Multiple molecules of APF-1/Ubiquitin are conjugated to the proteolytic substrate, probably signalling it for degradation.

To interpret the data described in the experiment depicted in Figure 2 and to test the hypothesis that APF-1 is conjugated to the target proteolytic substrate, ¹²⁵I-APF-1/ubiquitin was incubated along with crude Fraction II (Figure 3  and text) in the absence (lane 1) or presence (lanes 2–5) of ATP and in the absence (lanes 1,2) or presence (lanes 3–5) of increasing concentrations of unlabeled lysozyme. Reaction mixtures resolved in lanes 6 and 7 were incubated in the absence (lane 6) or presence (lane 7) of ATP, and included unlabeled APF-1/ubiquitin and ¹²⁵I-labeled lysozyme. C1–C6 denote specific APF-1/ubiquitin-lysozyme adducts in which the number of APF-1/ubiquitin moieties bound to a the lysozyme moiety of the adduct is increasing, probably from 1 to 6. Reactions mixtures were resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized following exposure to an X-ray film (autoradiography).

(with permission from Proceedings of the National Academy of the USA; published originally in Ref. ⁴⁰).