Isolation and characterization of an activator for Azotobacter vinelandii nicotinamide mononucleotide glycohydrolase.
Azotobacter vinelandii NMN glycohydrolase [EC 3.2.2.14] has been shown to require absolutely GTP or a high-molecular-weight and heat-stable component for its function. The intracellular activator could be purified from its sonicate by heat treatment, acetone precipitation, phenol extraction, and acid precipitation in a good yield. The purified activator showed high affinity and effectiveness for NMN glycohydrolase (KA = 0.012 optical density unit at 257 nm/ml; Vmax standardized by the activity at 1 mM GTP = 88%). Negative cooperativity of the enzyme activation with the activator was also shown. On treatment with either micrococcal nuclease or pancreatic RNase, the activator activity was completely abolished, whereas pronase and trypsin had no effect. The activator could be replaced by yeast RNA as well as calf liver RNA, whereas DNAs purified from Micrococcus lysodeikticus, T 7 and calf thymus had no effect on the enzyme. Furthermore, poly(G) and poly(I) could function as activators with the same effectiveness as the purified activator, and the enzyme activation with these RNA homopolymers was inhibited by poly(C), suggesting that the activation mechanism is specific with respect to base composition. Based on a kinetic analysis of the enzyme activation with commercial RNAs, together with the results from enzymatic digestion, specific inhibition of the enzyme by spermine, and its chemical properties, the activator was identified as an RNA. A model is described for NMN glycohydrolase regulation in which the RNA activator plays an important role in the NMN salvage cycles.
D001395:Azotobacter / Q000201:enzymology*; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D011066:Poly C / Q000494:pharmacology; D012329:RNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D013096:Spermine / Q000494:pharmacology
D012329:RNA, Bacterial; D013096:Spermine; D011066:Poly C; D009699:N-Glycosyl Hydrolases; C021881:NMN nucleosidase