Inactivation of yeast ornithine decarboxylase by polyamines in vivo does not result from the incorporation of polyamines into enzyme protein

The peptide mixture obtained from controlled proteolytic digestion of ligandin with proteinase K or subtilisin retained 40% of glutathione-S-transferase and steroid isomerase activities, immunological reactivity and lower affinity bilirubin binding but binding at the primary site was abolished. When these limited proteolytic digests, which had no intact ligandin as determined by SDS gel electrophoresis, were subjected to Sephadex G-75 column chromatography, 40–50% of the peptide fragments were recovered in fractions where intact ligandin eluted. The results suggest that intact ligandin is not required for enzymatic activities, binding of bilirubin at the secondary site, or immunological reactivity; steroid isomerase and glutathione-S-transferase activities are modulated in a parallel manner and may be mediated by the same region of the protein, and primary and secondary binding sites for bilirubin are distinct and independent, despite nicks introduced by proteolysis in ligandin's subunits, some of the fragments remain associated under non-denaturing conditions and the susceptibility of the two subunits to the proteases is different.