title: Metabolic turnover of methotrexate polyglutamates in lysosomes derived from S180 cells. Definition of a two-step process limited by mediated lysosomal permeation of polyglutamates and activating reduced sulfhydryl compounds.
abstract: Transport and metabolic turnover of methotrexate (MTX) polyglutamates were examined in lysosomes derived from S180 cells. These studies extend prior work from this laboratory (Barrueco, J. R., and Sirotnak, F. M. (1991) J. Biol. Chem 266, 11732-11737) which described basic properties of a facilitative transport system in lysosomes capable of mediating intralysosomal accumulation of MTX polyglutamates. In the present report, we show that the rate of turnover of MTX polyglutamates in lysosomes, which releases MTX in the extralysosomal space, is limited by the extent of mediated intralysosomal accumulation of the polyglutamate and reduced sulfhydryls that activate the enzyme folylpolyglutamate hydrolase. Evidence is presented that cysteine functions as the naturally occurring reduced sulfhydryl compound in lysosomes being equipotent to 2-mercaptoethanol as an activator of folylpolyglutamate hydrolase. Folylpolyglutamate hydrolase in permeabilized lysosomes from S180 cells exhibited a low pH optimum characteristic of a lysosomal enzyme, was activated at concentrations of reduced sulfhydryl at 0.1 mM and above, and exhibited Km values in the range of 0.2-3 microM that decreased with increase in polyglutamate chain length. Values for Km for MTX polyglutamates of folylpolyglutamate hydrolase activity were 100-200-fold lower than values for Km or Ki for facilitated intralysosomal transport, whereas capacities for both processes were similar. This relationship between the kinetic properties of each process ensures efficient hydrolysis of MTX polyglutamates within the lysosome.
authors.affiliation: Laboratory for Molecular Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York.
authors.forename: J R
authors.initials: JR
authors.lastname: Barrueco
authors.forename: D F
authors.initials: DF
authors.lastname: O'Leary
authors.forename: F M
authors.initials: FM
authors.lastname: Sirotnak
mesh_terms: D000818:Animals; D001692:Biological Transport; D003545:Cysteine / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism*; D008623:Mercaptoethanol / Q000494:pharmacology; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008954:Models, Biological; D010084:Oxidation-Reduction; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D012510:Sarcoma 180 / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism; D011623:gamma-Glutamyl Hydrolase / Q000378:metabolism*
chemical_list: D013438:Sulfhydryl Compounds; D011099:Polyglutamic Acid; D008623:Mercaptoethanol; C014085:methotrexate polyglutamate; D001619:beta-N-Acetylhexosaminidases; D011623:gamma-Glutamyl Hydrolase; D003545:Cysteine; D008727:Methotrexate