Takeshi TOMITA (Grad. Univ. for Advanced Studies), Shingo TSUTAMA (Osaka Prefecture Univ.), Yoshio IMAI (Osaka Prefecture Univ.) and Teizo KITAGAWA
[J. Biochem. (Tokyo) 122, 531 (1997)]
Soluble guanylate cyclase (sGC) consisting of two different subunits ((alpha); Mr =74,000, (beta); Mr = 69,000) was purified from bovine lung by more than 12,000-fold in terms of specific activity than that of the supernatant of homogenates and characterized. The heme content determined with the pyridine hemochromogen method and Bradford's protein assey was 0.8 heme per dimer. Cholera-, pertussis-, and botulinum C3 toxins modified exclusively the (beta)-subunit of sGC, yielding ADP-ribose-bound compound with 1:1 stoichiometry, and Vmax for the cyclase reaction increased by 10 times owing to this modification. When the ADP-ribosylation of sGC was performed simultaneously with two or three bacterial toxins which have distinct amino acid specificities, the resultant enzyme had only one ADP-ribose, and the activity was the same as that of the enzyme modified with one toxin. When NO was incorporated into the reaction mixture containing the ADP-ribosylated sGC, the cyclase activity noticeably increased approximately by the same amount as that seen for the unmodified enzyme. Such effects were not seen with CO. When ADP-ribosylated sGC was incubated with Mn2+, the enzyme activity was synergistically increased. The heme-deleted sGC was also ADP-ribosylated by bacterial toxins and its activity was raised. These findings suggest that sGC has an ADP-ribosylation site in the proximity of the GTP binding site similar to other GTP-binding proteins and that the (beta)-subunit regulates the activity.