Biochemical characterization of a Ca2+/NAD(P)H-dependent H2O2 generator inhuman thyroid tissue

An NAD(P)H-dependent H2O2 forming activity has been evidenced in thyroid ti ssue from patients with Grave's disease. Its biochemical properties were co mpared to those of the NADPH oxidase previously described in pig thyroid gl and. Both were Ca2+-dependent and activated by inorganic phosphate anions i n the same range of concentrations. Both are flavoproteins using FAD as cof actor, but the human enzyme was also able to utilize FMN. The apparent K-m for NADPH of the human enzyme (100 mu M) was 5-10 times higher than that of porcine enzyme. V-m was 3 to 10 times higher in pig (150 nmol x h(-1) x mg (-1)) than in man (14 to 45). Total content in human tissue was 7 to 9% of that in porcine tissue. An unidentified inhibitor has been detected in the 3000 g particulate fraction from most patients, which could account for thi s apparently low enzyme content. An NADH-dependent H2O2 production has also been observed in porcine and human thyroid tissues. This activity was only partly Ca2+-dependent (man, 50-70%; pig, 80-90%) and presented similar app arent K-m values for NADH (man, 100 mu M; pig, 200 mu M). In pig thyrocytes , the expression of the Ca2+-dependent part of the NADH-oxidase activity wa s induced by TSH and down-regulated by TGF beta, as was the NADPH oxidase a ctivity. Furthermore, NADPH and NADH-dependent activities were not additive . We conclude that a single, inducible, NAD(P)H-oxidase can use NADPH or NA DH as substrate to catalyse H2O2 formation, and that human and porcine NAD( P)H-oxidases are highly similar. Differences observed could be attributed t o minor differences in enzyme structure and/or in membrane microenvironment . The NADH-dependent Ca2+-independent activity observed in human and porcin e thyroid fractions could be attributed to a distinct and constitutive enzy me. (C) Societe francaise de biochimie et biologie moleculaire / Elsevier, Paris.