David E. Jensen

Quantitation of S-methylcysteine formed in O6-methylguanine-DNA: methyltransferase

The O6-methylguanine-DNA:methyltransferase is known to transfer the methyl group from O6-methylguanine (O6-mG) in DNA to the cysteinyl residue of the methyl acceptor protein which is the methyltransferase itself. We developed a novel method to estimate the amount of S-methylcysteine formed in the acceptor protein, utilizing O6-[methyl-3H]methylguanine-containing DNA as the methyl donor. Following hydrolysis of the methyl-incorporated macromolecules in formic acid--HCl, S-[methyl-3H]methylcysteine is chromatographically isolated on a small column of Dowex-50(H+). Utilizing this method, we measured O6-mG DNA:methyltransferase activity in rat liver during neonatal development; the activity increases from 0.013 pmol methyl transferred/mg protein at 2 days postnatally to 0.06 pmol/mg at 8 weeks, the latter being equal to the adult liver activity.

Evidence for metabolism of N-nitrosoproline

The metabolism of nitrosoproline (NPRO) was re-investigated in uni- and bilaterally nephrectomized rats that have reduced or absent ability to excrete urine. About 1% of the administered radioactivity from L-[U-14C]-NPRO appeared as 14CO2 in the expired air and the production of 14CO2 was time-dependent over a period of 23 h. As compared with sham-operation, uni- or bilateral nephrectomy did not significantly increase the amount of NPRO metabolism, though urinary excretion of radioactivity was decreased in the unilaterally nephrectomized animals. In microsome-mediated and in vitro enzyme-free (Udenfriend-hydroxylating) systems covalent binding of [2,3,4,5-3H]NPRO to exogenous calf thymus DNA was demonstrated. The above findings confirm that in vivo metabolism of NPRO is possible, albeit, to a very small extent.