/sup 125/I-ubiquitin is kinetically invalid as a tracer radiolabel for studies of ATP-dependent proteolysis

One implication of the fact that so many proteins are conjugated to Ub is that there is little selectivity to Ub dependent proteolysis. This can be questioned on two bases. First, an enzyme activity exists in chromatin and reticulocyte lysates which hydrolyzes conjugates and thio-esters of Ub to free Ub and the protein or thiol which was attached. Thus a futile cycle exists which may be a control mechanism similar to that of aminoacyl t-RNA synthetase. Since we have no idea how the balance of hydrolysis vs. proteolysis is altered on fractionation of the reticulocyte lysate, it is difficult to evaluate the significance of the large number of conjugates observed in the steady-state. Secondly, the iodination of Ub could change the rates of hydrolysis and proteolysis. It is assumed that tracer-labeled /sup 125/I-Ub is functional in all of the steps of this system, but the activity of iodinated Ub has not been examined. The authors have addressed this question by synthesizing monoiodotyrosyl-ubiquitin (I-Ub) and directly testing its ability to support protein degradation