Protein modification of glutamine transporters in SV40-transformed hepatocytes and immunodetection of proteins associated with hepatic system N transport activity.
The secondary active transport of glutamine into hepatocytes is mediated by the sodium-dependent System N amino acid transporter. In SV40-transformed fetal or adult hepatocytes, System N activity was increased by culturing the cells at the growth-permissive temperature of 33 degrees C compared with 40 degrees C. The sulfhydryl specific protein modification reagent p-chloromercuribenzene sulfonate (PCMBS) inactivated all of System N activity in fetal cells, whereas in the adult cells PCMBS inactivated only the elevated System N activity expressed at 33 degrees C; basal System N activity remained unaffected. Also, the inactivation of transport in the adult cells could be blocked by the presence of the substrate glutamine, whereas that in the fetal cells was completely resistant to protection. To characterize the System N transport, murine monoclonal antibodies System N transporter, murine monoclonal antibodies were generated. Two hybridomas (3E1-2 and 1E7-3) produced antibodies that inhibit System N activity in hepatocytes and can immunoprecipitate the activity from a solubilized protein fraction. Based on two-dimensional PAGE, the molecular size of the carrier protein has been shown to be approximately 100 kDa.
Tamarappoo
B K
BK
Department of Medicine, University of Florida College of Medicine, Gainesville 32610.
Singh
H P
HP
Kilberg
M S
MS
D000375:Aging / Q000378:metabolism; D026923:Amino Acid Transport Systems, Basic; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism*; D002461:Cell Line, Transformed; D004591:Electrophoresis, Polyacrylamide Gel; D005333:Fetus / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D006825:Hybridomas; D008099:Liver / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D011233:Precipitin Tests; D051381:Rats; D013539:Simian virus 40 / Q000502:physiology
D026923:Amino Acid Transport Systems, Basic; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; C077738:sodium-dependent glutamine transporter; D005973:Glutamine