Thermal inactivation of oligomeric enzymes is most often irreversible and is frequently accompanied by precipitation. We have engineered two symmetry related disulfide bridges (155-188' and 188-155') across the subunit interface of Lactobacillus camel thymidylate synthase, at sites chosen on the basis of an algorithm for the introduction of stereochemically unstrained bridges into proteins. In this communication, we demonstrate a remarkable enhancement in the thermal stability of the covalently cross-linked double disulfide containing dimeric enzyme. The mutant enzyme remains soluble and retains secondary structure even at 90 °C, in contrast to the wild-type enzyme which precipitates at 52°C. Furthermore, the mutant enzyme has a temperature optimum of 55°C and possesses appreciable enzymatic activity at 65°C. Cooling restores complete activity, in the mutant protein, demonstrating reversible thermal unfolding. The results suggest that inter-subunit crosslinks can impart appreciable thermal stability in multimeric enzymes.

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The expression of cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) genes has been studied as a function of development in rat liver. The levels of cytochrome P-450 (b+e) mRNAs and their transcription rates are too low for detection in the 19-day old fetal liver before or after phenobarbitone treatment. However, glutathione transferase (Ya+Yc) mRNAs can be detected in the fetal liver as well as their induction after phenobarbitone treatment can be demonstrated. These mRNAs contents as well as their inducibility with phenobarbitone are lower in maternal liver than that of adult nonpregnant female rat liver. Steroid hormone administration to immature rats blocks substantially the phenobarbitone mediated induction of the two mRNA families as well as their transcription. It is suggested that steroid hormones constitute one of the factors responsible for the repression of the cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) genes in fetal liver.