Tryptophan hydroxylase: cloning and expression of the rat brain enzyme in mammalian cells.
A cDNA encoding full-length tryptophan hydroxylase was produced by reverse transcriptase-PCR from rat brain mRNA and expressed transiently in a human fibroblast cell line. Catalytic activity was low unless transfected cells were grown in the presence of FeSO4. Recombinant tryptophan hydroxylase was found almost exclusively within the soluble compartment of the cell and was dependent on tryptophan and tetrahydrobiopterin for activity. The catalytic activity of recombinant tryptophan hydroxylase was stimulated > 25-fold by Fe(II) and to a somewhat lesser extent 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 gradient centrifugation and analytical gel filtration, the recombinant enzyme behaved the same as the native enzyme from brain. A monoclonal antibody against phenylalanine hydroxylase that cross-reacts with brain tryptophan hydroxylase was capable of immunoprecipitating the recombinant hydroxylase from solution. These data indicate that recombinant tryptophan hydroxylase expressed in mammalian cells is assembled into tetramers of approximately 220,000 daltons. Its catalytic and physical properties appear to be very similar to those of the native enzyme from brain.
D'Sa
C M
CM
Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, Detroit, Michigan, USA.
Arthur
R E
RE
States
J C
JC
Kuhn
D M
DM
D000818:Animals; D000975:Antioxidants / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line / Q000201:enzymology; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003429:Cross Reactions; D003676:Deferoxamine / Q000494:pharmacology; D004298:Dopamine / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006801:Humans; D007501:Iron / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D008322:Mammals; D008636:Mesencephalon / Q000166:cytology / Q000201:enzymology*; D008969:Molecular Sequence Data; D010651:Phenylalanine Hydroxylase / Q000276:immunology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics; D012701:Serotonin / Q000096:biosynthesis; D017262:Siderophores / Q000494:pharmacology; D014162:Transfection; D014364:Tryptophan / Q000378:metabolism / Q000494:pharmacology; D014365:Tryptophan Hydroxylase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*
D000975:Antioxidants; D011994:Recombinant Proteins; D017262:Siderophores; D001708:Biopterins; D012701:Serotonin; D014364:Tryptophan; D007501:Iron; D010651:Phenylalanine Hydroxylase; D014365:Tryptophan Hydroxylase; C003402:sapropterin; D003676:Deferoxamine; D004298:Dopamine