IN-VITRO EFFECTS OF OXYGEN-DERIVED FREE-RADICALS ON TYPE-I AND TYPE-II CAMP-DEPENDENT PROTEIN-KINASES

Oxygen free radicals may act as second messengers in signal transducti on pathways and contribute to inflammatory diseases. We studied the ac tion in vitro of radiolytically generated hydroxyl radicals ((OH)-O-.) and superoxide radicals (O(2)radical anion) on the cAMP-dependent pro tein kinases, I and II (PKAI and -II, respectively). The effects of th e gasses O-2, and N2O used to produce O(2)radical anion or (OH)-O-. ra dicals by gamma-radiolysis of the water were also studied. PKAI is mor e sensitive than PKAII to oxygen gas (10 mM sodium formate) and to hyd roxyl and superoxide radicals. Hydroxyl radicals decreased the kinase phosphotransferase activities stimulated either by cAMP or its site-sp ecific analogs for both PKAI and PKAII; however, PKAI was more affecte d. The binding of [H-3]cAMP and of 8-N-3-[P-32]cAMP to RI regulatory s ubunits was decreased. (OH)-O-. caused a loss of tryptophan 260 fluore scence at site A of PKAI and of bityrosine production. Superoxide radi cals affected only PKAI. O(2)radical anion modified both cAMP-binding sites A and B of the regulatory subunit but had a smaller effect on th e catalytic subunit, The catalytic subunit was more sensitive to radic als when free than when part of the holoenzymes during exposure to the oxygen free radicals. These results suggest that oxygen free radicals alter the structure of PKA enzymes. Thus, oxidative modifications may alter key enzymes, including cAMP-dependent protein kinases, in certa in pathological states.