Isolation, crystallization, and properties of Achromobacteraceae glutaminase-asparaginase with antitumor activity.

Crystalline glutaminase-asparaginase with antitumor activity was prepared from an Achromobacferaceae soil isolate organism. This enzyme has L-glutaminase and L-asparaginase activity in a ratio of 1.2:1. The purification procedure provides an over-all yield of 40 to 60% from crude cell-free extract to homogeneous glutaminase-asparaginase and is adaptable to large scale isolation of the enzyme. Glutaminase-asparaginase is crystallizable from aqueous alcohol solutions in the absence of heavy metal cations. The highest yields of enzyme were obtained when cells were grown aerobically in a basal synthetic medium composed of L-glutamic acid, ammonium sulfate, trace minerals, and phosphate buffer. Glutaminase-asparaginase content remained relatively constant when the organism was at temperatures between 15 and 25”. Above 25” the enzyme content decreased with increasing temperature. The isoelectric point by isoelectric focusing of glutaminaseasparaginase on ampholytes is 8.43. The specilic activity of homogeneous enzyme is 190 f 20 i.u. per mg of protein and the E$$ is 10.2. No carbohydrate or phospholipid was detected in the enzyme. No disuliide or sulfhydryl groups appear to be present on the enzyme. The K, values for L-glutamine and L-asparagine are 5.8 f 1.5 X 10m6 and 4.8 f 1.4 X 10m6 M, respectively. Glutaminase-asparaginase catalyzes the hydrolysis of the D isomers of glutamine and asparagine at about one-third the rate of the L isomers. The enzyme is not inhibited by ethylenediaminetetraacetate (0.1 mu), ammonia (10 mu), L-glutamate (30 mu), or L-aspartate (30 mu). 6-Diazod-oxo-Lnorleucine which is not a substrate for the enzyme irreversibly inactivates glutaminase-asparaginase at very low concentrations. In contrast the L and D isomers of 5-diazo-4-oxonorvaline are attacked by the enzyme and are considerably poorer inhibitors.