Abstract
This 14CO2-trapping microassay for tyrosine hydroxylase activity uses microtest tubes (1.5 or 2.0 ml) with pierceable caps for injecting the reaction mixture. A folded filter paper strip (1 × 4 cm) impregnated with Protosol is placed directly inside the top of the tube prior to capping in order to trap liberated 14CO2. The effects of several variables and components involved in the assay have been systematically studied. The tyrosine hydroxylation reaction may be optimized by incubating 300 μg protein with 150 μM l-Tyr, 0.8 mM 6MPH4, 1 mM FeSO4, and 0.12 M Tris-acetate buffer (pH 5.8) for 10 min at 37°C. The DOPA decarboxylation reaction may be optimized by continual incubation of the tyrosine hydroxylation medium with 175 μg hog kidney aromatic-l-amino acid decarboxylase, 6.25 mM 3-iodotyrosine, and 0.125 M potassium phosphate buffer (pH 8.0) for 30 min at 37°C. Under these conditions, the radioactivity of 14CO2 recovered after 1 h at 37°C may reach 14,000 dpm, whereas the blank only has 300 dpm (< 3% of test value). This microassay is fast (< 2 h to complete all reactions) and convenient for performing a large number of determinations.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 163-173 |
| Number of pages | 11 |
| Journal | Journal of Neuroscience Methods |
| Volume | 16 |
| Issue number | 2 |
| DOIs | |
| State | Published - 1986 |
| Externally published | Yes |
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Keywords
- CO
- microassay
- trapping
- tyrosine hydroxylase activity
ASJC Scopus subject areas
- Neuroscience(all)
Cite this
A simplified 14CO2-trapping microassay for tyrosine hydroxylase activity. / Shen, Rong sen; Hamilton-Byrd, Elizabeth L.; Vulliet, Philip R; Kwan, Sau Wah; Abell, Creed W.
In: Journal of Neuroscience Methods, Vol. 16, No. 2, 1986, p. 163-173.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A simplified 14CO2-trapping microassay for tyrosine hydroxylase activity
AU - Shen, Rong sen
AU - Hamilton-Byrd, Elizabeth L.
AU - Vulliet, Philip R
AU - Kwan, Sau Wah
AU - Abell, Creed W.
PY - 1986
Y1 - 1986
N2 - This 14CO2-trapping microassay for tyrosine hydroxylase activity uses microtest tubes (1.5 or 2.0 ml) with pierceable caps for injecting the reaction mixture. A folded filter paper strip (1 × 4 cm) impregnated with Protosol is placed directly inside the top of the tube prior to capping in order to trap liberated 14CO2. The effects of several variables and components involved in the assay have been systematically studied. The tyrosine hydroxylation reaction may be optimized by incubating 300 μg protein with 150 μM l-Tyr, 0.8 mM 6MPH4, 1 mM FeSO4, and 0.12 M Tris-acetate buffer (pH 5.8) for 10 min at 37°C. The DOPA decarboxylation reaction may be optimized by continual incubation of the tyrosine hydroxylation medium with 175 μg hog kidney aromatic-l-amino acid decarboxylase, 6.25 mM 3-iodotyrosine, and 0.125 M potassium phosphate buffer (pH 8.0) for 30 min at 37°C. Under these conditions, the radioactivity of 14CO2 recovered after 1 h at 37°C may reach 14,000 dpm, whereas the blank only has 300 dpm (< 3% of test value). This microassay is fast (< 2 h to complete all reactions) and convenient for performing a large number of determinations.
AB - This 14CO2-trapping microassay for tyrosine hydroxylase activity uses microtest tubes (1.5 or 2.0 ml) with pierceable caps for injecting the reaction mixture. A folded filter paper strip (1 × 4 cm) impregnated with Protosol is placed directly inside the top of the tube prior to capping in order to trap liberated 14CO2. The effects of several variables and components involved in the assay have been systematically studied. The tyrosine hydroxylation reaction may be optimized by incubating 300 μg protein with 150 μM l-Tyr, 0.8 mM 6MPH4, 1 mM FeSO4, and 0.12 M Tris-acetate buffer (pH 5.8) for 10 min at 37°C. The DOPA decarboxylation reaction may be optimized by continual incubation of the tyrosine hydroxylation medium with 175 μg hog kidney aromatic-l-amino acid decarboxylase, 6.25 mM 3-iodotyrosine, and 0.125 M potassium phosphate buffer (pH 8.0) for 30 min at 37°C. Under these conditions, the radioactivity of 14CO2 recovered after 1 h at 37°C may reach 14,000 dpm, whereas the blank only has 300 dpm (< 3% of test value). This microassay is fast (< 2 h to complete all reactions) and convenient for performing a large number of determinations.
KW - CO
KW - microassay
KW - trapping
KW - tyrosine hydroxylase activity
UR - http://www.scopus.com/inward/record.url?scp=0022555925&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0022555925&partnerID=8YFLogxK
U2 - 10.1016/0165-0270(86)90049-X
DO - 10.1016/0165-0270(86)90049-X
M3 - Article
VL - 16
SP - 163
EP - 173
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
SN - 0165-0270
IS - 2
ER -