Biochemical characterization of a Ca 2+/NAD(P)H-dependent H 2O 2 generator in human thyroid tissue

• Published in: Biochimie Vol. 81; no. 4; pp. 373 - 380
• Main Authors: Leseney, Anne-Marie, Dème, Danielle, Legué, Odette, Ohayon, Renée, Chanson, Philippe, Sales, Jean-Patrick, Pires de Carvalho, Denise, Dupuy, Corinne, Virion, Alain
• Format: Journal Article
• Language: English
• Published: Elsevier Masson SAS 1999
• Subjects: H 2O 2; hydrogen peroxide; NADPH oxidase; thyrocyte; thyroid; thyroxine; triiodothyronine; H 2O 2; thyroxine; HRP, horseradish peroxidase; thyroid; TGFβ, transforming growth factor β; NADPH oxidase; triiodothyronine; hydrogen peroxide; TSH, thyrotropin; DTT, DL-dithiothreitol; thyrocyte; PTU, propylthiouracil
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/S0300-9084(99)80084-4

An NAD(P)H-dependent H 2O 2 forming activity has been evidenced in thyroid tissue from patients with Grave's disease. Its biochemical properties were compared to those of the NADPH oxidase previously described in pig thyroid gland. Both were Ca 2+-dependent and activated by inorganic phosphate anions in the same range of concentrations. Both are flavoproteins using FAD as cofactor, but the human enzyme was also able to utilize FMN. The apparent K m for NADPH of the human enzyme (100 μM) was 5–10 times higher than that of porcine enzyme. V m was 3 to 10 times higher in pig (150 nmol × h -1 × 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 this apparently low enzyme content. An NADH-dependent H 2O 2 production has also been observed in porcine and human thyroid tissues. This activity was only partly Ca 2+-dependent (man, 50–70%; pig, 80–90%) and presented similar apparent K m values for NADH (man, 100 μM; pig, 200 μM). In pig thyrocytes, the expression of the Ca 2+-dependent part of the NADH-oxidase activity was induced by TSH and down-regulated by TGFβ, as was the NADPH oxidase activity. Furthermore, NADPH and NADH-dependent activities were not additive. We conclude that a single, inducible, NAD(P)H-oxidase can use NADPH or NADH as substrate to catalyse H 2O 2 formation, and that human and porcine NAD(P)H-oxidases are highly similar. Differences observed could be attributed to minor differences in enzyme structure and/or in membrane microenvironment. The NADH-dependent Ca 2+-independent activity observed in human and porcine thyroid fractions could be attributed to a distinct and constitutive enzyme.