Hydrolytic cleavage of methotrexate gamma-polyglutamates by folylpolyglutamyl hydrolase derived from various tumors and normal tissues of the mouse.
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 greater than bone marrow greater than small intestine approximately equal to liver) than from tumor cells (Sarcoma 180 greater than Ehrlich approximately equal to 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 Km values were proportional to gamma-glutamyl chain length in the case of L1210 cell-derived enzyme but not for enzyme derived from small intestine. Km values derived for 4-amino-10-methylpteroyldiglutamate were the same [Km = 80.4 +/- 9 (SE) microM] for small intestine and L1210 cells. However, with 4-amino-10-methylpteroyltetraglutamate Km values were 3-fold lower for tumor cell preparations and 8-fold lower for preparations derived from small intestine. Fourfold lower Km 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.
L L
LL
Samuels
L J
LJ
Goutas
D G
DG
Priest
J R
JR
Piper
F M
FM
Sirotnak
D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism*; D007421:Intestine, Small / Q000201:enzymology; D007700:Kinetics; D007939:Leukemia L1210 / Q000201:enzymology; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism; D051379:Mice; D010455:Peptides / Q000378:metabolism*; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D012510:Sarcoma 180 / Q000201:enzymology; D013379:Substrate Specificity; D014018:Tissue Distribution; D011623:gamma-Glutamyl Hydrolase / Q000378:metabolism*
D010455:Peptides; D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D002268:Carboxypeptidases; D011623:gamma-Glutamyl Hydrolase; D008727:Methotrexate