Figure 5.

HPLC separation of products and substrates from assays using purified recombinant guanylyl cyclase (RC1_3783). (a) Separation of adenine nucleotide standards AMP (1), ADP (2), cAMP (3) and ATP (4). (e) Separation of guanine nucleotide standards GMP (1), GDP (2), cGMP (3) and GTP (4). All reactions were incubated for 2 hr and contained 0.5 mM ATP (b–d) or GTP (f–h), 10 mM MnCl₂ (b, d, f, h) or MgCl₂ (c, g) and 0.5 μg/μl purified RC1_3783 (c, d, g, h). Control reactions (b, f) had buffer added in place of enzyme. A small amount of cGMP indicated by a black arrow (↓) is produced in reaction g containing GTP, Mg²⁺ and recombinant cyclase, with greater activity occurring in reaction h when Mn²⁺ is substituted for Mg²⁺. (i) Purified recombinant cyclase elutes as a dimer in the absence of NaCl (Blue line) but as a monomer when chromatographed on a Superose 6 column equilibrated in 100, 50 or 25 mM NaCl (Black, Green & Red lines, respectively).