J Korean Neurol Assoc.  2002 Jan;20(1):60-66.

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

• Yoo YS
• Yi SD
• Lim JK
• Kim YS
• Nam EJ
• Joo WS
• Lee U
• Lee YJ

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 recom-binant 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 nM) 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 inhibi-tion by the end product.

Keyword

Parkinson's disease; Tyrosine hydroxylase; Regulatory domain; Stability; L-dopa production; Feedback inhibition

MeSH Terms

Amino Acids
Blotting, Western
Cycloheximide
DNA, Complementary
Dopamine
Fruit
GTP Cyclohydrolase
Humans
Levodopa
Parkinson Disease
Polymerase Chain Reaction
Retroviridae
Staphylococcal Protein A
Tyrosine 3-Monooxygenase*
Tyrosine*
Zidovudine
Amino Acids
Cycloheximide
DNA, Complementary
Dopamine
GTP Cyclohydrolase
Levodopa
Staphylococcal Protein A
Tyrosine
Tyrosine 3-Monooxygenase
Zidovudine