Rat liver cytosol contains NADPH- and GSH-dependent factors able to restore ornithine decarboxylase inactivated by removal of thiol reducing agents.
Removal of dithiothreitol (DTT) from partially purified ornithine decarboxylase (ODC) led to an almost complete inhibition of enzymic activity. The inactivation was reversed by addition of millimolar concentrations of DTT, whereas natural reductants such as NADPH or NADH were ineffective, and GSH had only a limited effect. Addition of rat liver cytosol to the incubation mixture resulted in a noticeable re-activation of ODC; however, dialysed cytosol had little effect unless NADPH or GSH was present. Fractionation of rat liver cytosol by gel filtration on Sephadex G-75 yielded two fractions involved in the NADPH- and GSH-dependent re-activation of ODC: one designated 'A', eluted near the void volume (Mr greater than or equal to 60,000), and the other designated 'B', eluted later (Mr approx. 12,000). The NADPH-dependent mechanism required both fractions A and B for maximal ODC re-activation; the most effective concentration of NADPH was 0.15 mM, although a significant effect was observed at a concentration more than 10-fold lower. The GSH-dependent mechanism involved the mediation of Fraction B only, and operated at millimolar concentrations of GSH. These results suggest the existence of reducing systems in the cytosol, which may play a role in maintaining, and potentially in regulating, ODC activity by modulation of its thiol status.
Flamigni
F
F
Dipartimento di Biochimica, Università di Bologna, Italy.
Guarnieri
C
C
Caldarera
C M
CM
D000818:Animals; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology*; D008099:Liver / Q000378:metabolism*; D009249:NADP / Q000494:pharmacology*; D009955:Ornithine Decarboxylase / Q000378:metabolism*; D065108:Ornithine Decarboxylase Inhibitors; D051381:Rats; D011919:Rats, Inbred Strains
D065108:Ornithine Decarboxylase Inhibitors; D009249:NADP; D009955:Ornithine Decarboxylase; D005978:Glutathione; D004229:Dithiothreitol