Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: Involvement of a membrane receptor and a cytosolic protein

Journal Article · Tue Oct 04 00:00:00 EDT 1988 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00420a040· OSTI ID:7004412

Price, E M ^[1]; Ratnam, M; Rodeman, K M; Freisheim, J H ^[2]

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1. Univ. of Cincinnati College of Medicine, OH (USA)
2. Medical College of Ohio, Toledo (USA)

A radioiodinated photoaffinity analogue of methotrexate, N{sup {alpha}}-(4-amino-4-deoxy-10-methyl-pteroyl)-N{sup {epsilon}}-(4-azidosalicylyl)-L-lysine (APA-ASA-Lys), was recently used to identify the plasma membrane derived binding protein involved in the transport of this folate antagonist into murine L1210 cells. The labeled protein has an apparent molecular weight of 46K-48K when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no such labeling occurs in a methotrexate transport-defective cell line (L1210/R81). Labeling of the total cytosolic protein from disrupted cells, followed by electrophoresis and autoradiography, showed, among other proteins, a 21K band, corresponding to dihydrofolate reductase (DHFR), in both the parent and R81 cells and a 38K band only in the parent cells. However, when whole cells were UV irradiated at various times at 37{degree}C following addition of radiolabeled APA-ASA-Lys, the 38K protein and DHFR were the only cytosolic proteins labeled in the parent cells, while the intact R81 cells showed no labeled cytosolic protein, since the photoprobe is not transported. Further, when the parent cells were treated with a pulse of radiolabeled photoprobe, followed by UV irradiation at different times at 37{degree}C, the probe appeared sequentially on the 48K membrane protein and both the 38K cytosolic protein and dihydrofolate reductase. A 48K protein could be detected in both parent L1210 cells and the R81 cells on Western blots using antisera to a membrane folate binding protein from human placenta. These results suggest a vectorial transport of APA-ASA-Lys or methotrexate and reduced folate coenzymes into murine L1210 cells mediated by a 48K integral membrane protein and a 38K cytosolic or peripheral membrane protein. The 38K protein may help in the trafficking of reduced folate coenzymes, shuttling them to various cytosolic targets.

• OSTI ID: 7004412
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 27:20; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601 -- Medicine-- Unsealed Radionuclides in Diagnostics
560300* -- Chemicals Metabolism & Toxicology
62 RADIOLOGY AND NUCLEAR MEDICINE
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ANIMAL CELLS
ANIMALS
ANTIMETABOLITES
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
CHEMOTHERAPY
CYTOPLASM
DAYS LIVING RADIOISOTOPES
DISEASES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ENZYMES
HALOGENATION
HEMIC DISEASES
IMMUNE SYSTEM DISEASES
INTERMEDIATE MASS NUCLEI
IODINATION
IODINE 125
IODINE ISOTOPES
ISOTOPES
LABELLED COMPOUNDS
LEUKEMIA
MAMMALS
MEMBRANE PROTEINS
MEMBRANE TRANSPORT
MEMBRANES
METHOTREXATE
MICE
NEOPLASMS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PROTEINS
RADIOISOTOPES
RECEPTORS
RODENTS
THERAPY
TUMOR CELLS
VERTEBRATES