title: Purification and characterization of the major constitutive form of testicular heme oxygenase. The noninducible isoform.
abstract: Recently we reported on the presence of two isoforms of heme oxygenase in rat liver microsomes, referred to as HO-1 and HO-2, and that only HO-1 is inducible (Maines, M. D., Trakshel, G. M., and Kutty, R. K. (1986) J. Biol. Chem. 261, 411-419). Presently we report on the detection of two isoforms of the enzyme in rat testis and purification to near homogeneity of the noninducible isoform, HO-2. A comparative characterization of the liver HO-1 and the testicular HO-2 is also provided. The relative abundance of the isoforms in the two organs was dissimilar. In the testis, the predominant form was HO-2, and only minute amounts of HO-1 were detected. In the liver, however, a 1:2 ratio of HO-1 to HO-2 was noted. The activity of HO-2 in both organs was refractory to cadmium, an inducer of the hepatic HO-1. Under nondenaturing electrophoresis conditions, HO-2 showed a higher mobility than HO-1; on a sodium dodecyl sulfate-polyacrylamide gel, HO-2 displayed a higher monomeric Mr. The apparent Mr values for HO-2 and HO-1 were 36,000 and 30,000, respectively. The isoforms differed in immunochemical properties. Antiserum to the liver HO-1 did not recognize the testicular HO-2 when examined by double immunodiffusion or by Western immunoblotting. HO-2 was more sensitive to heat inactivation than HO-1. When exposed at 65 degrees C (10 min), 70% of HO-1 activity was retained; however, nearly 80% of HO-2 activity was lost. The apparent Km values for heme for HO-1 and HO-2 were 0.24 and 0.40 microM, respectively. HO-1 and HO-2 had similar requirements for cofactor and flavoprotein reductase and were inhibited by heme-ligands (CO, KCN, NaN3). HO-2 utilized as substrate, Fe-protoporphyrin, Fe-hematoporphyrin, and Fe-hematoporphyrin acetate; it did not degrade intact purified rat liver cytochromes b5 and P-450 LM2, catalase, cytochrome c, hemoglobin, or myoglobin.
mesh_terms: D000818:Animals; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006419:Heme Oxygenase (Decyclizing) / Q000032:analysis / Q000302:isolation & purification*; D006358:Hot Temperature; D007163:Immunosorbent Techniques; D007527:Isoenzymes / Q000032:analysis / Q000302:isolation & purification*; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000302:isolation & purification*; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D013737:Testis / Q000201:enzymology*
chemical_list: D007527:Isoenzymes; D006899:Mixed Function Oxygenases; D006419:Heme Oxygenase (Decyclizing)