Purification and Properties of Tryptophan 5‐Monooxygenase from Rat Brain‐Stem

Abstract

Tryptophan 5-monooxygenase was purified approximately 5500-fold, to apparent homogeneity with a specific activity of 374 nmol min-' mg-' at 30 "C, from rat brain-stem using Sepharose CL-6B, DEAE-Sepharose CL-6B and pteridine-agarose chromatography. Two distinct active forms were separable by DEAE-Sepharose CL-6B and designated as form I and form I1 based on their order of elution from the gel column. The apparent molecular weight of form I was determined to be 300000 by gel filtration on Ultrogel AcA 34 and 288000 by gradient polyacrylamide gel electrophoresis. The enzyme gave a single band on sodium dodecylsulfate/polyacrylamide gel electrophoresis, the molecular weight of which was estimated to be 59 000, indicating that the enzyme might be composed of four identical subunits. The tetrameric structure of the enzyme was further suggested by crosslinking studies using dimethyl suberimidate as a bifunctional reagent. The enzyme activity was stimulated approximately 3.5-fold by the addition of Fe2+. Kinetic studies revealed that this activation was associated with an increase of V value. The purified enzyme had an activity of phenylakdnine hydroxylation but not an activity of tyrosine hydroxylation.Tryptophan 5-monooxygenase [L-tryptophan, tetrahydropteridine : oxygen oxidoreductase (5-hydroxylating)] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the initial and rate-limiting step in the biosynthesis of the neurotransmitter serotonin [l, 21. Although much effort has been devoted to purification and characterization of tryptophan 5-monooxygenase [3 -1 I], relatively little progress has been made because of the extreme lability of the enzyme with the standard purification procedure. Tong and Kaufman [I 11 reported that they obtained an almost homogenous (85 -90 % pure) preparation of tryptophan 5-monooxygenase from rabbit hindbrain with a 25 yield. However, the specific activity of their final preparation, 2.1 nmol min-' mg-' at 37 "C, was only 58-fold higher than that of the crude enzyme. Recently we purified the rabbit brain tryptophan 5-monooxygenase to a specific activity of 15.9 nmol min-' mg-' at 30 "C with a 29 % yield but it was still far from a homogenous preparation [12], suggesting that much higher purification was required to attain homogenous enzyme.In the present study tryptophan 5-monooxygenase was purified to a specific activity of 374nmol min-' mg-' at 30°C from rat brain-stem and some physical and catalytic properties of the enzyme were examined. This is the first demonstration of the purification of mammalian tryptophan 5-monooxygenase to an apparent homogeneity. EXPERIMENTAL PROCEDURE MaterialsEscherichia coli P-galactosidase, rabbit muscle aldolase rabbit muscle lactate dehydrogenase, beef liver catalase and horse Abbreviations. Me~PteH4, 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine; MePteH4, 2-arnino-4-hydroxy-6-methyltetrahydropteridine; Hepes, 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid.Enzyme. Tryptophan 5-monooxygenase or tryptophan hydroxylase (EC ...

Proteasome‐driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell line

“…This reaction is the initial and rate-limiting step in the biosynthesis of serotonin [2][3][4][5]. TPH has been extensively purified from various sources such as bovine pineal gland [6], mouse mastocytoma [7,8], and mammalian brains [9][10][11]. Physicochemical, enzymic and immunochemical properties differed between TPHs of neural and non-neural tissue origin, and it is accepted that neural TPH might be a different entity from the non-neural enzyme [8,10,12,13].…”

“…TPH has been extensively purified from various sources such as bovine pineal gland [6], mouse mastocytoma [7,8], and mammalian brains [9][10][11]. Physicochemical, enzymic and immunochemical properties differed between TPHs of neural and non-neural tissue origin, and it is accepted that neural TPH might be a different entity from the non-neural enzyme [8,10,12,13]. Complimentary DNAs of TPH have been cloned from various sources but no differences or only trivial variation in amino acid sequences were found among them [14][15][16][17][18][19].…”

Stimulation of tryptophan hydroxylase production in a serotonin‐producing cell line (RBL2H3) by intracellular calcium mobilizing reagents

“…Studies on TPH have been hampered by difficulties in obtaining the pure enzyme in sufficient quantity and by the lack of an appropriate cell culture system, as compared with studies on phenylalanine or tyrosine hydroxylase which are also pterin-dependent monooxygenases. Rodent mast cells produce serotonin, and established cell lines of mast cell origin are available [4][5][6][7][8]. In our previous work, we found a very rapid turnover of TPH protein (half-life, 15-60 min) which was driven by ATP-dependent proteolysis in the mast cell lines, RBL2H3 and FMA3 [9].…”

“…Extended color development usually yielded nonspecific signals what were presumed to be proteolytic degradation products [13]. TPH was identified by simultaneous electrophoresis of the authentic enzyme which was purified from mouse mastocytoma P815 according to Nakata and Fujisawa [7].…”

Distribution and Some Properties of Tryptophan Hydroxylase from Liver in Several Fishes

“…This enzyme is of great interest be cause it is one of the three known pterin-requiring aro matic amino acid hydroxylases and catalyzes the rate-limit ing step in the biosynthesis of serotonin in the brain and other peripheral tissues.1-3) It was first discovered in mam malia,4) and purified from rat brain stem and mouse mastocytoma. 5,6) On the other hand, this enzyme in fish has not been stud ied in detail yet,7) and its distribution and characterization have not been clarified. As a part of studies on serotonin biosynthesis in fish, tryptophan hydroxylase activity in the liver of several fishes was surveyed in this paper.…”