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201.
Purification and characterization of methylmalonate-semialdehyde dehydrogenase from rat liver. Identity to malonate-semialdehyde dehydrogenase 总被引:1,自引:0,他引:1
G W Goodwin P M Rougraff E J Davis R A Harris 《The Journal of biological chemistry》1989,264(25):14965-14971
Methylmalonate semialdehyde dehydrogenase was purified from rat liver in order to define the distal portion of valine catabolism and related pathways in mammals. The purified enzyme is active with malonate semialdehyde and consumes both stereoisomers of methylmalonate semialdehyde, implicating a single semialdehyde dehydrogenase in the catabolism of valine, thymine, and compounds catabolized by way of beta-alanine. The oxidation of malonate and methylmalonate semialdehydes by this enzyme is CoA-dependent, the products being acetyl-CoA and propionyl-CoA, respectively. Expected activity with ethylmalonate semialdehyde as substrate was not found. Methylmalonate semialdehyde dehydrogenase was separated on DEAE-Sephacel into two isoforms which differ in mobility during nondenaturing polyacrylamide gel electrophoresis. The two forms are immunologically cross-reactive and exhibit the same N-terminal sequence, suggesting that one form is the product of the other. The monomer molecular mass, determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was 58 kDa. The native molecular mass, estimated by gel filtration, was 250 kDa, suggesting a tetrameric structure. 相似文献
202.
Defective H(+)-ATPase of hygromycin B-resistant pma1 mutants fromSaccharomyces cerevisiae 总被引:5,自引:0,他引:5
D S Perlin S L Harris D Seto-Young J E Haber 《The Journal of biological chemistry》1989,264(36):21857-21864
Mutations in the plasma membrane H(+)-ATPase gene (PMA1) of Saccharomyces cerevisiae that confer growth resistance to hygromycin B have been shown recently to cause a marked depolarization of whole cell membrane potential (Perlin, D. S., Brown, C. L., and Haber, J. E. (1988) J. Biol. Chem. 263, 18118-18122). In this report, the biochemical and genetic properties of H+-ATPases from four prominent hygromycin B-resistant pma1 mutants, pma1-105, pma1-114, pma1-147, and pma1-155, are described. Single base pair changes were identified in pma1-105, pma1-114, and pma1-147 that resulted in amino acid substitutions of Ser-368----Phe, Gly-158----Asp, Pro-640----Leu, respectively. An A----G transition mutation at -39 in the 5'-untranslated region of the mRNA of pma1-155 was also found. This mutation creates an out-of-Frame upstream AUG initiation codon that apparently reduces normal translation of PMA1. DNA sequence analysis of PMA1 from strain Y55 identified 9 base pair substitutions that resulted in 6 amino acid changes in nonconserved regions when compared to the published sequence for strain S288C. Plasma membranes of three of the four pma1 mutants contained normal amounts of H(+)-ATPase; membranes from pma1-155 contained enzyme at 62% of the wild-type level. The kinetics of ATP hydrolysis were most strongly altered for enzymes from pma1-105 and pma1-147 which showed changes in both Km and Vmax. A striking pH dependence for these parameters was found for enzyme from pma1-105 which resulted in a precipitous decline in Km and Vmax below pH 6.5. ATP hydrolysis by enzymes from pma1-105 and pma1-147 was insensitive to inhibition by vanadate. These enzymes, in contrast to wild-type and vanadate-sensitive mutant enzymes, were poorly protected from trypsin-induced inactivation by MgATP and vanadate or Pi alone. These results are pertinent to the mechanism of vanadate-induced enzyme inhibition and suggest that Ser-368 and Pro-640 influence the affinity of the phosphate-binding site for Pi. All mutant enzymes catalyzed ATP-induced pH gradient formation following purification and reconstitution into liposomes. Finally, these results further demonstrate the usefulness of hygromycin B as a generalized screening tool for isolating diverse plasma membrane ATPase mutants. 相似文献
203.
The effect of the bile salts, sodium cholate, deoxycholate, glycocholate and taurocholate, on the solubility in aqueous solution of the hydrophobic, environmental mutagen, 1,8-dinitropyrene (DNP), was examined. In the absence of bile salts, the DNP appeared to precipitate out of solution, whereas bile salts at a concentration of greater than or equal to 4 mM maintained the DNP in solution. In the presence of the model dietary fiber, alpha-cellulose, the DNP absorbed to this preferentially. Bile salts reduced this adsorption at low alpha-cellulose levels, but had little effect at high alpha-cellulose levels. The implication of these results is that bile salts have solubilising properties that could affect the distribution of hydrophobic molecules, including mutagens, in the digestive tract. 相似文献
204.
Between January 1985 and December 1986, 811 patients were treated for carcinoma of the breast at the Joint Center for Radiation Therapy by an identical protocol. Of these 811 patients, five patients (0.6%) were identified as having an unusually sensitive clinical response to routine external beam irradiation. This unusual clinical response was characterized by severe skin erythema and edema during the first few weeks of treatment, requiring treatment breaks. Skin fibroblast cell strains were established from these five women as well as from six women with a normal clinical response to breast irradiation (chosen at random from the population of 811 patients). Radiation survival parameters were determined by a colony formation assay from complete survival curves in coded and blinded samples. Cells from the sensitive patients were significantly more sensitive to the cytotoxic effects of radiation in vitro as determined by the parameters D0, D, D10, and n, than were the strains derived from patients with a normal response. We conclude that an unusually severe response to standard fractionated radiotherapy may be associated with greater intrinsic radiation sensitivity of the individual's somatic cells. 相似文献
205.
206.
Herpes simplex virus type 1 immediate-early protein Vmw110 reactivates latent herpes simplex virus type 2 in an in vitro latency system. 总被引:33,自引:27,他引:6
Reactivation of latent herpes simplex virus type 2 (HSV-2) by the immediate-early protein Vmw110 was studied by using an in vitro latency system. Adenovirus recombinants that express Vmw110 reactivated latent HSV-2. An HSV-1 mutant possessing a deletion in a carboxy-terminal region of Vmw110 reactivated latent HSV-2, whereas mutant FXE, which has a deletion in the second exon, did not. Therefore, Vmw110 alone is required to reactivate latent HSV-2 in vitro, and the region of Vmw110 defined by the deletion in FXE is important for this process. 相似文献
207.
Glycosphingolipids bearing GlcNAc beta 1----3 and GalNAc beta 1----4 linked to beta-Gal of lactosylceramide (lacto-ganglio hybrids), first isolated from a murine myelogenous leukemia cell line [Kannagi, R., Levery, S. B., & Hakomori, S. (1984) J. Biol. Chem. 259, 8444-8451], have since been found as normal components of mullet roe and English sole liver. In order to clarify the biosynthetic pathways responsible for its occurrence both as a product of normal tissues and as a possible mammalian cancer-associated antigen, the lacto-ganglio hybrid core structure LcGg4Cer was synthesized from Lc3Cer using a GalNAc beta 1----4 transferase preparation from English sole liver. A preliminary characterization of the enzyme, which may be identical to the GalNAc T-1 responsible for synthesis of GM2 ganglioside, is presented. The enzymatically synthesized product was analyzed by 1- and 2-D 1H NMR spectroscopy, confirmining its primary structure as GalNAc beta 1----4-(GlcNAc beta 1----3)Gal beta 1----4Glc beta 1----1Cer. In addition to assigning all nonexchangeable glycosyl proton resonances, measurements of several properties of the amide NH protons, including chemical shift, coupling constants, exchange rates, and temperature shift coefficients, were obtained and compared to those in the simpler constituent triglycosylceramides, Lc3- and Gg3Cer. An approximate three-dimensional structure for LcGg4Cer is proposed, consistent with all data obtained, which should be useful in discussing the results of 1H NMR analysis of compounds containing this core tetrasaccharide. The structure is characterized by an unusual arrangement of terminal N-acetylhexosamine residues, resulting in a pi-H hydrogen-bonding interaction between their acetamido groups. 相似文献
208.
Lucien J. Houenou Martine Pinon-Raymond Luis Garcia A. John Harris Franois Rieger 《Developmental neurobiology》1990,21(8):1249-1261
Developmental aspects of the neuromuscular system in mouse embryos chronically paralyzed in utero with tetrodotoxin (TTX) between embryonic days 14 and 18 were studied using biochemical and histological methods. The number of lumbar spinal motoneurons (MNs) was higher in inactive embryos than in controls suggesting a decreased motoneuron cell death. In association with the increase in MN number, choline acetyltransferase activity was significantly increased in both spinal cord and peripheral synaptic sites. Paralyzed muscles exhibited a decreased number of mature myofibers and the nuclei were centrally located. Creatine kinase activity was greatly decreased and total acetylcholine receptor and receptor cluster numbers per myofiber were significantly increased in paralyzed muscles. A similar pattern of changes occurs in the neuromuscular system of the mutant mouse muscular dysgenesis (mdg). However, in contrast to the mdg mutant, tetrodotoxin-treated muscles were similar to controls in their innervation pattern, in the ultrastructural aspects of the excitation–contraction coupling system (i.e., dyads and triads) and in the extent of dihydropyridine binding. Thus, neuromuscular inactivity is not sufficient to impair the pattern of muscle innervation or the appearance of either the triadic junctions or dihydropyridine receptors. These results indicate that alterations of dihydropyridine binding sites and triads in muscular dysgenesis cannot be accounted for by inactivity but rather must reflect a more primary defect involving the structural gene(s) regulating the development of one or more aspects of muscle differentiation. 相似文献
209.
William A. Harris 《Developmental neurobiology》1990,21(7):953-957
This article introduces this special issue of the Journal of Neurobiology by reviewing several basic issues in metamorphosis as they specifically relate to the nervous system. It promotes the idea that metamorphic changes in the nervous system (neurometamorphosis) represent adaptive restructurings rather than recapitulations of evolutionary transitions. It introduces, but leaves unresolved, the question of whether neurometamorphosis is achieved primarily as a delayed phase of embryonic neurogenesis or as a special neurogenic period. It points out that respecification of old neurons and the addition of new neurons are the main contributory pathway of neural restructuring at metamorphosis, that respecification can be dramatic and seems to be preferred over the elimination and replacement of particular neurons. It also highlights the question of how much the central rewiring during metamorphosis is driven by trophic interactions with the changing body of the metamorphic animal and to what extent neurometamorphosis is driven by the direct action of metamorphic hormones on the neural elements themselves. Finally, this article introduces the question of the cellular and molecular pathways of neurometamorphosis, from the role of the nervous system in triggering the event to the receptor mediated changes in gene expression. Further details on all of these issues are to be found in the articles that make up the rest of this special issue. 相似文献
210.
Microtubule Depolymerization Inhibits Ethanol-Induced Enhancement of GABAA Responses in Stably Transfected Cells 总被引:1,自引:0,他引:1
Valerie J. Whatley Susan J. Brozowski †Karen L. Hadingham †Paul J. Whiting ‡ R. Adron Harris 《Journal of neurochemistry》1996,66(3):1318-1321
Abstract: We studied whether microtubule organization is important for actions of ethanol on GABAA ergic responses by testing the effects of microtubule depolymerization on ethanol enhancement of GABA action in mouse L(tk− ) cells stably transfected with GABAA receptor α1 β1 γ2L subunits. The microtubule-disrupting agents colchicine, taxol, and vinblastine completely blocked ethanol-induced enhancement of muscimol-stimulated chloride uptake. β-Lumicolchicine, a colchicine analogue that does not disrupt microtubules, had no effect on ethanol action. Colchicine did not alter the potentiating actions of flunitrazepam or pentobarbital on muscimol-stimulated chloride uptake. Thus, colchicine specifically inhibited the potentiating action of ethanol. From these findings, we conclude that intact microtubules are required for ethanol-induced enhancement of GABAA responses and suggest that a mechanism involving microtubules produces posttranslational modifications that are necessary for ethanol sensitivity in this cell system. 相似文献