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Hydrolytic Cleavage of Methotrexate -Polyglutamates by Folylpolyglutamyl Hydrolase Derived from Various Tumors and Normal Tissues of the Mouse¹

Memorial Sloan-Kettering Cancer Center and Sloan-Kettering Division, Graduate School of Medical Sciences, New York, New York 10021 [L. L. S., L. J. G., F. M. S.]; Department of Biochemistry, Medical University of South Carolina, Charleston, South Carolina 29425 [D. G. P.]; and Organic Chemistry Research, Southern Research Institute, Birmingham, Alabama 35255 [J. R. P.]

Folylpolyglutamate hydrolase (folyl hydrolase) activity derived from murine tumors and various normal tissues was measured by means of high performance liquid chromatography using methotrexate polyglutamates as substrate. Enzyme-mediated hydrolysis was considerably greater (10–20-fold) on a specific activity basis in extracts from all normal mouse tissues (kidney > bone marrow > small intestine liver) than from tumor cells (Sarcoma 180 > Ehrlich L1210 cells). Enzyme preparations from purified absorptive and crypt cell epithelium from mouse small intestine exhibited comparable levels of specific activity and were greater than that derived from the total organ. Folyl hydrolase from mouse kidney showed mixed endo- and exopeptidase activity while that derived from all other normal tissues and tumor cells was consistent with endopeptidase activity. Levels of cell-free folyl hydrolase activity derived from tumor cells varied substantially with the phase of growth in vivo. Also, levels were appreciably lower from the same cells grown in vitro. Hydrolysis by crude or partially purified enzyme preparations from mouse small intestine or tumor cells conformed to Michaelis-Menten kinetics (single saturable component). Rates of hydrolysis and K_m values were proportional to -glutamyl chain length in the case of L1210 cell-derived enzyme but not for enzyme derived from small intestine. K_m values derived for 4-amino-10-methylpteroyldiglutamate were the same [K_m = 80.4 ± 9 (SE) µM] for small intestine and L1210 cells. However, with 4-amino-10-methylpteroyltetraglutamate K_m values were 3-fold lower for tumor cell preparations and 8-fold lower for preparations derived from small intestine. Fourfold lower K_m values for 4-amino-10-methylpteroyldiglutamate were obtained with enzyme derived from Sarcoma 180 cells as compared to the enzyme from L1210 or intestinal cells. Varying levels of folyl hydrolase activity for methotrexate polyglutamates in cell-free preparations from different tumor cells appeared to reflect differences in in situ hydrolytic activity shown for the same substrate when internalized. The relevance of these results to antifolate pharmacology and, specifically, to a role for polyglutamates of 4-aminofolate compounds in determining cytotoxicity and selective antitumor activity of these agents is discussed.

¹ Supported in part by Grants CA 08748, CA 18856, CA 22764, and CA 25236 from the National Cancer Institute, USPHS, and the Elsa U. Pardee Foundation.

² To whom requests for reprints should be addressed, at Laboratory for Molecular Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.

Received 9/ 9/85. Revised 1/14/86. Accepted 1/20/86.

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