Catalytic activity of type II iodothyronine 5'-deiodinase polypeptide is dependent upon a cyclic AMP activation factor.
Type II iodothyronine 5'-deiodinase is an approximately 200-kDa multimeric enzyme in the brain that catalyzes the deiodination of thyroxine (T4) to its active metabolite, 3,5,3'-triiodothyronine. In astrocytes, cAMP stimulation is required to express catalytically active type II iodothyronine 5'-deiodinase. The affinity ligand N-bromoacetyl-L-T4 specifically labels the 29-kDa substrate-binding subunit (p29) of this enzyme in cAMP-stimulated astrocytes. To determine the requirements for cAMP-induced activation of this enzyme, we optimized N-bromoacetyl-L-T4 labeling of p29 in astrocytes lacking type II iodothyronine 5'-deiodinase activity and examined the effects of cAMP on the hydrodynamic properties and subcellular location of the enzyme. We show that the p29 subunit is expressed in unstimulated astrocytes and requires 10-fold higher concentrations of N-bromoacetyl-L-T4 to achieve incorporation levels equal to those of p29 in cAMP-stimulated cells. Gel filtration showed that p29 was part of a multimeric membrane-associated complex in both cAMP-stimulated and unstimulated astrocytes and that cAMP stimulation led to an increase of approximately 60 kDa in the mass of the holoenzyme. In unstimulated astrocytes, p29 resides in the perinuclear space. Cyclic AMP stimulation leads to the translocation of p29 to the plasma membrane coincident with the appearance of deiodinating activity. These data show that cAMP-dependent activation of type II iodothyronine 5'-deiodinase activity results from the synthesis of additional activating factor(s) that associates with inactive enzyme and leads to the translocation of enzyme polypeptide(s) from the perinuclear space to the plasma membrane.
Safran
M
M
Molecular Endocrinology Laboratory, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Farwell
A P
AP
Leonard
J L
JL
D000345:Affinity Labels / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D003994:Bucladesine / Q000494:pharmacology*; D002384:Catalysis; D002478:Cells, Cultured; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000378:metabolism*; D004705:Endocytosis; D004789:Enzyme Activation; D007150:Immunohistochemistry; D007453:Iodide Peroxidase / Q000032:analysis / Q000378:metabolism*; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D051381:Rats; D013974:Thyroxine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*
D000345:Affinity Labels; D007527:Isoenzymes; D046911:Macromolecular Substances; D003994:Bucladesine; C015042:N-bromoacetylthyroxine; D000242:Cyclic AMP; D007453:Iodide Peroxidase; D013974:Thyroxine