A one-step procedure for the isolation of fructose 1,6-bisphosphatase and fructose 1,6-bisphosphate aldolase from rabbit liver |
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Authors: | H Kido A Vita B L Horecker |
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Institution: | Department of Biology, Indiana University, Bloomington, Indiana 47405 USA |
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Abstract: | Human ceruloplasmin, which is usually cleaved by limited proteolysis into three major fragments during preparation (, 50,000, and 70,000) was isolated in good yield as an undegraded single-chain protein (). The cryosupernatant from fresh frozen plasma (100 liters) was fractionated with polyethylene glycol (PEG 4000) at + 5°C yielding a ceruloplasmin-enriched fraction in the 20% PEG supernatant. Three steps of chromatography on DEAE-Sephacel, hydroxyapatite, and Sephadex G-200 produced a homogeneous protein with maximal enzymatic activity and the ratio of 0.046 corresponding to 98–100% purity. Two forms of ceruloplasmin having this absorbance ratio were obtained; Form I was predominant and was studied further. The procedure separated both forms from apoceruloplasmin and degraded ceruloplasmin. The single-chain ceruloplasmin (Form I) had an NH2-terminal sequence of Lys-Glu-Lys-His-Tyr-Tyr-Ile-, the same as for the 70,000 fragment, and is suitable for structural study by sequence analysis and physicochemical methods. |
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Keywords: | To whom reprint requests and correspondence should be addressed |
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