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Purification and Properties of a Mouse Ascites Tumor Dipeptidase, a Metalloenzyme

From the ¹ From the Institute for Cancer Research, Fox Chase, Philadelphia, Pennsylvania 19111

A dipeptidase that hydrolyzes l-Ala-Gly and a wide spectrum of other l--dipeptides has been purified 800-fold from the soluble fraction of Ehrlich-Lettré mouse actesis tumor cells. The highest specific activity (micromoles of dipeptide hydrolyzed at 40° per min per mg of protein) achieved was 2,600 with Ala-Gly, the substrate with which purification was followed. With the best substrate, Ala-Ile, this specific activity was equivalent to a molecular activity (moles of dipeptide hydrolyzed at maximum velocity at 40° per min per mole of enzyme) of 2 x 10⁶. Instability of the enzyme at this high activity prevented determination of homogeneity; a sample of molecular activity of 1 x 10⁶ (Ala-Gly specific activity, 1,360) was estimated to be 50% pure by acrylamide gel electrophoresis. Studies with metal chelators indicate that the dipeptidase is a metalloenzyme. For instance, o-phenanthroline completely inhibited the enzyme activity (50% at 0.1 mm), whereas m-phenanthroline had no effect. Although atomic absorption analyses showed a correlation of zinc content with enzyme activity in the final chromatographic step and a value of 0.9 ± 0.1 g atom of zinc per mole of enzyme of specific activity 2,600 (Ala-Gly), it has not been confirmed that the dipeptidase is a zinc metalloenzyme. The enzyme was separated from leucine aminopeptidase, prolidase, and tripeptidase by Sephadex G-150 filtration and, by comparison with standard proteins, was shown to have a molecular weight of 85,000 ± 5,000. The dipeptidase hydrolyzes only l--dipeptides with a free amino and carboxyl group. Kinetic studies of 15 dipeptides have shown the K_m values to vary between 0.4 and 22 mm and the relative V_max values (Ala-Gly standard, V_max = 1) to vary from 5.3 (Ala-Ile) to 0.01 (Gly-Gly). Inhibition by high substrate concentrations (3 to 50 mm) was observed in cases where the K_m was low. Peptides with small NH_2- terminal and bulky nonpolar COOH-terminal R groups were preferred. The hydrolyses of the relatively poor substrates, Pro-Gly and Gly-Gly, were activated by mm Mn²⁺ and Co²⁺, respectively, whereas that of Ala-Gly was inhibited by both these metals as well as by 10 other metals. No evidence was obtained that these hydrolyses were carried out by separate enzymes: the enzyme peaks were congruent, the ratios of activities were constant throughout purification, and the activities toward the three substrates decayed at the same rate on exposure to 30° for periods up to 24 hours.

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