Effect of Acinetobacter Glutaminase-Asparaginase Treatment on Free Amino Acids in Mouse Tissues1
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[CANCER RESEARCH 35. 1320 1325. May 1975] Effect of Acinetobacter Glutaminase-Asparaginase Treatment on Free Amino Acids in Mouse Tissues1 John S. Holcenberg, Esther Tang, and William C. Dolowy Departments of Medicine and Pharmacology. University of Washington School of Medicine. Seattle. Washington 9X195 [J. S. H.. E. T.}. and Chicago Medical School and Hiñes Vêlerons Administration Hospital. Chicago. Illinois 60612 [W. C. D.] SUMMARY LDH virus-infected mice (26) and are confined largely to the vascular space. As expected from these considerations, Acinetobacier glutaminase-asparaginase (AGA) and treatment of mice and humans with EC-2 produces pro Escherichia coli asparaginase were compared for their longed depletion of plasma asparagine but only transient effects on plasma and tissue levels of amino acids, am depletion of plasma glutamine (21, 25). AGA treatment in monia, and glutamyl transferase activity in the mouse. Free mice produces prolonged depletion of both plasma aspara asparagine was depleted similarly in plasma and tissues by gine and glutamine (26). both enzymes. AGA treatment produced partial depletion Several studies have reported changes in free amino acids of glutamine concentrations in muscle, spleen, small intes in tissues during treatment with asparaginase enzymes, but tine, and liver. Brain and kidney glutamine concentrations none of these enzymes has had appreciable glutaminase actually rose with treatment. Despite over 100-fold increase activity under physiological conditions (2, 7, 20, 30). The in plasma glutamate, only the kidney showed a substantial amino acid levels in tissues should depend on the delivery of increase in free glutamate levels during AGA treatment. substrates and enzymes by the vascular system, together Glutamine biosynthesis measured by glutamyl transferase with the rate of synthesis and utilization of the amino acid in activity showed an appreciable increase only in the kidney. the tissue. Ammonia levels in tissues and plasma rose 1.3- to 4.3-fold. This paper compares the effect of AGA and EC-2 In general, E. coli asparaginase treatment had much less treatment on the tissue concentration of several free amino effect on these measurements than did AGA. The changes in acids, ammonia, and glutamyl transferase activity. these levels are discussed in relation to sites of possible toxicity and antitumor effects. MATERIALS AND METHODS INTRODUCTION Materials. SSA, lithium hydroxide, lithium citrate, and ninhydrin were amino acid grade from Pierce Chemical Co., Rockford, 111.Nessler's reagent (64091) was from Harleco, Asparaginase and glutaminase enzymes have produced prolonged remissions of certain experimental tumors (40). Philadelphia, Pa. All other chemicals were reagent grade. During treatment, plasma asparagine and glutamine are AGA was isolated and purified as described previously depleted. The degree of amino acid depletion depends on the (27), except that potassium phosphate buffers were used in kinetic properties of the enzyme, its biological half-life in the final DE-Sephadex and lyophilization steps. This change the animal, and the rate of input of the amino acid into the provided much greater stability during shell freezing. In circulation. EC-23 catalyzes the hydrolysis of glutamine at addition, the sediment after sonic extraction was extracted only 3% the rate of hydrolysis of asparagine. In addition, the with 30% saturation ammonium sulfate to increase the Km for glutamine is over 100-fold that for asparagine (40). overall yield from each batch of bacteria. All AGA On the other hand, AGA has approximately equal hydro- preparations used had a specific activity greater than 120 lytic activity and an equally low Km with both amino acids lU/mg protein. Such preparations elute as a single peak on (27). The 2 enzymes have comparable biological half-lives in Sephadex G-200 chromatography and show a single band on sodium dodecyl sulfate gel electrophoresis and disc gel 1This work was supported by USPHS Research Grant C'A 11881 from electrofocusing (15). The enzyme was stored as a lyophilized the National Cancer Institute. A preliminary report of some of this work powder at 4°. was presented at the 65th Annual Meeting of the American Association for EC-2 was a gift from Dr. M. Zimmerman. Merck Sharp Cancer Research, at Houston. Texas. March 1974 (4). '' Research Career Development Awardee CA 25976 from National and Dohme, Inc., Westpoint, Pa., received as a lyophilized Cancer Institute. To whom reprint requests should be addressed, at powder (lot ABI-2) that had 200 lU/mg protein. This Department of Medicine RG-20. University of Washington. Seattle, Wash. preparation was homogeneous on disc gel electrophoresis 98195. (14). 3The abbreviations used are: EC-2. Escherichia coli asparaginase: Mice. CD-I female mice were supplied by Charles River AGA, Acinetobacter glutaminase-asparaginase. American Type Culture Collection No. 27197; LDH virus, láclatedehydrogenase-elevating virus: Breeding Laboratories, Wilmington, Mass. They were fed SSA. sulfosalicylic acid. Purina mouse chow and water ad libitum. Mice were stored Received September 13, 1974; accepted February 11, 1975. 5 to 6 in a filter-top cage. Cages, water bottles, filters, and 1320 CANCER RESEARCH VOL. 35 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1975 American Association for Cancer Research. AG A Treatment Sanicel bedding (Paxton Processing Company, Paxton, 111.) to 1 ml were pipeted into 50-ml serum bottles fitted with a were steam sterilized before use. rubber stopper and a 0.3- x 6-cm ground glass rod wetted Mice were infected with LDH virus 3 or more days before with 0.5 M H2SO4. Distillation was started by addition of I treatment. Virus was initially obtained from Dr. V. Riley. ml saturated solution of K2CO3. Bottles were rotated for 20 Pacific Northwest Research Foundation, Seattle, Wash., min. The ammonium sulfate on the glass rod was rinsed into and was later propagated in mice. The dose was 0.1 ml of a a test tube with 1 ml of water and assayed with a 1:10 dilution of pooled mouse plasma in Fisher's medium concentrated phenol-hypochlorite reagent (3). Solution 1 and contained about 10" 50% infectious dose units (26). contained 6.2 g phenol and 25 mg sodium nitroprusside per Sample Preparation. Three to 4 hr after the last enzyme 100 ml. Solution 2 contained lOgNaOH and 2.15 ml bleach treatment, blood was obtained by heart puncture from mice per 100 ml. One ml Solution I and 0.2 ml Solution 2 were anesthetized with ether. The blood was treated with sodium added in rapid sequence to the samples. Tubes were heated heparin. rapidly chilled, and centrifuged. Plasma for en at 37°for 15 min and cooled, and the absorbance was read zyme assay was tested immediately or stored at - 100°. at 640 nm. Standards were NH4C1 with and without SSA. Storage for several weeks did not decrease the enzyme SSA decreased the slope of standard ammonia curves by activity. Samples for amino acid analysis were deprotein- 23%. Ammonia added to tissue samples or glutamine ized within 1 hr with 10% SSA. The precipitate was washed solutions was completely recovered, and glutamine added to 2 times with 5% SSA. The combined supernatants were tissue samples was not hydrolyzed. titrated to pH 2.6 to 2.7 with LiOH and diluted with lithium Asparaginase and glutaminase activity was determined citrate buffer, pH 2.76. by ammonia formation by direct nesslerization, as previ Tissue samples were obtained from mice anesthetized ously described (27). Glutamyl transferase activity was with ether. They were rapidly trimmed of fat, blotted dry, determined by hydroxamate formation, as previously de and weighed. The sample of small intestine was obtained scribed (33). from the 1st 8 cm of duodenum and jejunum after the lumen Treatments. All enzyme preparations were injected i.p. had been rinsed with cold 0.9% NaCl solution. Whole EC-2 and AGA were dissolved in water and administered samples or aliquots less than 0.5 g were immediately daily at doses of 250 to 350 IU/kg of body weight. Previous plunged in 2 ml 10% SSA and minced. The mince was studies showed that these maximally tolerated doses of homogenized in a Potter-Elvehjem tissue grinder with a AGA (27) maintained asparaginase activity above 1.0 Teflon plunger. The homogenate was transferred with 1 ml ID/ml of plasma. 10% SSA and centrifuged at 27,000 x g for 15 min. The pellet was rinsed with 1 ml 5% SSA and recentrifuged. The combined supernatants were titrated like the plasma sam RESULTS ples and diluted to approximately 5 ml. Exceptions to this method were the spleen, which was homogenized and Weight. The effects of enzyme treatment on animal and washed in one-half of the standard volume, and muscles, spleen weight are shown in Chart 1. AGA treatment caused which were homogenized in a Sorval Omnimix microattach- a progressive loss of up to 35% of the initial body weight by ment at full speed for 2 min. Samples were stored at - 100°. the 11th day of treatment. This weight loss was accompa Tissues for glutamyl transferase activity were homoge nied by marked loss of s.c. tissue and fat and moderately nized in Medium A (17) and centrifuged at 100,000 x g for ruffled hair. In contrast, animals treated with equal doses of 60 min or at 48,000 x g for 90 min (33). EC-2 based on asparaginase activity had less than 15% Amino Acid Determinations. Aliquots of plasma and weight loss, which tended to stabilize by 7 days of treatment. tissue were analyzed on a JEOLCO 5AH amino acid Spleen weight of animals treated with AGA decreased analyzer with a dual channel integrator Model DK. Neutral markedly and remained less than one-half normal on all 4 and acidic amino acids were analyzed through the glutamine days sampled.