Glutamine transport by vesicles isolated from tumour-cell mitochondrial inner membrane.
Mitochondrial-inner-membrane vesicles, isolated from Ehrlich ascites carcinoma cells by titration with detergents, accumulated L-glutamine by a very efficient transport system. The vesicles lack any phosphate-activated glutaminase activity, allowing measurement of transport rates without interference by L-glutamine metabolism. The time course of the transport was linear for the first 60 s, reaching a steady state after 120 min. L-Glutamine transport showed co-operativity, with a Hill coefficient of 2.2; the kinetic parameters S0.5 and Vmax had values of 5 mM and 26 nmol/30 s per mg of protein respectively. The pH-dependence curve showed a bell shape, with a pH optimum about 8.0. The uptake of L-glutamine was not affected by the presence of a 50-fold molar excess of D-glutamine, L-cysteine, L-histidine, L-alanine, L-serine and L-leucine, whereas L-glutamate behaved as a poor inhibitor. The structural analogue L-glutamate gamma-hydroxamate (5mM) inhibited the net uptake by 68%; interestingly, other analogues (6-diazo-5-oxo-L-norleucine, acivicin and L-glutamate gamma-hydrazide) were ineffective. The impermeant thiol reagent p-chloromercuriphenylsulphonic acid (0.5mM) completely abolished the mitochondrial L-glutamine uptake; in contrast, other thiol reagents (mersalyl and N-ethylmaleimide) did not significantly affect the transport. These data confirm the existence of a specific transport system with high capacity for L-glutamine in the mitochondrial inner membrane, a step preceding the highly operative glutaminolysis in tumour cells.
Molina
M
M
Departament de Bioquímica y Biología Moleuclar. Facultad de Ciencias, Universidad de Málaga, Spain.
Segura
J A
JA
Aledo
J C
JC
Medina
M A
MA
Núnez de Castro
I
I
Márquez
J
J
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000596:Amino Acids; D013439:Sulfhydryl Reagents; D005973:Glutamine