Immunochemical characterization of brain and pineal tryptophan hydroxylase.
Recombinant mouse tryptophan hydroxylase (TPH) was expressed in Escherichia coli, using a bacterial expression vector and has been purified to homogeneity by sonication followed by Sepharose 4B column chromatography and native slab gel electrophoresis. This purified enzymatically active TPH protein was used for production of a specific antiserum. This antiserum identified the predicted TPH band (molecular weight, 54 kDa) on Western blot of crude extracts from the rat and mouse dorsal raphe, and the rat pineal gland. However, this antiserum recognized an additional protein band of lower molecular weight (48 kDa) in pineal extract. It is not clear whether the 48 kDa TPH band represents an isozyme or a protease cleavage product of TPH. Since the pineal gland contains higher TPH mRNA and lower TPH activity when it is compared with dorsal raphe nucleus enzyme, this lower molecular weight TPH may participate in the reduced TPH specific activity. In addition, there are no specific TPH inhibitors in the pineal gland and this lower molecular weight TPH is inactive or has a very low specific activity. This antiserum specifically immunostained serotonergic cell bodies in the dorsal raphe nuclei, some large caliber serotonergic processes in the dorsal raphe area as well as terminals in the olfactory bulb. It also immunolabeled the pineal gland and immunoprecipitated equally well TPH protein from the dorsal raphe nucleus and the pineal gland in a concentration-dependent manner.
Chung
Y I
YI
Department of Psychiatry, Pusan National University Medical College, Seo-gu, Pusan, Korea. yichung@hyowon.pusan.ac.kr
Park
D H
DH
Kim
M
M
Baker
H
H
Joh
T H
TH
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D003429:Cross Reactions; D004586:Electrophoresis; D007106:Immune Sera / Q000276:immunology; D007150:Immunohistochemistry; D051379:Mice; D010870:Pineal Gland / Q000201:enzymology*; D011817:Rabbits; D051381:Rats; D014365:Tryptophan Hydroxylase / Q000276:immunology / Q000378:metabolism*
D007106:Immune Sera; D014365:Tryptophan Hydroxylase