Molecular size of a Na(+)-dependent amino acid transporter in Ehrlich ascites cell plasma membranes estimated by radiation inactivation.
Radiation inactivation was used to estimate the molecular size of a Na(+)-dependent amino acid transport system in Ehrlich ascites cell plasma membrane vesicles. Na(+)-dependent alpha-aminoisobutyric acid uptake was measured after membranes were irradiated at -78.5 degrees C in a cryoprotective medium. Twenty-five percent of the transport activity was lost at low radiation doses (less than 0.5 Mrad), suggesting the presence of a high molecular weight transport complex. The remaining activity (approximately 75% of total) decreased exponentially with increasing radiation dose, and a molecular size of 347 kDa was calculated for the latter carrier system. Vesicle permeability and intravesicular volume were measured to verify that losses in transport activity were due to a direct effect of radiation on the transporter and not through indirect effects on the structural integrity of membrane vesicles. Radiation doses 2-3-fold higher than those required to inactivate amino acid transport were needed to cause significant volume changes (greater than 15%). Vesicle permeability was unchanged by the irradiation. The structural integrity of plasma membrane vesicles was therefore maintained at radiation doses where there was a dramatic decrease in amino acid transport. The relationship between the fragmentation of a 120-130-kDa peptide, a putative component of the Na(+)-dependent amino acid carrier [McCormick, J. I., & Johnstone, R. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7877-7881], and loss of transport activity in irradiated membranes was also examined. Peptide loss was quantitated by Western blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)
McCormick
J I
JI
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Jetté
M
M
Potier
M
M
Béliveau
R
R
Johnstone
R M
RM
D026905:Amino Acid Transport Systems; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002286:Carcinoma, Ehrlich Tumor / Q000188:drug therapy / Q000378:metabolism* / Q000532:radiotherapy; D002352:Carrier Proteins / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000528:radiation effects; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D011829:Radiation Dosage; D012964:Sodium / Q000494:pharmacology*; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism; D014407:Tumor Cells, Cultured
D026905:Amino Acid Transport Systems; D000596:Amino Acids; D002352:Carrier Proteins; D013868:Thioinosine; D012964:Sodium; C001789:4-nitrobenzylthioinosine