FIG. 6.

Time courses of in situ activation of the enzyme-CN-Cbl complex in recombinant E. coli. Permeabilized cells of E. coli carrying pSTV(eutABC)3 (A) or pSTV(eutBC)3 (B) (each, 1.6 × 10⁸ cells) were incubated at 37°C for 1 min with (triangles and inverted triangles) or without (circles) 7.5 μM CN-Cbl in 40 mM potassium phosphate buffer (pH 8.0) containing 50 mM KCl and 5 mM ethanolamine in a total volume of 6.0 ml. The 0.2-ml aliquots were drawn from the mixtures and added to the following mixtures: 30 mM potassium phosphate buffer (pH 8.0), 60 mM KCl, 10 mM ethanolamine, and 15 μM AdoCbl, in the presence (solid symbols) or absence (open symbols) of ATP plus MgCl₂ (3 mM each), to a final volume of 1.0 ml. In one control experiment, ATP and MgCl₂ were added but AdoCbl was not added (solid inverted triangles). The reaction mixtures were incubated at 37°C for the indicated times, and the acetaldehyde formed was determined as described in the text.