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Purification and Characterization of L-Asparaginase with Anti-lymphoma Activity from Vibrio succinogenes*

Homogeneous L-asparaginase with anti-lymphoma activity was prepared from Vibrio succinogenes, an anaerobic bacterium from the bovine rumen. An overall yield of pure L-asparaginase of 40 to 45% and a specific activity of 200 + 2 IU per mg of protein were obtained. The pure enzyme can be stored at ~ 20” for at least 3 months with no loss of activity. The isoelectric point of the L-asparaginase is 8.74. No carbohydrate, phosphorus, tryptophan, disulfide, or sulfhydryl groups were detected. The enzyme has a molecular weight of 146,000 and a subunit molecular weight of approximately 37,000. The K, of the enzyme for L-asparagine is 4.78 x 10m5 M and the pH optimum of the L-asparaginase reaction is 7.3. D-Asparagine was hydrolyzed at 6.5% of the rate found with the L isomer. I,-Glutamine and a variety of other amides were not hydrolyzed at significant rates; the activity of the enzyme for L-glutamine was 130to 600-fold less than that of other therapeutically effective L-asparaginases of bacterial origin. The L-asparaginase from V. succinogenes is immunologically distinct from the L.-asparaginase (EC-2) of Escherichia coli.

The K, of the enzyme for L-asparagine is 4.78 x 10m5 M and the pH optimum of the L-asparaginase reaction is 7.3. D-Asparagine was hydrolyzed at 6.5% of the rate found with the L isomer. I,-Glutamine and a variety of other amides were not hydrolyzed at significant rates; the activity of the enzyme for L-glutamine was 130to 600-fold less than that of other therapeutically effective L-asparaginases of bacterial origin. The L-asparaginase from V. succinogenes is immunologically distinct from the L.-asparaginase (EC-2) of Escherichia coli.

RESULTS
Purification of L-Asparaginase from Vibrio succinogenes-A homogeneous preparation of L-asparaginase was obtained by ammonium sulfate fractionation followed by chromatography on columns of hydroxylapatite, CM-Sephadex, and DEAE-Sephadex, respectively. The results of a typical purification are presented in Table IP. Details of this procedure and information concerning some of the characteristics of L-asparaginase are presented in the miniprint supplement immediately following the parent article.
Purity of L-Asparaginase-The sulfuric acid-phenol colorimetric determination for carbohydrate (12) and the total phosphorus determination (13) were negative. Disc gel electrophoresis in the presence of sodium dodecyl sulfate (14) yielded a single protein band (Fig. 1P). The native enzyme does not migrate into 7.5% acrylamide gels (15) at pH 7.9 or 4.3.
Isoelectric Point-The isoelectric point of L-asparaginase as determined by isoelectrofocusing on 1% ampholytes was 8.72 with a pH 3.5 to 10 carrier and 8.75 with a pH 7 to 9 carrier. There was one protein peak in each of the electrofocusing columns as determined by A,,,. The single protein peak from each column coincided with the L-asparaginase activity (Fig.  2P).
Effect of Substrute Concentration on Actiuity-The assay for L-asparaginase at low concentrations of substrate is linear with enzyme concentration up to 1.0 IU per reaction tube and is linear with time between 0 and 4 min.
The initial reaction rates of the deamidation of L-asparagine at varying substrate concentrations were determined at pH 7.0. Double reciprocal plots of these data fit a straight line between 0.015 and 40 mM L-asparagine. Two determinations at different enzyme concentrations of L-asparaginase are shown in Fig. 3P. The average K, value and standard deviation is 4.78 f 0.21 x lo-d M (eight determinations).
Enzyme Specificity-The enzyme catalyzes the hydrolysis of both the D and L isomers of asparagine, but its activity with the L isomer as substrate is more than 15 times that obtained with 3 Some of the data are presented in a miniprint format immediately following this paper. Details of the Materials and Methods, References, Tables IS and IIS, and Figs. 1s to 4s. will be found in the miniprint section. For the convenience of those who prefer to obtain this material in the form of full size photocopies, it is available as JBC Document 76M-584. Orders should specify the title, authors and reference to this paper, the JBC Document number, and the number of copies desired. Orders should be addressed to The Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Maryland 20014, and must be accompanied by a remittance to the Journal of $1.95 per set of photocopies. the D isomer as substrate (Table IIP). P-Cyanoalanine is hydrolyzed at a rate of 3.1% compared to Lasparagine. The hydrolosis of Lglutamine, L-analyl-casparagine, N-carbobenzoxy-L asparagine, and L and n-5-diazo-4-oxonorvaline proceeds at an extremely slow rate. GAspartic acid P-hydroxamate is hydrolyzed about 4 times more slowly than L-asparagine. Also, the hydroxamate of Lasparagine is formed when the enzyme is incubated with 40 mM L-asparagine, 60 mM neutralized hydroxylamine, and 0.5 mM Tris buffer, pH 8.0. Formation of the hydroxamate proceeds at a rate about g-fold slower than the rate for Gasparagine hydrolysis, and is proportional to enzyme concentration.  Procedure") except for r,-glutamine hydrolysis which was measured by the NADH-coupled assay for ammonia-producing systems (see under "Experimental Procedure"), 5-diazo-4-oxonorvaline breakdown which was determined by the decrease in absorbance at 274 nm, and L-aspartic acid P-hydroxamate hydrolysis which was measured by loss of absorbance at 540 nm of the iron hydroxamate complex 07).

Substrate
Concentration E. coli and V. succinogenes, respectively, an immunodiffusion experiment was performed (Fig. 4P). Antiserum prepared against r.,-asparaginase from E. coli (EC-2) was used. In immunodiffusion with this antiserum, the enzyme from E. coli predictably formed precipitin bands after 2 days at each dilution of enzyme. There was no detectable reaction with the antiserum and L-asparaginase from V. succinogenes at any dilution. Three concentrations of antiserum were used in replicate sets of immunodiffusion slides. In each case the results were the same. The lack of cross-reactivity between the L-asparaginase of V. succinogenes and antiserum prepared against EC-2 suggests that these enzymes are immunologically distinct.
L-Asparaginase therapy selectively inhibits certain neoplasms in man by hydrolyzing L-asparagine and thus starving tumor cells of a required nutrient.
Even though normal mammalian cells do not require an exogenous source of L-asparagine for growth, several problems occur when this enzyme is used chemotherapeutically for the treatment of acute lymphoblastic leukemia (18). Two of these problems, host toxicity and immunosuppression, are at least partially due to the cross-reactivity of L-asparaginase with the substrate 11, 22-24), the enzyme from V. succinogenes has nearly undetectable L-glutaminase activity. This enzyme hydrolyzes L-glutamine at a rate of 0.015% compared to the hydrolysis of L-asparagine. The L-asparaginase (EC-2) from E. coli hydrolyzes L-glutamine at a rate more than 130-fold faster (25) than the L-asparaginase from V. succinogenes. Similarly, Lasparaginase from E. carotooora hydrolyzes L-glutamine at a rate more than 600-fold faster (26) than the L-asparaginase from V. succinogenes. Most L-asparaginases can hydrolyze the C-N bond of several amides, hydroxamates, hydrazides, diazoketones, and nitriles of L-asparagine and its analogs (22)(23)(24). When most of these substrates were tested with I,-asparaginase from V. succinogenes, minimal activities were observed.
Substrate specificity studies with L-asparaginase from V. succinogenes indicate that the oc-carboxyl group of L-asparagine is required for enzymatic activity, since no hydrolysis occurs when /+alanine amide (the decarboxylation product of Lasparagine) is provided as a substrate. The a-carboxyl group may serve to stabilize substrate binding to enzyme. An alternative hypothesis is that the cr-carboxyl group participates in the catalysis by forming an aspartic acid-anhydride-activated intermediate as suggested for other Lasparaginases (27). It has been shown that L-asparaginase from V. succinogenes is sensitive to steric hindrance at the site of binding of the a-amino group. Specific interactions that involve the amino group may facilitate stability of the enzyme'substrate complex when it is in the proper orientation to the a-carboxyl group.
I.-Asparaginase from V. succinogenes has some biochemical characteristics similar to the L-asparaginase from E. carotouoru (4,(26)(27)(28). Both enzymes have chemotherapeutic utility, low K, values, basic isoelectric points, and similar molecular weights. In addition, the amino acid composition of both enzymes is similar with the exception of glutamic acid and alanine which are present in 1.5-fold greater quantities in the enzyme from Vibrio. Although these enzymes have several definite similarities, the active sites must have some distinct differences as evidenced by substrate specificity. Unlike the L-asparaginase from V. succinogenes, the enzyme from E. carotouora is capable of hydrolyzing the amide groups of D-and L-glutamine, and m-analyl-m-asparagine at a significant rate. More extensive substrate specificity studies and experiments to determine the differences of the active sites of these two enzymes are planned.
An effective inhibitor for L-asparaginase from V. succinogenes was not found even when potential inhibitors were used at high concentrations. Enzyme activity is not inhibited by the hydrolysis products of L-asparagine:Laspartate and ammonia. Ammonia has been shown to be an effective inhibitor of the EC-2 enzyme from E. coli (29). Sulfhydrylblocking or reducing agents do not affect enzyme activity, suggesting that the active site of the enzyme does not require a reduced sulfhydryl group for activity. This agrees with the results obtained from amino acid analysis indicating that the enzyme does not have disulfide or sulfhydryl groups.
In summary, L-asparaginase from V. succinogenes has the necessary characteristics of chemotherapeutically active enzymes. It has been shown to be a potent anti-lymphoma agent and has a half-life of 26 to 31 h in 6C3HED mice.' It is obtained from a nonpathogenic organism and is relatively easy to purify to homogeneity on a large scale. High concentrations of the hydrolysis products of L-asparagine are not inhibitory. The enzyme hydrolyzes L-glutamine at a nearly undetectable rate, and should, therefore, overcome glutaminase-associated toxicity problems. These characteristics make the L-asparaginase from V. succinogenes an ideal candidate for thorough clinical evaluation.