純化方法
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.]
