Resumen
Phosphomevalonate kinase is eluted with a linear gradient of 60-250 mM potassium phosphate buffer pH 7.5 containing 10 mM mercaptoethanol and 0. l mM EDTA (3 liters total volume) at a flow rate of 150 ml/ hr. The active fractions are pooled and the enzyme is precipitated with solid ammonium sulfate added up to 80% saturation. After centrifuging for 15 min at 16,000 g, the precipitate is resuspended in about 60 ml of 10 mM potassium phosphate buffer, pH 7.5, l0 mM mercaptoethanol, 100 mM KC1. At this stage, the enzyme is free of phosphatase and NADH oxidase activities. Phosphomevalonate kinase catalyzes the phosphorylation of phosphomevalonate (MVAP) by MgATP to pyrophosphomevalonate (MVAPP) and ADP. Only the 3R isomer of MVAP is phosphorylated and the reaction is reversible. The equilibrium constant is near unity at pH 7.5 and 30°. Percentage transformation of MVAP to MVAPP is calculated and the values obtained at different times (usually 1, 2, and 3 min) are used for initial velocity calculations. Other radioassay methods for phosphomevalonate kinase using paper and column chromatography to separate labeled substrates and products are described by Tchen.
| Idioma original | English |
|---|---|
| Páginas (desde-hasta) | 78-85 |
| Número de páginas | 8 |
| Publicación | Methods in Enzymology |
| Volumen | 110 |
| N.º | C |
| DOI | |
| Estado | Published - 1 ene 1985 |
Huella dactilar
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
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[9] Phosphomevalonate kinase from pig liver. / Eyzaguirre, Jaime; Bazaes, Sergio.
En: Methods in Enzymology, Vol. 110, N.º C, 01.01.1985, p. 78-85.Resultado de la investigación: Article
TY - JOUR
T1 - [9] Phosphomevalonate kinase from pig liver
AU - Eyzaguirre, Jaime
AU - Bazaes, Sergio
PY - 1985/1/1
Y1 - 1985/1/1
N2 - Phosphomevalonate kinase is eluted with a linear gradient of 60-250 mM potassium phosphate buffer pH 7.5 containing 10 mM mercaptoethanol and 0. l mM EDTA (3 liters total volume) at a flow rate of 150 ml/ hr. The active fractions are pooled and the enzyme is precipitated with solid ammonium sulfate added up to 80% saturation. After centrifuging for 15 min at 16,000 g, the precipitate is resuspended in about 60 ml of 10 mM potassium phosphate buffer, pH 7.5, l0 mM mercaptoethanol, 100 mM KC1. At this stage, the enzyme is free of phosphatase and NADH oxidase activities. Phosphomevalonate kinase catalyzes the phosphorylation of phosphomevalonate (MVAP) by MgATP to pyrophosphomevalonate (MVAPP) and ADP. Only the 3R isomer of MVAP is phosphorylated and the reaction is reversible. The equilibrium constant is near unity at pH 7.5 and 30°. Percentage transformation of MVAP to MVAPP is calculated and the values obtained at different times (usually 1, 2, and 3 min) are used for initial velocity calculations. Other radioassay methods for phosphomevalonate kinase using paper and column chromatography to separate labeled substrates and products are described by Tchen.
AB - Phosphomevalonate kinase is eluted with a linear gradient of 60-250 mM potassium phosphate buffer pH 7.5 containing 10 mM mercaptoethanol and 0. l mM EDTA (3 liters total volume) at a flow rate of 150 ml/ hr. The active fractions are pooled and the enzyme is precipitated with solid ammonium sulfate added up to 80% saturation. After centrifuging for 15 min at 16,000 g, the precipitate is resuspended in about 60 ml of 10 mM potassium phosphate buffer, pH 7.5, l0 mM mercaptoethanol, 100 mM KC1. At this stage, the enzyme is free of phosphatase and NADH oxidase activities. Phosphomevalonate kinase catalyzes the phosphorylation of phosphomevalonate (MVAP) by MgATP to pyrophosphomevalonate (MVAPP) and ADP. Only the 3R isomer of MVAP is phosphorylated and the reaction is reversible. The equilibrium constant is near unity at pH 7.5 and 30°. Percentage transformation of MVAP to MVAPP is calculated and the values obtained at different times (usually 1, 2, and 3 min) are used for initial velocity calculations. Other radioassay methods for phosphomevalonate kinase using paper and column chromatography to separate labeled substrates and products are described by Tchen.
UR - http://www.scopus.com/inward/record.url?scp=0021894247&partnerID=8YFLogxK
U2 - 10.1016/S0076-6879(85)10062-5
DO - 10.1016/S0076-6879(85)10062-5
M3 - Article
VL - 110
SP - 78
EP - 85
JO - Methods in Enzymology
JF - Methods in Enzymology
SN - 0076-6879
IS - C
ER -