Purification and kinetic properties of galactokinase from human placenta

Satish Srivastava, Karl Georg Blume, Cécile Van Loon, Ernest Beutler

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Galactokinase from human placenta was purified about 350-fold using DEAE-Sephadex-A-50 chromatography followed by Sephadex-G-200 and CM-Sephadex-C-50 filtration. The final steps of purification involved electrofocusing and ammonium sulfate precipitation. In analytical disc electrophoresis the purified enzyme moved as a single protein band. Crude enzyme is extremely unstable. Purification of the enzyme increases the stability slightly. The activation energy of the enzyme was found to be 9500 Cal. Galactokinase has an absolute requirement for a divalent ion. The enzyme is maximally active in the presence of Mg2+, 5 mm. However, Mg2+ could be partially replaced by Ca2+, Co2+, and Mn2+. The enzyme is almost completely inhibited by p-chloromercuribenzoate and partially inhibited by N-ethylmaleimide. The Km of placental galactokinase for galactose and ATP is 250 μm and 100 μm, respectively. Galactose-1-P appears to inhibit the enzyme in a competitive fashion with ATP with a Ki of 2.0 mm. The isoelectric point of galactokinase is 5.7 and the molecular weight is 58,000. The kinetic properties of placental galactokinase have been compared to the red cell galactokinase. The Km of the placental enzyme for galactose is about twice as high as that of the red cell enzyme whereas the affinity of the placental enzyme for ATP is twice as high as that of the red cell enzyme. Co-chromatography of placental and adult erythrocyte extract resulted in the separation of two peaks of galactokinase activity, one peak with the kinetic characteristics of the purified placental enzyme and the other peak with the kinetic characteristics of the red cell enzyme.

Original languageEnglish (US)
Pages (from-to)191-198
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume150
Issue number1
DOIs
StatePublished - 1972
Externally publishedYes

Fingerprint

Galactokinase
Placenta
Purification
Kinetics
Enzymes
Galactose
Cells
Adenosine Triphosphate
Chromatography
Chloromercuribenzoates
Disc Electrophoresis
Enzyme Stability
Ethylmaleimide
Enzyme Activation
Isoelectric Point
Ammonium Sulfate
Isoelectric Focusing
Electrophoresis

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Purification and kinetic properties of galactokinase from human placenta. / Srivastava, Satish; Blume, Karl Georg; Van Loon, Cécile; Beutler, Ernest.

In: Archives of Biochemistry and Biophysics, Vol. 150, No. 1, 1972, p. 191-198.

Research output: Contribution to journalArticle

Srivastava, Satish ; Blume, Karl Georg ; Van Loon, Cécile ; Beutler, Ernest. / Purification and kinetic properties of galactokinase from human placenta. In: Archives of Biochemistry and Biophysics. 1972 ; Vol. 150, No. 1. pp. 191-198.
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