Facilitated transport of methotrexate polyglutamates into lysosomes derived from S180 cells. Further characterization and evidence for a simple mobile carrier system with broad specificity for homo- or heteropeptides bearing a C-terminal glutamyl moiety.
Studies are presented further characterizing a facilitative system transporting methotrexate (MTX) polyglutamates into lysosomes derived from S180 cells. Initial influx of [3H]MTX + G1 (MTX with 1 additional glutamyl residue) exhibited a slightly alkaline pH optimum (pH 7.7) and was moderately temperature-dependent (Q10 27-37 degrees C = 3.1 +/- 0.1). An analysis of the kinetics of intralysosomal accumulation of [3H]MTX + G1 showed saturation kinetics for initial influx, but linear kinetics for the steady-state level of exchangeable [3H]MTX + G1 at different external concentrations of [3H]MTX + G1. In addition, the system exhibited substantial directional asymmetry with respect to the interaction with MTX + G1 during influx and efflux. Accelerated homo- and heteroexchange diffusion was demonstrated for influx of [3H]MTX + G1, while decelerated homoexchange diffusion was demonstrated for efflux of [3H]MTX + G1 following trans-positioning of MTX + G1 or glutamyl-gamma-glutamate in the opposite compartment. These observations were consistent with a single mobile carrier system mediating influx and efflux of this polyglutamate. Based upon an analysis of competitive interactions with [3H] MTX + G1, this system displayed specificity for MTX-gamma-glutamates, folyl-gamma-polyglutamates, alpha- or gamma-glutamyl peptides and heteropeptides bearing a C-terminal gamma-glutamate but not for MTX or glutamate, themselves. Among polyglutamates, gamma-glutamyl chain length was not a significant factor for transport except in the case of MTX polyglutamates. Overall, our results appear to delineate in the lysosomal membrane a simple mobile carrier system with broad specificity for folyl- or non-folyl-bearing peptides responsible for the transport of MTX polyglutamates.
Barrueco
J R
JR
Laboratory for Molecular Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
O'Leary
D F
DF
Sirotnak
F M
FM
D000818:Animals; D001667:Binding, Competitive; D001693:Biological Transport, Active; D005971:Glutamates / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism*; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D012510:Sarcoma 180; D013696:Temperature
D005971:Glutamates; D005973:Glutamine; D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D008727:Methotrexate