Solubilization of aminopeptidase from cell membranes of Mycoplasma salivarium.

概要

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Attempts were made to liberate aminopeptidase from cell membranes of Mycoplasma salivarium ATCC 23064. The membranes were separated from the organism cells sonicated and treated with DNase and RNase ; and incubated at 37°C with varying amounts of DOC, SDS, Triton X-100, Tween 80, Brij 35, CHAPSO, digitonin or EDTA for 15 min, and trypsin or papain for 2 h. SDS seemed to be most effective.<BR>Approximately 80% of aminopeptidase activity and 90% of membrane proteins were solubilized by treating 1 part of the membranes with 2.5 parts of SDS by weight. Triton X-100 solubilized the activity only slightly but 66% of the protein s.<BR>Based on the results, the following procedure for partial purification of the enzyme was devised. First of all, the membranes (1 part) were treated with Triton X-100 (5 parts), and centrifuged at 40, 000 g for 1 h. The sediment (1 part) was treated with SDS (3 parts) or papain (1/20 parts), and recentrifuged. The supernatants were chromatographed on a Sephacryl S-300 column. Thus, partially purified specimens were obtained. However, the specimen solubilized with SDS was water-insoluble, whereas papain produced a water-soluble specimen, the activity of which accounted for about 50% of the activity of the membranes. Therefore, it seems that treatment with Triton X-100 and papain is a favorable procedure for liberation of the enzyme from the membranes.
歯科基礎医学会の論文