By Jack Barchas (Eds.)
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The most useful assay in this category was proposed by Lovenberg, Jequier and Sjoerdsma (1967). 4 χ 10" M). After tryptophan hydroxylation, a second enzymatic step using a purified decarboxylase preparation i s necessary. Following the hydroxylation step, a known amount of cold 5-HTP i s added. This assay requires measurement of the specific a c t i v i t y of 5-HT enzymatically formed and isolated by column chromatography. Although this method gives invaluable information, i t i s time consuming; it involves two separate enzymic incubations — the assay of 5-HT by fluorescence, and the counting of radioactive 5-HT isolated by column chromatography.
5 L-Tryptophan (8) 200 mg/kg i . p . 4 ± DL-5-HTP (8) 200 mg/kg i . p . p. p. 08 ± 0 . 2 ± 3 . 61 ± 0 . 05 + Number of animals in parentheses. 31 Jean Renson CONCLUSION Several rapid and sensitive assays for brain tryptophan hydroxy lase are now available which have p a r t i a l l y unraveled several i n t r i g uing properties of this enzyme. The molecular mechanisms by which cerebral 5-HT biosynthesis i s regulated i s s t i l l obscure. The complexi ty of the regulation of hepatic phenylalanine hydroxylase, another en zyme using tetrahydrobiopterin as an electron donor source, i l l u s t r a t e s and provides an excellent model to pursue the purification of trypto phan hydroxylase in order to elucidate i t s regulatory properties.
Properties 1. of Tryptophan Hydroxylase. Kinetic Properties of Tryptophan Hydroxylase 23 Jean Renson Several groups have estimated a f f i n i t y constants of tryptophan hydroxylase from the brain and the pineal gland of a few species. Some of these data are summarized in Table 1 . Only values reported for enzy matic preparations in cell-free systems, and sometimes after some de gree of p u r i f i c a t i o n , are meaningful. Data on tryptophan hydroxylase activity in particulate fractions (mostly synaptosomes) represent more complex events involving the uptake and compartmentation of the sub strate, making the results more d i f f i c u l t to interpret.