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1.
Of the two mitochondrial enzymes of the urea cycle, carbamoyl phosphate synthetase (CPS) was and ornithine transcarbamylase (OTC) was not inactivated by the Fe3+-oxygen-ascorbate model system for mixed-function oxidation [R. L. Levine, (1983) J. Biol. Chem. 258, 11828-11833]. The susceptibility of OTC was not increased by its substrates, products, or inhibitors, whereas that of CPS was markedly increased by acetylglutamate (its allosteric activator) when ATP was absent. Thus, acetylglutamate binds in the absence of ATP and exposes to oxidation essential groups of the enzyme. We estimate for this binding a KD value of 1.6 mM, which greatly exceeds the KD values (less than 10 microM) determined in the presence of ATP and bicarbonate. ATP, and even more, mixtures of ATP and bicarbonate protected CPS from inactivation. Acetylglutamate exposes the site for the ATP molecule that yields Pi, and it appears that ATP protects by binding at this site. Experiments of limited proteolysis with elastase suggest that oxidation prevents this binding of ATP and show that it accelerates cleavage of CPS by the protease, thus supporting the idea that oxidation may precede proteolysis. Trypsin, chymotrypsin, and papain also hydrolyze the oxidized enzyme considerably faster than the native enzyme. Our results also support the idea that oxidative inactivation is site specific and requires sites on the enzyme for Me2+ and, possibly, for a nucleotide. 相似文献
2.
Carbamoyl-phosphate synthetase was inactivated by elastase with first-order kinetics, and N-acetyl-L-glutamate speeded inactivation. From the dependence of the t1/2 value for inactivation on the concentration of acetylglutamate we estimate a Kd value for binding of the activator of 0.365 mM, which is approximately 600 times greater than in the presence of ATP, HCO3-, K+ and Mg2+. K+ and Mg2+ are not required for binding with low affinity, and in the absence of ATP they do not appear to increase the affinity for acetylglutamate. In the presence of acetylglutamate, mixtures of ATP, K+ and Mg2+ protect the enzyme from inactivation. ADP or AdoPP[NH]P partly replaced ATP in protecting the enzyme and thus binding of the nucleotide without further reaction is enough for protection. Two partial activities of the enzyme were inactivated by elastase to the same extent as the overall reaction, and thus elastase affects some property of the enzyme which is essential for catalysis. With other proteinases tested, inactivation was also accelerated by acetylglutamate and was slowed by mixtures of ATP, K+, Mg2+ and acetylglutamate, suggesting that changes in the accessibility of susceptible bonds are responsible for the changes in the degree of inactivation. It is concluded that elastase attacks at or close to the binding sites for ATP, and that exposure of the binding site for the ATP molecule that yields Pi (ATPA) upon binding of acetylglutamate causes the acceleration of the proteolytic inactivation. 相似文献
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F Sánchez R González J Carretero M Rubio J M Riesco E Blanco J A Juanes R Vázquez 《Histology and histopathology》1992,7(4):719-724
Using the peroxidase-antiperoxidase immunocytochemical technique, a morphometric study of the magnocellular neurons of the Paraventricular nucleus of the rat hypothalamus, reactive to specific anti-vasopressin rabbit serum, was made. Following systemic and chronic administration of met-enkephalin the number of immunoreactive neurons was higher, especially in females. Additionally, in the females, it was possible to observe an increase in the immunoreactivity and the presence of well-stained fibres. These findings suggest, especially in females, a blockage in the release of vasopressin, facilitating its immunocytochemical visualization. 相似文献
5.
Flora Sánchez Angeles Touriño Susana Traseira Agustín Pérez-Aranda Víctor Rubio Miguel A. Peñalva 《Molecular & general genetics : MGG》1986,205(2):248-252
Summary We cloned the Penicillium chrysogenum trpC gene from a genomic library by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase activity. The gene ecodes a 2.7 kb poly(A)+ RNA. We localized the gene by sequence analysis in a 2.9 kb DNA insert found in the smallest plasmid selected from the library. Sequence data strongly suggest that the organization of the gene is similar to that described in other Ascomycetes. We found that a DNA fragment which codes only for the carboxy-terminal protion of the polypeptide is sufficient for complementation of the E. coli trpC9830 mutation. 相似文献
6.
Microbial metabolism of chlorosalicylates: effect of prolonged subcultivation on constructed strains
Miguel Angel Rubio Karl-Heinrich Engesser Hans-Joachim Knackmuss 《Archives of microbiology》1986,145(2):123-125
The hybrid strain Pseudomonas sp. WR4016 was subcultivated with increasing concentrations of 5-chlorosalicylate (510 mM) as sole carbon source over a period of 9 months. At intervals of approximately 3 months derivative strains WR4017, WR4018 and WR4019 were isolated which exhibited higher growth rates and increased substrate tolerance. Comparative analysis of the turnover rates of the key enzymes in chlorosalicylate degradation showed that the adaptation process did not result from structural modifications of these proteins. Instead, balanced over-production of the salicylate hydroxylase and catechol 1,2-dioxygenase prevented the accumulation of toxic chlorocatechols and accounted for the reduction of the doubling times with 4- or 5-chlorosalicylate. A comparative analysis of a genetically engineered chlorosalicylate degrader PL300-1 showed similar regulatory patterns as the most advanced isolate WR4019 from the adaptation series. 相似文献
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An improved medium for the isolation of Cryptococcus neoformans from pigeon droppings 总被引:1,自引:0,他引:1
Isolation of Cryptococcus neoformans is enhanced when methyl violet at 2 mg l-1 is added to the usual Guizotia abyssinica medium. In preliminary tests the medium has also proved useful for isolating C. neoformans from other sources. 相似文献
10.
Recombination experiments using radioactive mitochondria and mitoplasts, and nonradioactive lysosomes or digitonin-soluble fraction of mitochondria, show equal rates of proteolysis and of inactivation of carbamyl phosphate synthetase; the amount of lysosomal protein was equal in both cases on the basis of N-acetyl-beta-glucosaminidase activity. Therefore, lysosomes seem to be responsible for all the proteolytic activity exhibited by the digitonin soluble fraction of mitochondrial preparations. Since this fraction contains ca. 90% of the proteolytic activity present in mitochondrial preparations, most of the proteolysis can be attributed to lysosomal contamination. These findings and stability characteristics "in vitro" and "in vivo" of some matrix enzymes are presented and discussed in relation to protein turnover. 相似文献