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 Journal Article
| Authors: | Barrueco, J. R.; O'Leary, D. F.; Sirotnak, F. M. |
| Article 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 μM 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. |
| Keywords: | controlled study; methotrexate; animal; mice; models, biological; ph; enzyme activation; kinetics; enzyme kinetics; membrane transport; hydrolysis; biological transport; oxidation-reduction; cysteine; membrane permeability; hydrogen-ion concentration; lysosome membrane; lysosomes; cell strain; turnover time; lysosome enzyme; gamma-glutamyl hydrolase; thiol derivative; polyglutamic acid; hydrolase; beta-n-acetylhexosaminidase; sulfhydryl compounds; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.; mercaptoethanol; folylpolyglutamate synthase; sarcoma 180; methotrexate polyglutamate; triton x 100 |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 267 |
| Issue: | 22 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 1992-08-05 |
| Start Page: | 15356 |
| End Page: | 15361 |
| Language: | English |
| PUBMED: | 1379226 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Source: Scopus |
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