Stephen Roth

A new assay and cellular localization for an inducible glucuronyltransferase in the embryonic chick liver

A direct, radioisotopic assay is described for the uridine diphosphate glucuronic acid (UDPGA): p-aminophenol glucuronyltransferase. The assay uses solid phase p-aminophenol-Sephadex as the glucuronyl acceptor and UDP-[14C]GA as the glucuronyl donor. After incubation with the enzyme, the derivatized Sephadex beads are washed in SDS-urea or with high salt concentrations to remove all labeled material except for that covalently attached to the beads. Sonicated livers from chick embryos exposed to phenobarbital for at least 5 days transfer more than ten times the glucuronic acid to derivatized beads than do uninduced livers of the same developmental age. Glucuronyl-transferase activity can be detected on intact, living cells after 5 days of phenobarbital induction, whereas sonicate activity is detectable within 3 days of induction. Suspensions of living cells can show 25% the activity found in the same suspension after all the cells are lysed by sonication.

Change of isozyme pattern during activation of a transforming gene product: its relation to other biochemical markers of cellular transformation and differentiation.

Isozyme patterns of leucine aminotransferase were studied in connection with glucose transport and DNA synthesis during the activation and deactivation of the transforming gene product in rat kidney cells transformed by one Rous sarcoma virus mutant (which has a temperature-sensitive lesion in its transforming gene. On temperature shift-down of confluent transformed cells grown at 40 degrees C in the presence of fresh serum, isozyme III of leucine aminotransferase appeared in 12--20 h, with increasing amounts from 24 to 48 h. Upon temperature shift-up, isozyme I became the predominant form in these cells within 4 days, the major change occurring within the first 24 h. The rate of protein turnover was similar to the rate of loss of isozymes I and III during temperature shift-down and shift-up, respectively. A stimulation of incorporation of [3H]thymidine into DNA was observed within 8--12 h after temperature shift-down of the transformed cells. For the maintenance of stimulated DNA synthesis for at least 16 h, continued exposure to the permissive temperature is not necessary. Stimulation of glucose transport occurred prior to the stimulation of [3H]thymidine incorporation. The isozymes of leucine aminotransferase also changed during the in vitro differentiation of Yaffee L6A cells in such a way that isozyme I represented the major part of this enzyme in the fused myotube, and isozyme III was more predominant in the less differentiated state (mononucleated cells).