bluuebunny/crossref_metadata_2025_split huggingface dataset:
Endogenous Role of Epoxide Hydratase
Authors: BINDEL Ulla , SPARROW Alan , SCHMASSMANN Hansueli , GOLAN Mario , BENTLEY Philip , OESCH Franz 
Published: March 2005

A highly sensitive and rapid radiometric assay for the determination of specific epoxide hydratase activity with a steroid epoxide (16α, 17α‐epoxy‐1,3,5(10)‐estratrien‐3‐ol, ‘estroxide’) has been developed. The unreacted substrate was separated from the product 1,3,5(10)‐estratrien‐3,16β,17α‐triol by extraction into light petroleum. The product was then extracted into ethyl acetate and measured by scintillation spectrometry. Radiochromatography established that after subtraction of the blank the entire radioactivity measured in the ethyl acetate phase resulted from the product 1,3,5(10)‐estratrien‐3,16,17‐triol, whilst high performance liquid chromatography with the four possible isomers of 1,3,5(10)‐estratrien‐3,16,17‐triols as standards showed that the product was the 3,16β,17α‐triol. Limits of linearity with time and protein were established with microsomal fractions from liver, testes and ovaries.The apparent Km and V values were determined with liver and testes microsomes. The apparent Km for estroxide was very similar in both organs (liver 10 μM; testes 6.3 μM) which was similar to the value obtained for the series of epoxides derived from polycyclic hydrocarbons but substantially lower than that for styrene oxide. The specific activity of epoxide hydratase with estroxide as substrate was about twice as high as with benzo(a)pyrene 4,5‐oxide. The ratio of the specific epoxide hydratase activity with the two substrates was similar (1.6–2.2) in all investigated organs [liver, kidney, lung, testes (ovaries) and adrenals] of either sex of rats. Immunoprecipitation of solubilized liver microsomes by antibodies raised against epoxide hydratase purified to homogeneity was measured with estroxide, benzo(a)pyrene 4,5‐oxide and styrene oxide as substrates. The extent of precipitation of activity towards all three substrates with increasing amounts of added antibody was the same which suggests that all three reactions are catalysed by the same microsomal enzyme.
Subcellular Distribution of Membrane‐Bound Glycosyltransferases from Pea Stems
Authors: DÜRR Mathias , BAILEY David S. , MACLACHLAN Gordon 
Published: March 2005

Particulate and subcellular membrane preparations from growing regions of etiolated pea stems catalyse the transfer of sugars from UDP‐[14C]glucose and GDP‐[14C]mannose to a variety of endogenous lipid, glycoprotein and polysaccharide acceptors. Glycolipids were fractionated and identified by chromatography on DEAE‐cellulose and silica gel. They included neutral components, e.g. sterylglycosides, and polar lipids, comprising polyprenylmonophospho‐monosaccharides and polyprenyldiphospho‐oligosaccharides. High‐molecular‐weight material was partially hydrolyzed with pronase or acidic cyanogen bromide in order to solubilize glycoproteins and products bound to proteins, and to separate these from insoluble polysaccharide.Pea membranes with densities at which endoplasmic reticulum equilibrates in linear sucrose gradients contained most of the recovered capacity for glycosylation of endogenous polyprenyl monophosphate. When dolichyl phosphate was added to the membrane preparations, it was readily glycosylated in the presence of lysophosphatidylcholine but there was no indication that dolichylphospho‐monosaccharide served as an intermediate for synthesis of other products. In contrast, glycosyltransfer to endogenous neutral lipids, polyprenyl diphosphate and polymeric products occurred to a limited extent in the endoplasmic reticulum region and was most extensive in membranes which equilibrate at higher densities, i.e. in regions containing Golgi and possibly plasma membrane vesicles. The data are consistent with the conclusion that glycosylation of polymeric products occurs throughout the pea endomembrane system, with polyprenylmonophospho‐monosaccharide available to act as an intermediate (precursor) primarily in the endoplasmic reticulum.
Cytochrome c Oxidase Subunits in Nuclear and Extranuclear Cytochrome‐aa3‐Deficient Mutants of Neurospora crassa
Authors: BERTRAND Helmut , WERNER Sigurd 
Published: March 2005

The mitochondria of cytochrome‐aa3‐deficient Neurospora crassa mutants were screened for the seven polypeptide constituents of cytochrome c oxidase. The polypeptides of the holoenzyme and the unassembled or partially assembled subunits were detected by sodium dodecyl sulfate/acrylamide gel electrophoresis of immunoprecipitates obtained with antiserum to the holoenzyme as well as to several individual subunits. With respect to the mitochondrially synthesized polypeptides of the oxidase, subunits 1 to 3, the results obtained from the analysis of immunoprecipitates were confirmed through the direct electrophoretic analysis of mitochondrial translation products. The results were as follows.

The mitochondria of the cya‐2‐8 and cya‐3‐16 nuclear mutants and the [exn‐5] cytoplasmic mutant contained a protein complex immunoprecipitated by anti‐holoenzyme antibody and composed of the complete set of the seven cytochrome oxidase polypeptides. Only the oxidase sub‐units 5 and 6 were immunoprecipitated by anti‐holoenzyme antibody from the mitochondria of the cyt‐2‐1 and 299‐1 nuclear mutants, even though at least some of the mitochondrially synthesized polypeptides were detected in both mutants by subunit specific immunoprecipitation.
A ‘subunit 1’ polypeptide larger than the authentic subunit‐1 polypeptide of wild‐type cytochrome oxidase was found in the mitochondria from two nuclear mutants, cyt‐2‐1, and 299‐1 and the [mi‐3] cytoplasmic mutant. This larger polypeptide may be an unprocessed precursor of the ‘mature’ subunit 1 protein of the holoenzyme. No changes in the apparent molecular weights were found for the polypeptide subunits of cytochrome oxidase in mitochondria of the [exn‐5] cytoplasmic mutant and the cya‐2‐8 and cya‐4‐23 nuclear mutants.
A nuclear mutant, 299‐1, lacks the mitochondrially synthesized subunit‐2 polypeptide of cytochrome oxidase. When cells were labelled in the presence of cycloheximide, the subunit 2 content of mitochondria from mutants [exn‐5], cya‐2‐8, cya‐3‐16 and cya‐4‐23 was lower than in mitochondria from wild‐type. This deficiency, however, does not appear to be sufficiently severe to fully account for the lack of cytochrome aa3 in these mutants. The cya‐4‐23 nuclear mutant either is severely deficient in or lacks cytochrome oxidase subunits 5 and 6.On the basis of these and previously reported observations, it is proposed that the cytochrome oxidase deficiencies of as many as seven of the eight N. crassa cytochrome‐aa3‐deficient mutants could be caused by genetically imposed alterations in regulatory systems controlling the production of different components of the enzyme.
RNA Polymerase from the Fungus Aspergillus nidulans
Authors: STUNNENBERG Hendrik G. , WENNEKES Lambertus M. J. , VAN DEN BROEK Hendrikus W. J. 
Published: March 2005

The DNA‐dependent RNA polymerase I (or A) from the lower eukaryote Aspergillus nidulans has been purified on a large scale to apparent homogeneity by homogenizing the fungal hyphae in liquid nitrogen, extraction of the enzyme at high salt concentration, precipitation of RNA polymerase activity with polymin P (a polyethylene imine), elution of the RNA polymerase from the polymin P precipitate, ammonium sulphate precipitation, molecular sieving on Bio‐Gel A‐1.5m, binding to ion‐exchangers and DNA‐cellulose affinity chromatography. By this procedure 1.6 mg of RNA polymerase I can be purified over 2000‐fold from 500 g wet weight of starting material with a yield of 30–35%. The isolated RNA polymerase I is stable for several months at −20°C.The subunit composition has been resolved by polyacrylamide gel electrophoresis on two‐dimensional gels, using either non‐denaturing or 8 M urea (pH 8.7) cylindrical gels in the first dimension and sodium dodecyl sulphate slab gels in the second dimension. The putative subunits have molecular weights of 190000, 135000, 63000, 62000, 43000, 29000, 29000, (28000), 16000 and probably 13000 and 12000.Two distinct forms of RNA polymerase I (Ia and Ib) have been resolved by DEAE‐Sephadex A‐25 chromatography showing ample differences in enzymatic properties and subunit pattern.Additional information is given on RNA polymerase II (or B) which appears to be highly insensitive to α‐amanitin at concentrations up to 400 μg/ml.
Correlation between the Induction of Mouse Interferon and the Amount of Its mRNA
Authors: RAJ N. Babu Kishan , FERNIE Bruce F. , PITHA Paula M. 
Published: March 2005

Injection into Xenopus oocytes of polyadenylated cytoplasmic RNA from mouse C‐243‐3 cells induced with poly(rI) · poly(rC) and DEAE‐dextran resulted in the production of biologically active interferon. The synthesis of interferon by oocytes is linearly related to the amount of mRNA injected. Thus a titre of 160 reference units/oocyte was obtained by injecting 250 ng of total cytoplasmic poly(A)‐containing RNA.The kinetics of interferon production in mouse C‐243‐3 cells induced with poly(rI) · poly(rC) and DEAE‐dextran are characterized by an initial lag of 3–4 h and a burst of interferon secretion between 5–8 h with a maximum accumulation at 7 h. Comparison of the relative amount of interferon mRNA at various time intervals of induction with the cellular synthesis of interferon showed a general correlation. At 9 h after induction, i.e. during the shut off phase of interferon production, the cells contained no detectable amount of interferon mRNA (< 20 units/ml).Attempts to superinduce interferon production in mouse C‐243‐3 cells by using the cycloheximide/actinomycin D protocol as in human fibroblast cells were not successful. α‐Amanitin at concentrations similar to those required to inhibit cellular DNA‐dependent RNA polymerase form II completely inhibited the interferon production when applied during the first 3 h of induction. Addition of α‐amanitin at 3, 5 or 7 h after induction stimulated the subsequent interferon production by 2–4‐fold.
Inhibition of Protein Synthesis and Activation of Nuclease in Rabbit Reticulocyte Lysates by the Unusual Oligonucleotide pppA2′pSA2′p5′A
Authors: VAQUERO Catherine M. , CLEMENS Michael J. 
Published: March 2005

The oligonucleotide 5′‐triphosphoadenylyl(2′‐5′)adenylyl(2′‐5′)adenosine (pppA2′p5′A2′p5′A) is synthesised by reticulocyte lysates and extracts from interferon‐treated cells in the presence of double‐stranded RNA. It inhibits the translation of both viral and non‐viral mRNAs and of poly‐(rU,rC) in the micrococcal‐nuclease‐treated reticulocyte lysate system. The translation of poly(rU) is insensitive in this assay, however. Inhibition develops in vitro after a lag period, the length of which depends on the nature of the mRNA present and its concentration.The degradation of radioactive Mengo virus RNA or L cell cytoplasmic RNA is accelerated in reticulocyte lysates incubated with the oligonucleotide. This is due to activation of one or more endonucleases which degrade(s) RNA to large oligonucleotide fragments. Our results suggest that the endonuclease is responsible for most (if not all) of the inhibitory effects of the oligonucleotide on protein synthesis in the reticulocyte lysate.In the presence of pppA2′p5′A2′p5′A, protein synthesis can be reactivated after it has ceased by addition of fresh mRNA. In contrast, initiation factor eIF‐2 is unable to prevent inhibition by the oligonucleotide under conditions where it protects the system from the effects of the haem‐controlled repressor and partially protects against inhibition by double‐stranded RNA.These findings are discussed in relation to the regulation of protein synthesis by double‐stranded RNA and by interferon.
Characterization of a Thermosensitive Sporulation Mutant of Bacillus subtilis Affected in the Structural Gene of an Intracellular Protease
Authors: KERJAN Pierre , KERYER Eliane , SZULMAJSTER Jekisiel 
Published: March 2005

A thermosensitive sporulation mutant (ts‐15) of Bacillus subtilis has been isolated. This mutant when grown at the restrictive temperature (42°C) is unable to sporulate, shows no intracellular protease activity and no protein turnover. These three traits were recovered in two revertants (ts‐15R1 and ts‐15R2) and were also transmitted together by transformation into the wild type.Immunological studies have shown that when ts‐15 is grown at 42°C it synthesizes a ‘cryptic’ protein with apparently the same antigenic properties as the wild type or as ts‐15 mutant grown at the permissive temperature (30°C). The intracellular proteases from the wild type and from ts‐15 grown at 30°C and 42°C were completely purified and their properties were studied with respect to their molecular weights, substrate specificity, inhibition pattern, heat inactivation and antigenicity. The molecular weight of the enzyme from the wild type or ts‐15 grown at 30°C was 64000–65000 in the absence of sodium dodecylsulfate and 31000–32000 in the presence of sodium dodecylsulfate. It was assumed therefore that the active enzyme is formed from two similar subunits. However, the intracellular protease from ts‐15 grown at 42°C showed the same molecular weight of 32000–34000 in the presence or in the absence of sodium dodecylsulfate. On the basis of this experiment and others described in the paper we concluded that the mutation in ts‐15 is most likely a point mutation in a structural gene of an intracellular protease and results in an inability to assemble the two subunits into an active form.
Studies on a Proteinase B Mutant of Yeast
Authors: WOLF Dieter H. , EHMANN Claudia 
Published: March 2005

Yeast mutants lacking proteinase B activity have been isolated [Wolf, D. H. and Ehmann, C. (1978) FEBS Lett. 92, 121–124]. One of these mutants (HP232) is characterized in detail. Absence of the vacuolar localized enzyme is confirmed by checking for proteinase B activity in isolated mutant vacuoles. Defective proteinase B activity segregates 2:2 in meiotic tetrads. The mutation is shown to be recessive. Mutant proteinase B activity is not only absent against the synthetic substrate, Azocoll, but also against the physiological substrates pre‐chitin synthetase, cytoplasmic malate dehydrogenase and fructose‐1,6‐bisphosphatase. The mutant shows normal vegetative growth, a phenomenon not consistent with the idea that proteinase B might be the activating principle of chitin synthetase zymogen in vivo. Fluorescence microscopy shows normal chitin insertion. Enzymes underlying carbon‐catabolite inactivation in wild‐type cells (a mechanism proposed to be possibly triggered by proteinase B) such as cytoplasmic malate dehydrogenase, fructose‐1,6‐bisphosphatase, phosphoenolpyruvate carboxykinase and isocitrate lyase, are inactivated also in the mutant. NADP‐dependent glutamate dehydrogenase, which is found to be inactivated in glucose‐starved wild‐type cells, proceeds normally in the mutant. Mutant cells show more than 40% reduced protein degradation under starvation conditions. Sporulating diploids, homozygous for proteinase B absence, also exhibit an approximately 40% reduced protein degradation as compared to homozygous wild‐type diploids or diploids heterozygous for the mutant gene.The time of the appearance of the first ascospores of diploid cells, homozygous for proteinase B deficiency, is delayed about 50% and sporulation frequency is reduced to about the same extent as compared to homozygous wild‐type diploids or diploids heterozygous for the mutant gene.
Purification and Some Characteristics of the Human Coagulation Factor VII
Authors: FLENGSRUD Ragnar 
Published: March 2005

1. A purification procedure for factor VII (proconvertin) from human plasma is described. The procedure involves barium sulphate adsorption and elution, DEAE‐Sephadex column chromatography, barium sulphate adsorption and elution, heparin‐Sepharose column chromatography, preparative disc gel electrophoresis and finally adsorption with antiserum to prothrombin coupled to Sepharose and antiserum to albumin coupled to Sepharose. This procedure gave an approximately 8 · 105‐fold purification.2. The factor VII obtained from the electrophoresis step was mainly a single‐chain protein with an apparent molecular weight of 53000 ± 2000.3. After the final purification step, additional forms of factor VII, resulting from a fragmentation of the factor VII molecule were detected.4. Amino acid composition data of the purified factor VII are given.5. Antisera were raised in two different rabbits by injection of the purified factor VII. The antisera obtained gave a good titre against the factor VII activity and were not directed against any of the three other vitamin‐K‐dependent coagulation factors.
Site‐Specific Substituted Cobalt(II) Horse Liver Alcohol Dehydrogenases
Authors: MARET Wolfgang , ANDERSON Inger , DIETRICH Helmut , SCHNEIDER‐BERNLÖHR Helga , EINARSSON Roland , ZEPPEZAUER Michael 
Published: March 2005

The specific substitution, using highly selective techniques, of catalytic and/or noncatalytic zinc ions by cobaltous ions in horse liver alcohol dehydrogenase (EC 1.I.1.1) has been studied with dissolved, crystalline and agaroseimmobilised enzyme, in order to examine the effect of protein structure on the specificity of the metal exchange. The different binding sites can be clearly distinguished by the absorption spectra of their cobalt derivatives.In solution an anaerobic column chromatographic method made it possible to exchange half of the zinc in the enzyme by cobalt ions in a much shorter time than previous procedures. By raising the temperature in the exchange step, even the slowly exchanging zinc ions were substituted by cobalt, yielding products similar to cobalt alcohol dehydrogenases described earlier.Treatment of crystal suspensions of the enzyme with chelating agents (preferentially dipicolinic acid) gave an inactive protein with two zinc ions remaining bound. The enzyme could be reactivated by treatment of the crystalline protein with 5 mM zinc or cobaltous ions or by dialysis of dissolved inactive protein against 20 μM zinc or 1 mM cobaltous ions. Higher metal concentrations led to denaturation but the inactive protein could be crystallized from solution and then reactivated completely at higher metal concentrations. The preparation and absorption spectrum show that cobalt is bound specifically at the catalytic sites. Since metal substitution at these sites critically depends on the maintenance of the correct tertiary and quaternary structure, these must be preserved in the crystal lattice and partially altered in solution when the catalytic zinc ions are removed (or when excess of metal ions is applied), thus demonstrating the structure‐stabilizing rôle of the catalytic metal ions.The enzyme immobilised on agarose, with unchanged content of active sites [Schneider‐Bernlöhr et al. (1978) Eur. J. Biochem. 41, 475–484], was treated like the crystal suspensions. Although half of the zinc was removed, some activity remained. After reactivation with cobaltous ions, a loss of about 30% active sites was measured. Thus the apparently homogeneous bound enzyme was rather heterogeneous in the properties of its catalytic metal binding sites. These results are taken as further proof for the dependence of the metal substitution on the proper tertiary and quaternary structure which is strained by multiple interactions in the covalently immobilised enzyme.
Correlation between Rates and Enzyme Levels of Increased Gluconeogenesis in Rat Liver and Kidney after Partial Hepatectomy
Authors: KATZ Norbert 
Published: March 2005

The metabolic rates and key enzymes of gluconeogenesis and glycolysis were determined and correlated in perfused livers, isolated hepatocyte suspensions and kidney cortex slices of rats after two‐thirds hepatectomy. Fed unoperated, sham operated and fasted animals served as controls.In liver the following results were obtained. Firstly, after partial hepatectomy [14C]glucose formation from [14C]lactate was increased in perfused livers to 223% after 24 h and to 173% after 48 h and in hepatocyte suspensions to 222% after 48 h. [14C]Glucose production from [14C]fructose, however, was unchanged. These metabolic rates correlated well with an increase of the level of phosphoenolpyruvate carboxykinase to 206% after 24 h and to 184% after 48 h. The levels of fructose‐1,6‐bisphosphatase and glucose‐6‐phosphatase were unaltered. Secondly, after a 24‐h fast gluconeogenesis from lactate was enhanced to 282% in perfused livers and to 300% in hepatocyte suspensions. Gluconeogenesis from fructose was slightly increased to 140%. These altered rates were accompanied by an increase of phosphoenolpyruvate carboxykinase to 213% and of glucose‐6‐phosphatase to 190%. Thirdly, after partial hepatectomy [14C]lactate formation from [14C]glucose was decreased in hepatocyte suspensions to less than 20% after 48 h. [14C]Lactate production from [14C]fructose was unchanged. These rates coincided with a decrease of the level of glucokinase to 40% and with unaltered levels of phosphofructokinase and pyruvate kinase. Fourthly, after a 24‐h fast glycolysis from glucose was lowered to below 30%. This rate was paralleled by a decrease of the level of glucokinase to 60%.In kidney cortex the following results were obtained. Firstly, after partial hepatectomy gluconeogenesis from lactate was enhanced in tissue slices to 217% after 24 h. Gluconeogenesis from fructose was unchanged. These rates coincided with an increase of the level of phosphoenolpyruvate carboxykinase to 193% and of glucose‐6‐phosphatase to 151%. Secondly, after a 24‐h fast similar changes for the gluconeogenic rate to 200% and of phosphoenolpyruvate carboxykinase to 240% as well as of glucose‐6‐phosphatase to 136% were observed. Thirdly, neither the rates of glycolysis nor the levels of hexokinase, phosphofructokinase and pyruvate kinase were altered after partial hepatectomy or a 24‐h fast.No significant changes neither in liver nor in kidney cortex occurred after sham operation. Thus the drastic reduction of liver tissue after partial hepatectomy was compensated by an increase of gluconeogenesis in the liver remnant and in kidney cortex, both accompanied by an enhancement of the phosphoenolpyruvate carboxykinase level, in order to maintain the glucose homeostasis.
Enrichment and Characterization of the DNA Coding for Vitellogenin in Xenopus laevis
Authors: WIDMER Heinrich J. , JAGGI Rolf B. , WEBER Rudolf , RYFFEL Gerhart U. , MUELLENER Daniel 
Published: March 2005

Purified vitellogenin mRNA of Xenopus laevis was incubated with mechanically sheared DNA in high concentrations of formamide and the resulting R‐loops (i.e. RNA · DNA hybrid fragments) separated from the bulk DNA by caesium chloride buoyant density centrifugation. Hybridization with 125I‐labeled vitellogenin mRNA revealed a 15–30‐fold enrichment of the DNA coding for vitellogenin.Restriction analysis of the R‐loop‐enriched DNA demonstrated that all known endonuclease HindIII fragments coding for vitellogenin of unfractionated Xenopus DNA were also present in the enriched material, including the specific fragments for the oligo(A)‐containing segment of the RNA.Comparison of these restriction data with the structure found in cloned vitellogenin cDNA, indicates the presence of at least one intervening sequence in the genomic DNA coding for vitellogenin.