GLYCEROKINASE

Chemical properties

Beige powder

The Uses of GLYCEROKINASE

Glycerol kinase (glpK) was used to study the effects of pain controlling neuropeptides on human fat cell lipolysis.

The Uses of GLYCEROKINASE

This enzyme is useful for enzymatic determination of glycerol and triglyceride when coupled with glycerol-3-phosphate dehydrogenase (=G-3-P DH, G3D-301), glycerol-3-phosphate oxidase (=G-3-P oxidase, G3O-301, G3O-311, G3O-321) or pyruvate kinase (PYK-301) and lactate dehydrogenase (LCD-209, LCD-211), lipoprotein lipase (LPL-311, LPL-314) in clinical analysis.

The Uses of GLYCEROKINASE

Glycerol kinase has been used along with adenosine triphosphate (ATP) for the conversion of glycerol to glycerol-3 phosphate (glycerol-3P) in white adipose tissue (WAT) adipocyte samples by label distribution method. It has also been used along with adenosine triphosphate (ATP) in the derivatization of glycerol to sn-glycerol-3 phosphate (glycerol-3P).

Biochem/physiol Actions

Glycerol kinase catalyzes tge MgATP-dependent phosphorylation of glycerol to produce sn-glycerol-3-phosphate and is the rate limiting enzyme in the utilization of glycerol. It is also subject to feedback regulation by fructose-1,6-bisphosphate.

Purification Methods

Commercial enzyme has been dialysed against 2mM Hepes, 5mM dithiothreitol and 0.3mM EDTA, followed by several changes of 20mM Hepes and 5mM dithiothreitol prior to storage under N2 at -20o. [Knight & Cleland Biochemistry 28 5728 1989.] The enzyme from pigeon liver is purified by acid-precipitation (acetate buffer at pH 5.1), (NH4)2SO4 fractionation, heat treatment (60o/ 1hour), calcium phosphate gel filtration, a second (NH4)2SO4 fractionation, dialysis, elution of inert proteins and crystallisation. This is done by repeatedly extracting the precipitate from the last step with 0.05M sodium pyrophosphate (pH 7.5) containing 1mM EDTA and 0.2M (NH4)2SO4 is added. Careful addition of solid (NH4)2SO4 to this solution leads to crystallisation of the enzyme. Recrystallisation is repeated. The enzyme is activated by Mg2+ and Mn2+ ions and is most stable in solutions in the pH 4.5-5.5 range. The stability is greatly increased in the presence of glycerol. It has Km for glycerol of 60WM and for ATP 9WM in glycine buffer pH 9.8 and 25o. [Kennedy Methods Enzymol 5 476 1962.]