You are trying to isolate an enzyme that catalyzes the conversion of A ? B, and
ID: 1061880 • Letter: Y
Question
You are trying to isolate an enzyme that catalyzes the conversion of A ? B, and you have a sensitive assay for this enzyme. After lysing open the cells, you find that the activity is associated with the membrane fraction, not the soluble fraction (which you throw away). You then follow the procedure shown below. Explain the sudden re-occurrence of activity at the end of your protocol.
You are trying to isolate an enzyme that catalyzes the conversion of A ? B, and you have a sensitive assay for this enzyme. After lysing open the cells, you find that the activity is associated with the membrane fraction, not the soluble fraction (which you throw away). You then follow the procedure shown below. Explain the sudden re-occurrence of activity at the end of your protocol.
lyse cells soluble membrane no activity activity change pH from 7 to 6 soluble membrane no activity no activity treat with mild detergent soluble membrane no activity no activity combine fractions activityExplanation / Answer
when the first step lyse cells, the membrane has the Enzyme. hence it get the activity. But when the pH changes 7---> 6 (neutral-----> acidic) The optimum ph for the catalytic reaction is missed for the enzyme or the membrane bounded co-factor is dissolved in the acidic pH level. that"s the reason soluble and the membrane fractions get no activity. After that it membrane fraction treat with mild detergent. But still the both soluble fraction and membrane fraction didn't meet the activity. But at last the pH changed soluble fraction and the finally treated with mild detergent soluble fraction mixed together it forms the enzymatic activity.....
That because, it need the co-factor to catalyse the enzymatic reaction. The cofactor binded with the membrane and dissolved in the acidic pH levels. After adding the mild detergent to the membrane fraction, it crack the membrane fraction and dissovle the enzyme in the soluble fraction.. finally that detergent treated soluble fraction and pH changed soluble fraction mixed with together it forms the activity of the enzyme... that is the re-occurence of the enzyme activity.
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