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

• Published in: The Journal of biological chemistry 1971-02, Vol.246 (3), p.660-669
• Main Authors: Hayman, Selma, Patterson, Elizabeth K.
• Format: Article
• Language: English
• Subjects: Alanine; Animals; Carcinoma, Ehrlich Tumor - enzymology; Chromatography, DEAE-Cellulose; Cobalt; Dipeptidases - analysis; Dipeptidases - antagonists & inhibitors; Dipeptidases - isolation & purification; Dipeptides
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)62465-2

A dipeptidase that hydrolyzes l-Ala-Gly and a wide spectrum of other l-α-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-α-dipeptides with a free amino and carboxyl group. Kinetic studies of 15 dipeptides have shown the Km values to vary between 0.4 and 22 mm and the relative Vmax values (Ala-Gly standard, Vmax = 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 Km was low. Peptides with small NH2- 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.