Phosphate → L-Glutamic Acid

Participants

Reaction Steps

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 mM NAD+ solution (available from Oriental Yeast Co.) and 50 μl of 500 U/ml L-glutamate dehydrogenase solution (available from SIGMA Co.), 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).

Provenance & Evidence