title: 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.
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002999:Clone Cells / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D009447:Neuroblastoma / Q000201:enzymology*; D009535:Nicotinamidase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
chemical_list: D002241:Carbohydrates; D013439:Sulfhydryl Reagents; D000581:Amidohydrolases; D009535:Nicotinamidase