Fig. 5.

In vitro effects of chaperone addition. (a) DMSO and glycerol do not restore activity when added directly to purified recombinant I278T CBS. Recombinant I278T CBS was purified from E. coli as previously described [14]; 220 ng of purified enzyme was used in an in vitro reaction containing 10 mM serine, 10 mM l-homocysteine, and 100 mM AdoMet and the indicated amount of each chaperone. Results are expressed as a percentage of the control reaction (no chaperones). (b) Wild-type and I278T CBS were produced in an in vitro transcription/translation wheat germ extract either in the presence or absence of 0.9 M glycerol or 0.3 M DMSO, as described in ‘Materials and methods’. The presence of equal amounts of CBS protein produced was determined by western blot. Enzyme activity was measured in triplicate and error bars indicate standard deviation. Activity is expressed as amount of cystathionine produced in nanomoles. (c) Identical to (b) except that either proline or TMAO were added to the in vitro reactions.