Purification of bovine soluble guanylate cyclase and ADP-ribosylation on its small subunit by bacterial toxins.
Soluble guanylate cyclase (sGC) consisting of two different subunits (alpha: Mr = 74,000, beta: Mr = 69,000) was purified more than 12,000-fold in terms of specific activity from the supernatant of bovine lung homogenates and characterized. The heme content determined with the pyridine hemochromogen method and Bradford's protein assay was 0.8 heme per dimer. Cholera, pertussis, and botulinum C3 toxins modified exclusively the beta-subunit of sGC, yielding the ADP-ribose-bound compound with 1:1 stoichiometry, and Vmax for the cyclase reaction was increased 10 times by 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 by approximately 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 near the GTP binding site, like other GTP-binding proteins, and that the beta-subunit regulates the activity.
Tomita
T
T
The Graduate University for Advanced Studies and Institute for Molecular Science, Okazaki National Research Institute.
Tsuyama
S
S
Imai
Y
Y
Kitagawa
T
T
D000244:Adenosine Diphosphate / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology*; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000302:isolation & purification* / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008168:Lung / Q000737:chemistry / Q000201:enzymology; D046911:Macromolecular Substances; D008345:Manganese / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology; D012680:Sensitivity and Specificity; D012995:Solubility
D001427:Bacterial Toxins; D046911:Macromolecular Substances; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D009569:Nitric Oxide; D006418:Heme; D008345:Manganese; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase; D006152:Cyclic GMP