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Purification and characterization of a nicotinamide deamidase released into the growth medium of neuroblastoma in vitro.

Abstract
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.
AuthorsM Wintzerith, A Dierich, P Mandel
JournalBiochimica et biophysica acta (Biochim Biophys Acta) Vol. 613 Issue 1 Pg. 191-202 ( 1980) ISSN: 0006-3002 [Print] Netherlands
PMID7378417 (Publication Type: Journal Article)
Chemical References
  • Carbohydrates
  • Sulfhydryl Reagents
  • Amidohydrolases
  • Nicotinamidase
Topics
  • Amidohydrolases (metabolism)
  • Animals
  • Carbohydrates (analysis)
  • Clone Cells (enzymology)
  • Electrophoresis, Polyacrylamide Gel
  • Hot Temperature
  • Kinetics
  • Mice
  • Molecular Weight
  • Neuroblastoma (enzymology)
  • Nicotinamidase (analysis, isolation & purification, metabolism)
  • Sulfhydryl Reagents (pharmacology)

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