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Alkaline phosphatases ec 3.1.3.1 of brush border and basolateral membranes isolated from the rat jejunum mucosa 1. purification by chromatofocusing



Alkaline phosphatases ec 3.1.3.1 of brush border and basolateral membranes isolated from the rat jejunum mucosa 1. purification by chromatofocusing



Molecular Physiology 6(5-6): 355-364



Villous cells and crypt cells, isolated from rat jejunum mucosa, were digested with 0.5% Triton X-100 for 2 h at 37.degree. C. The solubilized enzymes and proteins were submitted to chromatofocusing. Numerous forms of alkaline phosphatase (EC 3.1.3.1) with different isoelectric points were present. But when brush border and basolateral membranes were prepared and digested in the same way, chromatofocusing revealed 1 single form of alkaline phosphatase for brush border membranes with an isoelectric point close to 5.6 and 1 single form for basolateral membranes with an isoelectric point close to 4.7. Moreover, most proteins of the membrane samples were either eluted before alkaline phosphatase or remained strongly bound to the polybuffer exchanger. The enrichment factor of purification of alkaline phosphatase by this chromatofocusing technique is > 10,000 for brush border membranes. These results point to the potentiality of the chromatofocusing technique for separation and purification of alkaline phosphatase from various sources.

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