Tryptophan 2,3-dioxygenase in chick embryo hepatocytes: studies in ovo and in culture.
Tryptophan dioxygenase is a hemoprotein in its active form, which has a relatively low affinity for heme. From previous studies in rats, the ratio of holoenzyme/total enzyme activity of tryptophan dioxygenase has been proposed to reflect the size of a "free" heme pool in hepatocytes. Chick embryo hepatocytes in ovo and in culture are other systems that have proven useful for study of hepatic heme metabolism and its control. Heretofore, there have been few studies of tryptophan dioxygenase activity in chick embryo hepatocytes. As part of studies on hepatic heme metabolism, using two different assays, we have measured tryptophan dioxygenase activity and percentage of heme saturation of the enzyme in chick embryo livers cells in ovo and in culture. One method of assay relies on endogenous formamidase to generate the final product, kynurenine, which is measured directly, whereas the other method uses a chemical hydrolysis step to form kynurenine which is further diazotized prior to measurement. The latter method is shown to be preferable for studies with chick embryo hepatocytes. In addition, we show that (i) tryptophan dioxygenase activity is present and can be increased by tryptophan and phenobarbital-like drugs in chick embryo hepatocytes in ovo; (ii) total enzyme activity falls markedly in cultured hepatocytes despite the presence of high concentrations of glucocorticoids in the culture medium; and (iii) under all conditions studied thus far in the cultures, the enzyme is nearly saturated with heme. Results are discussed in relation to regulation of heme metabolism in chick embryo hepatocytes.
Lincoln
B C
BC
Department of Biochemistry, Emory University, Atlanta, Georgia 30322.
Alvarado
A
A
Setty
N
N
Bonkovsky
H L
HL
D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005984:Glutethimide / Q000494:pharmacology; D006418:Heme / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007737:Kynurenine / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000196:embryology* / Q000201:enzymology; D008297:Male; D008706:Methemoglobin / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D014364:Tryptophan / Q000378:metabolism / Q000494:pharmacology; D014366:Tryptophan Oxygenase / Q000378:metabolism*
D007737:Kynurenine; D006418:Heme; D014364:Tryptophan; D008706:Methemoglobin; D003577:Cytochrome P-450 Enzyme System; D005984:Glutethimide; D014366:Tryptophan Oxygenase