"Conformational" isoenzymes of ascarid enolase.

The present studies were initiated to determine the biochemical basis fo r the existence of multiple, electrophoretically distinct enolase activities in the parasitic round worm Ascaris suum. A total of five activities were resolved by electrophoresis of worm body fluids on cellulose polyacetate strips followed by staining the strips specifically for enolase activity. These activities are referred to as enolases 1 t h rough 5, with enolase 1 exhibiting the highest mobility toward the anode. Enolases 1, 4, and 5 were found to be very unstable and disappeared completely during storage or during the purification procedures. The two relatively stable forms (enolases 2 and 3) were purified and partially characterized. Both isoenzymes are composed of subunits of approximately 50,000 daltons and both isoenzymes behaved as dimers during centrifugation in sucrose gradients under nondenaturing conditions. The two enzymes were distinguished on the basis of their sensitivities to heat denaturation but not on the basis of electrophoretic analysis in the presence of 8 M urea nor by the size distribution of peptides generated by treating the enzymes with Staphylococcus aureus V8 protease. Treatment of enolase 2 with a number of diverse agents in vitro (H+, OH-, urea, guanidine hydrochloride, and iodide ions) resulted in the conversion of this isoenzyme to a species similar to enolase 3 on the basis of electrophoretic mobility and sensitivity to heat denaturation. O u r observations are consistent with the view that enolases 2 and 3 m a y represent two different thermodynamically metastable conformations of the same protein species. Within this view, the thermodynamically least stable form (enolase 2) would be the most enzymatically active one and the one most resistant to heat denaturation. Several scenarios are given which m a y explain how multiple, thermodynamically metastable conformations of ascarid enolases could be generated in vivo.