반응 #1350568

반응 방정식

hydrochloric acid hydroxylamine

L-glutamate

L-glutamine

phosphate

→

glutamic acid

용매

반응 조건

후처리

1. 1
   기타the mixture was reacted at 37° C. for 30 minutes
2. 2
   기타the reaction was terminated
3. 3
   workup.ADDITIONby adding 250 μl of 0.75N perchloric acid solution, and 125 μl of 1.5N sodium hydroxide solution
4. 4
   workup.ADDITIONwas added
5. 5
   기타The above reaction mixture
6. 6
   기타(10,000 rpm, 10 minutes)
7. 7
   기타the mixture was reacted at 37° C., for 30 minutes

실험 절차

Glutaminase activity was measured by partly modifying the method described in Japanese Provisional Patent Publication No. 332553/1999. That is, to 250 μl of 2% (w/v) L-glutamine solution were added 500 μl of 0.2M phosphate buffer (pH 6.5) and 250 μl of an enzyme solution, and the mixture was reacted at 37° C. for 30 minutes, and then the reaction was terminated by adding 250 μl of 0.75N perchloric acid solution, and 125 μl of 1.5N sodium hydroxide solution was added thereto to neutralize the reaction mixture. The above reaction mixture was then centrifuged (10,000 rpm, 10 minutes). To 100 μl of the resultant supernatant were added 1.0 ml of 0.1M hydrochloric acid-hydroxylamine buffer (pH 8.0), 1.0 ml of 20 mMNAD+ solution (manufactured by Oriental Yeast Co.), and 50 μl of 500 U/ml L-glutamate dehydrogenase solution, and the mixture was reacted at 37° C., for 30 minutes and the absorbance at 340 nm was measured with a spectrophotometer. An amount of the enzyme forming 1 μmol of glutamic acid per one minute under the above conditions is determined as 1 unit (U).

출처