The Preparation and Characterization of Pure Rat Liver Glucokinase

Glucokinase from rat liver has been purified over 10,000-fold and appears homogeneous by the criterion of sodium dodecyl sulfate gel electrophoresis. The enzymatically active protein represents 0.007 % of the total liver protein and possesses a specific activity of 80 units per mg for purified enzym...

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Bibliographic Details
Published in:The Journal of biological chemistry 1974-05, Vol.249 (10), p.3055-3060
Main Authors: Grossman, Steven H., Dorn, Carl G., Potter, Van R.
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
Ammonium Sulfate
Animals
Binding Sites
Cellulose
Chromatography, DEAE-Cellulose
Chromatography, Gel
Chromatography, Ion Exchange
Dietary Carbohydrates - administration & dosage
Drug Stability
Electrophoresis, Polyacrylamide Gel
Glucokinase - antagonists & inhibitors
Glucokinase - isolation & purification
Hexoses - metabolism
Hot Temperature
Hydrogen-Ion Concentration
Kinetics
Liver - enzymology
Molecular Weight
Polysaccharides
Rats
Sodium Dodecyl Sulfate
Spectrophotometry, Ultraviolet
Structure-Activity Relationship
Ultracentrifugation
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Summary:Glucokinase from rat liver has been purified over 10,000-fold and appears homogeneous by the criterion of sodium dodecyl sulfate gel electrophoresis. The enzymatically active protein represents 0.007 % of the total liver protein and possesses a specific activity of 80 units per mg for purified enzyme. The use of potassium phosphate buffer gradients greatly enhances enzyme resolution during ion-exchange chromatographic procedures. In addition, use of substrate and sulfhydryl protecting reagents in all buffers prevents significant losses of activity. To our knowledge this is the highest degree of purity ever achieved for a mammalian hepatic ATP:d-hexose-6-phosphotransferase. Rat liver glucokinase has been characterized with respect to stability, Km, Vmax, and phosphorylation coefficient toward several substrates as well as behavior in the presence of several inhibitors. Spectral analysis fails to reveal the presence of a prosthetic group. The molecular weight determined by sodium dodecyl sulfate acrylamide gel electrophoresis and Sephadex G-100 gel filtration is 53,000 and 57,000, respectively. The turnover number is calculated to be 4,346 moles of d-glucose phosphorylated by ATP per min per mole of enzyme at 25°.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(19)42636-7