Solubilization of a phospholipid-requiring enzyme, iodothyronine 5'-deiodinase, from rat kidney membranes.
Enzymic activities catalyzing the reductive 5'-deiodination of thyroxine and 3,3',5'-triiodothyronine were solubilized from rat kidney microsomes by treatment with 0.2% deoxycholate. Deoxycholate reversibly inhibited the enzyme(s); removal of detergent restored activity and resulted in the formation of enzymatically active aggregates with a buoyant density of 1.17 g/ml resembling that of membranes. Fractionation of the solubilized membrane components in the presence of 0.2% deoxycholate by either gel filtration or sucrose gradient centrifugation inactivated the enzyme(s) and activity could be restored by the addition of partially purified soybean phospholipids; this allowed some of the physical properties of the enzyme(s) to be determined. 5'-Deiodinating activity of both thyroxine and 3,3',5'-triiodothyronine was associated with protein(s) with S20,W of 3.5 S, Stokes' radius of 32 A, and a calculated molecular weight of 49 900. A partial specific volume of 0.74 cm3/g was calculated from sedimentation in 2H2O and H2O sucrose gradients. Phospholipid reactivation of lipid-depleted enzyme preparations was concentration-dependent, with near maximal restoration when sufficient phospholipid was added to restore the phospholipid:protein ratio to that of thyroxine and 3,3',5'-triiodothyronine could not be resolved by sedimentation or molecular sieving and showed similar behavior toward deoxycholate solubilization and phospholipid reconstitution.
D000818:Animals; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003840:Deoxycholic Acid / Q000494:pharmacology; D007453:Iodide Peroxidase / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D010544:Peroxidases / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D012995:Solubility; D013974:Thyroxine / Q000378:metabolism; D014285:Triiodothyronine, Reverse / Q000378:metabolism
D010743:Phospholipids; D003840:Deoxycholic Acid; D014285:Triiodothyronine, Reverse; D010544:Peroxidases; D007453:Iodide Peroxidase; D013974:Thyroxine