Solubilization and Characterization of a Thyroid Ca²⁺‐Dependent and NADPH‐Dependent K₃Fe(CN)₆ Reductase

Abstract

The thyroid plasma membrane contains a Ca"-regulated NADPH-dependent H,O,-generating system which provides H,O, for the thyroid-peroxidase-catalyzed biosynthesis of thyroid hormones. The molecular nature of the membrane-associated electron transport chain that generates H,O, in the thyroid is unknown, but recent observations indicate that a flavoprotein containing a FAD prosthetic group is involved. Solubilization was reinvestigated using 3-[(3-cholamidopropyl)dimethylammonio]-l -propanesulfonate (Chaps), Triton X-100, and high salt concentrations. Chaps eliminated about 30% of the proteins, which included a ferricyanide reductase, without affecting the H,O,-generating system. Similarly, Triton X-100 alone did not extract the NADPH oxidase. An NADPH-oxidase activity, which was measured in the presence of the artificial electron acceptor potassium ferricyanide, was solubilized by increasing the ionic strength to 2 M KCI. This NADPH-ferricyanide reductase activity was shown to belong to the H,O,-generating system, although it did not produce H,O,. It was still Ca*+ dependent and H,O, production was restored by decreasing the ionic strength by overnight dialysis. No H,O, production activity was detected after sucrose density gradient centrifugation of the dialyzed solubilized enzyme, but a welldefined peak of NADPH oxidation activity with a sedimentation coefficient of 3.71 S was found in the presence of K,Fe(CN),. These results suggest that some unknown component(s) (phospholipid or protein) is removed during sucrose density gradient centrifugation. Finally, thyrotropin, which induces NADPH oxidase and regulates H,O, production in porcine thyrocytes in primary culture, also induced the NADPH-K,Fe(CN), reductase activity associated with the H,O,-generating system. Thus, this enzyme seems to be another marker of thyroid differentiation.

Purification of a Novel Flavoprotein Involved in the Thyroid NADPH Oxidase

“…These data are in good agreement with those of Raspé and Dumont (10), who reported that TSH induces H 2 O 2 generator activity in dog thyrocytes by activating the adenylate cyclase cascade. They also corroborate our results showing that the TSH and/or forskolin stimulate NADPH oxidase activity in pig thyrocytes (11), which seems to be correlated with a higher expression of the flavoprotein (16).…”

“…However, the partially purified flavoprotein is still able to catalyze the NADPH-dependent reduction of the electron scavengers nitroblue tetrazolium and potassium ferricyanide (K 3 Fe(CN) 6 ). We used this property to detect and quantify the flavoprotein activity during its purification (16). We have now purified the thyroid oxidase flavoprotein as a first step in defining the molecular structure of the enzyme.…”

“…A functional NAD(P)H oxidase, generating H 2 O 2 in a Ca 2ϩ -dependent manner, has been solubilized by treating pig thyroid plasma membranes with detergents at a high salt concentration (16). The flavoprotein became unable to reduce molecular oxygen after the first step of its purification by octyl-Sepharose chromatography, suggesting that it requires one or more additional component(s) to generate H 2 O 2 (16). However, the partially purified flavoprotein is still able to catalyze the NADPH-dependent reduction of the electron scavengers nitroblue tetrazolium and potassium ferricyanide (K 3 Fe(CN) 6 ).…”

Cloning of Two Human Thyroid cDNAs Encoding New Members of the NADPH Oxidase Family

“…Despite these convincing arguments, the definite proof that ThOX1 and ThOX2 are components of the thyroid H 2 O 2 generating system will await reconstitution of H 2 O 2 production in transfected nonthyroid cells. This may require co-transfection of constructs encoding additional components that remain to be identified (10).…”

“…H 2 O 2 is formed from the oxidation of NADPH by an NADPH oxidase, a flavoprotein that has been partially purified from solubilized pig thyroid plasma membrane (10). It is known that the enzyme requires Ca 2ϩ to be fully active, but the precise molecular structure of the H 2 O 2 generating system operating in the thyroid remains ill defined.…”