[9] Phosphomevalonate kinase from pig liver

Jaime Eyzaguirre, Sergio Bazaes

Resultado de la investigación: Article

13 Citas (Scopus)

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 originalEnglish
Páginas (desde-hasta)78-85
Número de páginas8
PublicaciónMethods in Enzymology
Volumen110
N.ºC
DOI
EstadoPublished - 1 ene 1985

Huella dactilar

Liver
Swine
Mercaptoethanol
Buffers
Paper Chromatography
Column chromatography
Phosphorylation
Equilibrium constants
Ammonium Sulfate
Enzymes
Phosphoric Monoester Hydrolases
Edetic Acid
Isomers
Adenosine Diphosphate
Precipitates
Adenosine Triphosphate
Flow rate
Substrates
phosphomevalonate kinase
potassium phosphate

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Citar esto

Eyzaguirre, Jaime ; Bazaes, Sergio. / [9] Phosphomevalonate kinase from pig liver. En: Methods in Enzymology. 1985 ; Vol. 110, N.º C. pp. 78-85.
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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

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AU - Bazaes, Sergio

PY - 1985/1/1

Y1 - 1985/1/1

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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.

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