TRYPTOPHAN-HYDROXYLASE - CLONING AND EXPRESSION OF THE RAT-BRAIN ENZYME IN MAMMALIAN-CELLS

A cDNA encoding full-length tryptophan hydroxylase was produced by rev erse transcriptase-PCR from rat brain mRNA and expressed transiently i n a human fibroblast cell line, Catalytic activity was low unless tran sfected cells were grown in the presence of FeSO4. Recombinant tryptop han hydroxylase was found almost exclusively within the soluble compar tment of the cell and was dependent on tryptophan and tetrahydrobiopte rin for activity. The catalytic activity of recombinant tryptophan hyd roxylase was stimulated >25-fold by Fe(II) and to a somewhat lesser ex tent by-the polyanions heparin and phosphatidylserine, The enzyme was inhibited by desferrioxamine and dopamine, both of which complex iron. When extracts from transfected cells were subjected to sucrose gradie nt centrifugation and analytical gel filtration, the recombinant enzym e behaved the same as the native enzyme from brain. A monoclonal antib ody against phenylalanine hydroxylase that cross-reacts with brain try ptophan hydroxylase was capable of immunoprecipitating the recombinant hydroxylase from solution. These data indicate that recombinant trypt ophan hydroxylase expressed in mammalian cells is assembled into tetra mers of similar to 220,000 daltons. Its catalytic and physical propert ies appear to be very similar to those of the native enzyme from brain .