Substrate protection against inactivation of the mammalian polyamine-transport system by 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide.
Mammalian polyamine transporters have not thus far been biochemically characterized. Since essential carboxy groups in the polyamine carrier might participate in the transport process, the ability of two different carbodi-imides to affect [3H]spermidine uptake was assessed in Chinese hamster ovary cells. Both the hydrophobic 1,3-dicyclohexylcarbodi-imide (DCC) and the more polar 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (EDC) irreversibly inhibited spermidine transport with EC50 values of 11 +/- 4 and 96 +/- 16 microM after 30 min at 22 degrees C respectively. Prior treatment with EDC in the absence of substrate decreased both the Vmax and K(m) for spermidine uptake in a time- and concentration-dependent manner. Spermidine-transport inactivation by EDC (1 mM) was temperature-dependent, with 60 and 90% inhibition observed after 10 min at 22 and 37 degrees C respectively. Spermine (10 microM) almost fully protected against spermidine-transport inactivation by EDC at 22 degrees C, and decreased the rate of inactivation at 37 degrees C by about 80%. Putrescine, spermidine and spermine were all effective in protecting against EDC-mediated inactivation of [3H]spermidine and [3H]putrescine uptake at 22 degrees C with EC50 values estimated at 10, 1 and less than 1 microM respectively. The nucleophile glycine ethyl ester (up to 50 mM) prevented the inhibition brought about by 1 mM EDC. Inhibition by 1 mM EDC was greater at pH 7.2 than at pH 5.8 (89 +/- 3 compared with 44 +/- 5%), whereas the converse was true for 100 microM DCC (81 +/- 3 compared with 92 +/- 5%). On the other hand, spermine did not protect against inactivation of spermidine uptake by DCC. Moreover, DCC, but not EDC, inhibited Na(+)-dependent amino acid uptake. The present data indicate that (i) EDC and DCC inhibit polyamine transport through distinct mechanisms, (ii) substrate binding occludes one or several carboxy groups lying in a polar environment of the carrier and (iii) these carboxyl residues might be activated by EDC to crosslink a neighbouring nucleophile side group, resulting in a conformation of the polyamine carrier which is inactive for transport.
Torossian
K
K
Laboratory of Molecular Endocrinology, Laval University Medical Research Center, Ste. Foy, Quebec, Canada.
Audette
M
M
Poulin
R
R
D000621:Aminoisobutyric Acids / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010053:Ovary / Q000187:drug effects / Q000378:metabolism; D011700:Putrescine / Q000378:metabolism; D013095:Spermidine / Q000378:metabolism*; D013096:Spermine / Q000378:metabolism
D000621:Aminoisobutyric Acids; D013096:Spermine; D004024:Dicyclohexylcarbodiimide; D005022:Ethyldimethylaminopropyl Carbodiimide; D013095:Spermidine; D011700:Putrescine