Unfolding and refolding occur much faster for a proline-free proteins than for most proline-containing proteins.

The kinetics for unfolding and refolding of a parvalbumin (band 5) have been examined as a function of pH near the transition region, using stopped-flow techniques. This protein is rather unusual in that it has no proline residues, and therefore serves as a good example to test the hypothesis that the rate-limiting step seen in denaturation reactions is due to the cis-trans isomerization of proline peptide bonds in the denatured state. The kinetics for parvalbumin unfolding and refolding are complex, with the data being resolvable into two fast phases at 25 degrees. The slower of the two phases seen for the parvalbumin is about 100 to 500 times faster than the slow phase seen for proline-containing proteins under the same conditions! These results argue strongly in support of the proline isomerization hypothesis. It is also suggested that the slower phase seen for parvalbumin and the second-slowest phase seen for proline-containing proteins might be due to the cis-trans isomerization of peptide bonds of non-proline residues.     

The effects of methapyrilene hydrochloride on hepatocarcinogenicity and pentobarbital-induced sleeping time in rats and mice

In a 6- to 8-month dose-response toxicity study, methapyrilene hydrochloride (HCl) was administered in the feed to $\text{Fischer-}\text{344}\text{N}$ rats and B6C3F1 mice at concentrations of 125, 250, 500, 1000, or 2000 ppm. After 26 weeks, rats fed the higher dose levels of methapyrilene HCl developed cholangiocellular carcinomas in addition to severe hepatotoxic lesions. After 31 weeks, mice exhibited only mild hepatotoxicity from ingesting methapyrilene HCl, even at the higher dose levels. The duration of sodium pentobarbital-induced sleeping time (PEN-induced ST) is determined by the rate of hepatic metabolism of PEN and can therefore be used as an in vivo measurement of the level of hepatic detoxifying enzymes. A subsequent study was undertaken with PEN-induced ST to investigate differences in the effect of methapyrilene HCl on hepatic detoxifying enzymes between the two species; in this study, prior treatment with methapyrilene HCl significantly increased PEN-induced ST of rats but not of mice, indicating that methapyrilene HCl had a suppressive effect on detoxifying enzymes in rats but not in mice. In other groups in this study, phenobarbital, a known inducer of hepatic detoxification enzymes, was administered simultaneously with methapyrilene HCl to both rats and mice. Methapyrilene HCl plus phenobarbital exerted a synergistic effect of enzyme induction in mice. In rats, the phenobarbital-induced hepatic enzymes increased the rate of hepatic detoxification of methapyrilene HCl, as measured by a reduction of PEN-induced ST as compared with that in control rats or rats receiving methapyrilene HCl alone. The difference in the effects of methapyrilene HCl on hepatic detoxifying mechanisms of rats and mice, as indicated by the metabolism of PEN, may explain the difference between the two species in their susceptibility to the hepatocarcinogenic effects of methapyrilene HCl.