J Korean Neurol Assoc > Volume 20(1); 2002 > Article

Journal of the Korean Neurological Association 2002;20(1): 60-66.

"Tyrosine Hydroxylase 조절부위의 인위적 변이가 단백질의 안정성과 효소적 특성에 미치는 영향"

유영수, 이상도 임정근 김용식 남은주 주완석 이이언 이영재

계명대학교 의과대학 신경과학교실, 서울대학교 의과대학 약리학교실, 가천의과대학 신경과학연구소

"Effects of Deletions in the Regulatory Domain on the Stability and Enzymatic Characteristics of Tyrosine Hydroxylase"

Young-Soo Yoo

"Department of Neurology, Keimyung University, Department of Pharmacology, Seoul National University, Neuroscience Research Institute, Gachon Medical School"

Abstract

"Background : Various vectors have been developed and tried for the delivery of tyrosine hydroxylase (TH) in order to supplement dopamine, which is severely deficient in Parkinson s disease, however, none of the protocols tried have yielded fruitful results that can be applied directly to humans. One of the problems revealed from previous trials was a short duration of expression of the delivered gene, that is, tyrosine hydroxylase. Methods : To extend the stability and to improve the enzymatic characteristics of the protein, part of the regulatory domain was deleted via PCR technique. The cDNA for regulatory domain-deleted THs (dTH) were sub-cloned into a retroviral vector and the resulting recombinant retrovirus was used to infect NIH-3T3. After selection, expression levels of TH were determined by Western blot analysis and the enzymatic characteristics were examined. Results : The deletion increased steady state expression level of TH protein by 7-fold for d19TH (TH with amino acids #2-19 are deleted) and 3-fold for d31TH (TH with amino acids #2-31 are deleted. The elevated expression level of d19TH is likely due to the enhanced stability of the protein as determined by a treatment of cycloheximide. The activity of d19TH was also increased approximately by 3-fold but no increase of the L-dopa production was observed. However, the production of L-dopa was dramatically increased when GTP cyclohydrolase I (GTPCH I) was co-transfected suggesting that the activity of d19TH is dependent on the presence of cofactor. d19TH seem to be free of feedback inhibition at low concentration of dopamine (10 nM~1 M) but more sensitive to the inhibition at high concentration of dopamine (10 mM). Conclusions : The deletion of 18 amino acids on the regulatory domain increases the stability of the protein, reduces the activity, and frees it from the feedback inhibition by the end product.Key Words : Parkinson s disease, Tyrosine hydroxylase, Regulatory domain, Stability, L-dopa production, Feedback inhibition."