Expression and characterization of hydroxyindole O-methyltransferase from a cloned cDNA in Chinese hamster ovary cells.
We have used two kinds of expression systems to test whether the cloned cDNA encoding hydroxyindole O-methyltransferase (HIOMT) of the bovine pineal gland was functional or not. First, when mRNA was synthesized in vitro by the SP6 system and injected into Xenopus oocytes, the enzymatic activity was expressed in the oocytes. Second, the cloned cDNA was recombined to a vector under the control of the simian virus 40 early promoter and transfected to Chinese hamster ovary (CHO) cells. The enzymatic activity of the crude supernatant of transfected cells (CHO-HT2) reached to 400 pmol melatonin formed per min per mg of protein, which value was approximately 9% of that of bovine pineal supernatant. The amounts of enzyme protein estimated by immunoblotting were proportional to the enzymatic activity in both CHO and pineal gland. The content of HIOMT protein was 8- to 30-fold larger in pineal gland compared to CHO cells. On the other hand, the content of mRNA encoding the enzyme measured by dot hybridization with [32P]cDNA, was in the same range in both CHO cells and pineal glands. These data suggest that the 11-fold higher enzymatic activity in pineal gland is due to an accumulation of the enzyme protein, not to a high level of the mRNA and also indicate that the cloned cDNA can express an intact hydroxyindole O-methyltransferase enzyme in CHO cells.
Ishida
I
I
Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neurosciences, Japan.
Ohsako
S
S
Nakane
M
M
Deguchi
T
T
D000121:Acetylserotonin O-Methyltransferase / Q000235:genetics*; D000818:Animals; D006224:Cricetinae / Q000378:metabolism*; D003412:Cricetulus / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004274:DNA, Recombinant; D005260:Female; D005786:Gene Expression Regulation; D066298:In Vitro Techniques; D008780:Methyltransferases / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D009865:Oocytes / Q000378:metabolism; D010053:Ovary / Q000166:cytology / Q000378:metabolism*; D010870:Pineal Gland / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic; D014982:Xenopus laevis
D004274:DNA, Recombinant; D012333:RNA, Messenger; D004247:DNA; D008780:Methyltransferases; D000121:Acetylserotonin O-Methyltransferase