Biochemical characterization of a Ca2+/NAD(P)H-dependent H2O2 generator in human thyroid tissue.
An NAD(P)H-dependent H2O2 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 Ca2+-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 Km for NADPH of the human enzyme (100 microM) was 5-10 times higher than that of porcine enzyme. Vm 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 this 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 apparent Km values for NADH (man, 100 microM; pig, 200 microM). In pig thyrocytes, the expression of the Ca2+-dependent part of the NADH-oxidase activity was induced by TSH and down-regulated by TGFbeta, 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 H2O2 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 Ca2+-independent activity observed in human and porcine thyroid fractions could be attributed to a distinct and constitutive enzyme.
Leseney
A M
AM
Unité 486 INSERM, IFR-ISIT, Faculté de Pharmacie, Châtenay-Malabry, France.
Dème
D
D
Legué
O
O
Ohayon
R
R
Chanson
P
P
Sales
J P
JP
Carvalho
D P
DP
Dupuy
C
C
Virion
A
A
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D002458:Cell Fractionation; D002478:Cells, Cultured; D046508:Culture Techniques; D005260:Female; D005486:Flavin Mononucleotide / Q000378:metabolism / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism / Q000494:pharmacology; D006111:Graves Disease; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism; D013552:Swine; D013961:Thyroid Gland / Q000378:metabolism* / Q000473:pathology
D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D002118:Calcium