It was concluded that R. communis agglutinin polypeptides were synthesized in precursor form, possibly as a ‘giant’ precursor in the case of the B chain, on membrane‐bound polysomes. Contranslational translocation across the endoplasmic reticulum membrane was accompanied by proteolysis to remove leader sequences and, where appropriate, core glycosylation. Even after contranslational processing agglutinin polypeptides were still in precursor form. Processing to authentic size appeared to occur posttranslationally.
The Regulation of Potassium Fluxes for the Adjustment and Maintenance of Potassium Levels in Escherichia coli
Authors: MEURY Jean , KEPES Adam 
Published: March 2005

The regulation of K+ fluxes has been studied in Escherichia coli after depletion of K+ by an osmotic shock or at steady state of potassium accumulation.In the absence of a carbon source, bacteria accumulate K+ to an intracellular level near 0.1 M about half of actively metabolizing cells. During uptake the rate of net unidirectional influx decreased with time down to zero as a plateau was reached and no efflux was observed. Accumulated K+ was not exchanged with external K+ under these conditions. In the presence of a carbon source bacteria took up potassium to an intracellular concentration near 0.2 M. Efflux was delayed: it started only when 50% of final cellular K+ has been taken up. At steady state cellular K+ was exchangeable with external K+. The removal of the carbon source or the addition of respiratory inhibitors immediately stopped the K+ influx but did not affect efflux until the cellular K+ concentration has dropped to a level near 0.1 M; residual potassium was no longer exchangeable with external potassium. Maintaining such an impermeability to K+ ion does not require energy (ΔΨ or ATP), even though residual K+ is concentrated 100‐fold compared to K+ in the medium. These results suggest that K+ efflux is dependent on active metabolism and on the concentration of intracellular K+ above a threshold. Unidirectional influx is also regulated by intracellular K+ according to a different concentration dependence. It appears that the trkA system, which is the only functional system under these experimental conditions is not a ‘pump and leak’ type of transport system.