RESUMEN
Nicotinamidase (NAMase) from the budding yeast, Saccharomyces cerevisiae, was purified by Ni(2+) affinity chromatography and gel filtration. N-terminal microsequencing revealed sequence identity with a hypothetical polypeptide encoded by the yeast YGL037C open reading frame sharing 30% sequence identity with Escherichia coli pyrazinamidase/nicotinamidase. A yeast strain in which the NAMase gene, hereafter named PNC1, was deleted shows a decreased intracellular NAD(+) concentration, consistent with the loss of NAMase activity in the null mutant. In wild-type strains, NAMase activity is stimulated during the stationary phase of growth, by various hyperosmotic shocks or by ethanol treatment. Using a P(PNC1)::lacZ gene fusion, we have shown that this stimulation of NAMase activity results from increased levels of the protein and requires stress response elements in the 5'non-coding region of PNC1. These results suggest that NAMase helps yeast cells to adapt to various stress conditions and nutrient depletion, most likely via the activation of NAD-dependent biological processes.
Asunto(s)
ADN de Hongos/genética , Nicotinamidasa/genética , Saccharomyces cerevisiae/genética , Amidohidrolasas/análisis , Secuencia de Aminoácidos , ADN de Hongos/química , ADN de Hongos/aislamiento & purificación , Datos de Secuencia Molecular , Mutación , NAD/análisis , Nicotinamidasa/análisis , Nicotinamidasa/metabolismo , Reacción en Cadena de la Polimerasa , Recombinación Genética , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de AminoácidoAsunto(s)
Lípidos/análisis , Mycobacterium/clasificación , Oxazinas/farmacología , Fosfatasa Ácida/análisis , Amidohidrolasas/análisis , Bleomicina/farmacología , Cromatografía en Capa Delgada , Farmacorresistencia Microbiana , Mycobacterium/análisis , Mycobacterium/efectos de los fármacos , Mycobacterium/enzimología , Nicotinamidasa/análisis , OfloxacinoRESUMEN
Nicotinamide deamidase (nicotinamide amidohydrolase, EC 3.5.1.19) has been demonstrated in the conditioned growth medium of the M1 clonal cell line of mouse C1300 neuroblastoma. The enzyme has been purified 1200-1500-fold by Sephadex G25, hydroxyapatite, DEAE-cellulose, Sephadex G200 and NAD-Sepharose column chromatographies. The purified protein was characterized by polyacrylamide gel electrophoresis under non-denaturing and denaturing conditions. The apparent molecular weight has been estimated to be 230,000, and the subunits had respective molecular weights of 65,000 and 50,000. Histidine was the only NH2-terminal amino acid found. The enzyme is a glycoprotein; mannose and N-acetyl-glucosamine have been identified. The effects of various ions on its activity have been investigated. The enzyme has a Km for nicotinamide in the order of 10(-6) M, a pH optimum of 7.2 and a pHi of 5.4. It is inhibited by heating and by sulfhydryl reagents. The existence of a nicotinamide deamidase with a high affinity for nicotinamide favors the operation of the Preiss-Handler pathway in M1 cells cultured in vitro. We found an induction of nicotinamide deamidase and a cellular increase of NAD with a higher nicotinamide supply and a repression of the released enzyme with supplying NAD in the nutrition medium of M1 cell cultures.
Asunto(s)
Amidohidrolasas/metabolismo , Neuroblastoma/enzimología , Nicotinamidasa/metabolismo , Animales , Carbohidratos/análisis , Células Clonales/enzimología , Electroforesis en Gel de Poliacrilamida , Calor , Cinética , Ratones , Peso Molecular , Nicotinamidasa/análisis , Nicotinamidasa/aislamiento & purificación , Reactivos de Sulfhidrilo/farmacologíaRESUMEN
The adrenal homologue of C. batrachus is distributed around the postcardinal vein in the pronephric head kidney. The cortical cells are round or oval in shape. They showed positive reaction for total lipid, glycogen and ascorbic acid. Their intense delta5-3beta HSDH activity indicates their capacity for steroid biosynthesis. In addition, the cortical cells of C. batrachus exhibited strong G-6-PD, NADPH diaphorase, NADH diaphorase, MAO and weak SDH and LDH activity. The presence of MAO suggests the aminergic control of the adrenal in this species and the silver positive fibres seen the cortical cells were hypertrophied, degranulated and the lipid content was also decreased. The chromaffin or medullary cells were distributed in groups among the cortical cells. They are largely oval or angular in shape. They react positively to ferric ferricyanide, chromaffin and argentaffin reactions and ascorbic acid test.