Protein kinase C modulation of rhodanese-catalyzed conversion of cyanide to thiocyanate.
Detoxification of cyanide is catalyzed by a sulfurtransferase, rhodanese, a phosphoprotein regulated by unknown protein kinases. In this study, we determined if a Ca2+/phospholipid-modulated phosphotransferase, protein kinase C (PKC) could modify rhodanese activity. Thiocyanate (SCN-) production as an estimate of rhodanese activity in vitro was measured in the presence or absence of exogenously added purified PKC, or 12-O-tetradecanoylphorbol acetate (TPA), a pharmacologic activator of the endogenous PKC. HI-6 (1-(2-(hydroximino)methyl))pyridinium-2-(4-(aminocarbonyl) pyridinium dimethylether) is an oxime that may dephosphorylate phosphoproteins due to the proposed phosphatase-like activity of the oximes. We examined HI-6's effect on rhodanese-catalyzed SCN- production. Bovine kidney rhodanese (0.40 mg/ml protein) was reacted with 4 mM KCN and SCN- production determined spectrophotometrically following the method of Westley (1981). Preincubating rhodanese with 20 or 100 ng of purified PKC (alpha, beta, gamma isozymes) for 5 min before initiating the reaction with 4 mM KCN as the substrate increased SCN- production by 17 or 40%, respectively, over the control (P < 0.05). Rhodanese formation of SCN- decreased when the preincubation was conducted with 1 nM or 100 nM of TPA. With HI-6 at 1 or 10 microM used in place of PKC, or TPA, rhodanese activity was increased by 6 or 14% (P < 0.05), respectively, compared to control. Under the conditions examined, exogenous PKC acting as a possible phosphate acceptor, and HI-6, a potential dephosphorylating compound, increased rhodanese activity. These data are consistent with the observation that rhodanese can exist as a phosphorylated enzyme which is not active and a dephosphorylated form which is active. It is suggested that addition of purified, exogenous PKC may accept phosphate from phosphorylated rhodanese or HI-6 may dephosphorylate rhodanese, both of which stimulate the conversion of cyanide anion to the less toxic SCN-. These observations support the possibility that rhodanese may be regulated by protein phosphorylation and treatments that alter the phosphorylation state of rhodanese may affect cyanide detoxification via SCN- formation.
Maduh
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Biochemical Pharmacology Branch, United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010-5425.
Baskin
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D000818:Animals; D002384:Catalysis / Q000187:drug effects; D002417:Cattle; D007668:Kidney / Q000201:enzymology; D010091:Oximes; D010766:Phosphorylation; D011190:Potassium Cyanide / Q000378:metabolism*; D011493:Protein Kinase C / Q000494:pharmacology*; D011726:Pyridinium Compounds / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013861:Thiocyanates / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000302:isolation & purification / Q000378:metabolism*
D010091:Oximes; D011726:Pyridinium Compounds; D013861:Thiocyanates; D011493:Protein Kinase C; D013884:Thiosulfate Sulfurtransferase; C022870:asoxime chloride; D011190:Potassium Cyanide; D013755:Tetradecanoylphorbol Acetate; C031760:thiocyanate