| Research Abstract |
At first, soluble guanylate cyclase (sGC) was purified more than 12,000-fold in term of specific activity from the supernatant of bovine lung homogenates. In the course of purification, the chromatographs of sGC were improved, because the enzyme could not eluted from blue Sepharose and hydroxyapatite column chromatographs. In spite of these resin, we used a Gigapite and GTP-Sepharose. About 4-10 mg of purified sGC was prepared from bovine lungs (2 bodies). Since it is well known that sGC is one of GTP-binding protein, we have checked the effects of mono-ADP-ribosylation by cholera, botulinum, and pertussis toxin on sGC. Earth mono-ADO-ribosylation by cholera, botulinum, and petussis toxin on xEG. Each modified exclusively the small-subunit of sGC, yielding the ADP-ribose-bound compound with 1 : 1 stoichiometry, and Vmax for the sGC reaction was increased 10 times by this modification (J. Biochem., 122, 1997, 531). The resonance Raman spectra of sGC and carbon monoxide (CO)-sGC including the Fe-His stretch at 203 cmィイD1-1ィエD1 and the Fe-CO stretch at 473 cmィイD1-1ィエD1 were unaltered by binding of GTP and cyclic GMP, but apparent resonance Raman spectra of nitric oxide (NO)-sGC in the presence of GTP changed with time and concentrations of GTP (Biochemistry, 36, 1997, 10155). Two monoclonal antibodies against bovine lung soluble guanylate cyclase were prepared and characterized. One of monoclonal antibody, mAb 3221 recognized both of the small- and large-subunits of soluble guanylated cyclase and had greater binding affinity to the enzyme in the presence of nitric oxide (FEBS Lett., 455, 1999, 291).
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