Purification and properties of a highly potent antitumor glutaminase-asparaginase from Pseudomonas 7Z.
Crystalline glutaminase-asparaginase which is effective against solid as well as ascites tumors was prepared from soil isolate organism Pseudomonas 7A. This enzyme has a ration of Vmax for L-glutamine and L-asparagine of 2.0. The presence of glutamic acid in the growth medium is essential for optimal enzyme production and glucose inhibits the production of glutaminase-asparaginase. The purification procedure provides an overall yield of 40 to 45% from crude cell extract to homogeneous glutaminase-asparaginase and is adaptable to large scale production of the enzyme. The specific activity of homogeneous enzyme is 160 +/- 15 i.u./mg of protein and the E1% 280 is 9.8. No disulfide or sulfhydryl groups appear to be present on the enzyme. The isoelectric point of glutaminase-asparaginase by isoelectric focusing on ampholine polyacrylamide gel plates is 5.8. The Km values for L-glutamine and L-asparagine are 4.6 and 4.4 X 10(-6) M, respectively. The enzyme catalyzes the hydrolysis of the D isomers of glutamine and asparagine at 87 and 69% the rate of the respective L isomers. L-Glutamic acid gamma-monohydroxamate is hydrolyzed at approximately the same rate as L-glutamine. The enzyme is not inhibited by ethylenediaminetetraacetate (0.1 mM), L-glutamate (30 mM), or L-aspartate (30 mM). Ammonium sulfate (10 mM) inhibits the enzymatic activity. The plasma half-life of Pseudomonas 7A glutaminase-asparaginase if 13 hours in normal mice and 43 hours in mice infected with the lactate dehydrogenase-elevating virus.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000970:Antineoplastic Agents; D001215:Asparaginase / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D003460:Crystallization; D005972:Glutaminase / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D008815:Mice, Inbred Strains; D011549:Pseudomonas / Q000201:enzymology*; D013329:Structure-Activity Relationship
D000596:Amino Acids; D000970:Antineoplastic Agents; D001215:Asparaginase; D005972:Glutaminase