Effect of (6R)- and (6S)-tetrahydrobiopterin on L-3,4-dihydroxyphenylalanine (DOPA) formation in NRK fibroblasts transfected with human tyrosine hydroxylase type 2 cDNA.
l-erythro-5,6,7,8-Tetrahydrobiopterin (BH(4)), which is the cofactor of aromatic amino acid hydroxylases, plays an important role in the biosyntheses of monoamine neurotransmitters. BH(4) exists as natural (6R)- and unnatural (6S)-isomers. In our previous reports, only (6R)-isomer significantly stimulated cofactor activity for tyrosine, tryptophan and phenylalanine hydroxylases (TH, TPH, PAH) in whole animals or in tissue slices. In this study we have compared the in situ cofactor activity on TH between natural (6R)- and unnatural (6S)-isomers in clonal cells. We have transfected human TH type 2 cDNA into the normal rat kidney (NRK) fibroblasts. These cells expressed TH protein, but had neither DOPA decarboxylase (DDC) nor BH(4). Thus, TH activity was observed only in the presence of exogenous BH(4). We compared the difference in in situ DOPA formation by TH activity in the presence of (6R)- or (6S)-BH(4) in the human TH-transfected cells. The effect of exogenous BH(4) was also compared between (6R)- and (6S)-isomers in rat pheochromocytoma PC12h cells, which contained approximately 100 ?M endogenous (6R)-BH(4). The rate of uptake of both BH(4) isomers into these cells increased in proportion to the pterin cofactor concentrations in the incubation medium up to 400 ?M but was nearly saturated at 1 mM BH(4). TH-transfected NRK fibroblasts formed DOPA only in the presence of exogenously added (6R)- or (6S)-BH(4) dose-dependently and released DOPA into the medium. At a saturating concentration of 1 mM, (6R)-BH(4) was approximately three times as active as (6S)-BH(4). In contrast, in PC12h cells which contained endogenous (6R)-BH(4) (approximately 100 ?M), exogenous (6R)-BH(4) activated DOPA formation maximally at 500 ?M about 10-fold, while (6S)-BH(4) activated it only slightly, about 2.5-fold. These results suggest that (6S)-isomer has lower cofactor activity with TH in the cells than (6R)-isomer. This TH transfected fibroblasts should be useful to assess cofactor activities of tetrahydropteridines in the cell.
Ishii
A
A
Department of Biochemistry, Nagoya University School of Medicine, Nagoya 466, Japan.
Hagihara
M
M
Matsuura
S
S
Uchida
K
K
Kiuchi
K
K
Kaneda
N
N
Toya
S
S
Kohsaka
S
S
Nagatsu
T
T