반응 #1467997

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phosphate

streptomycin sulfate

→

streptomycin

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후처리

1. 1
   기타After the removal of insoluble materials
2. 2
   기타by centrifugal separation
3. 3
   추출a crude extract
4. 4
   workup.ADDITIONcontaining adenylate kinase
5. 5
   기타was obtained
6. 6
   추출extract
7. 7
   기타Precipitates
8. 8
   기타formed
9. 9
   기타were removed by centrifugal separation

실험 절차

The thus-obtained cell was suspended in 600 ml of a 0.1M phosphate buffer solution, and broken by the use of a Dyno-mill. After the removal of insoluble materials by centrifugal separation, a crude extract containing adenylate kinase was obtained. Then, 200 ml of a 10% streptomycin sulfate solution was added to 400 ml of the crude extract. Precipitates formed were removed by centrifugal separation to thereby obtain a streptomycin supernatant liquid. This supernatant liquid was subjected to ammonium sulfate fractionation, and a fraction from 30% saturation (4° C.) to 60% saturation (4° C.) was obtained. This fraction was dissolved in a 50 mM Tris-hydrochloric acid buffer solution (pH 8.0) and passed through a DEAE-Sephadex column which had been equilibrated with the same buffer solution as above, and thereafter it was eluted with a solution prepared by adding sodium chloride to the above buffer solution. Near a concentration of sodium chloride of 0.2M, the desired adenylate kinase was eluted. The thus-eluted fraction was subjected to hydroxyapatite column chromatography under the same conditions as in Example 1, passed through a Sephadex G-75 column, and eluted with a 30 mM Tris-hydrochloric acid buffer solution (pH 8.0) containing 0.1M sodium chloride to thereby obtain an adenylate kinase sample which provided a single band by acrylamide disc electrophoresis as in the case of Example 1. Furthermore, as in the case of Example 1, the adenylate kinase sample provided a single peak at a molecular weight of about 22,000 as determined by Sephadex G-100 chromatography.

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