Immunochemical Characterization of Brain and Pineal Tryptophan Hydroxylase

Abstract

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.

Expression of tryptophan 5‐hydroxylase gene during sea urchin neurogenesis and role of serotonergic nervous system in larval behavior

“…Serotonin, on the contrary, spread in the entire cytoplasm of cells, including the axons (Nakajima, 1986;Yaguchi et al, 2000). This observation is consistent with that in mammals, where TPH and TPH were expressed in a cell body (Kim et al, 2002), and TPH and serotonin were detected in an entire neuron (Chang et al, 2001), indicating that the transcription and translation of TPH occur at the cell body, and yet only serotonin is transported into the axons. HpTPH-positive cells in sea urchin embryo formed a cluster at the apical ganglion region, and they were separated by a few non-HpTPH cells located in between, which are consistent with previous immunohistochemical observation of serotonin cell distribution (Yaguchi et al, 2000).…”

“…cDNAs of the enzyme have been cloned in human (Boularand et al, 1990), chick (Florez et al, 1996), and fruit fly (Neckameyer and White, 1992). Because TPH is present exclusively in the cells that produce serotonin (Chang et al, 2001), it would be a more competent marker than serotonin, an end product that is contained in serotonin-producing cells as well as in serotonin storage cells (Lebrand et al, 1996;Galter et al, 1999). Thus, the end product probe is not always adequate to specify SAG cell differentiation.…”