Transport of L-asparagine in Tetrahymena pyriformis ecto-L-asparaginase is not related to L-asparagine-protein transport system.
L-Asparaginase of T. pyriformis is a membrane-bound enzyme with an active site situated on the outside surface of the membrane. When radioactive L-asparagine was incubated with T. pyriformis cells in the L-asparaginase assay medium, the hydrolysis was 240 higher than the uptake of this amino acid. In a similar experiment performed in salt medium (Wagner's solution), the hydrolysis was linearly increased and reached after one hour of incubation a value of 60 nmol/10(6) cells, while the uptake after 20 min of incubation reached a plateau with a value of 15 nmol/10(6) cells. The uptake of L-leucine under these conditions was 44 nmol/10(6) cells/hr, while no measurable transport of aspartic acid was observed. That L-aspartic acid is not migrated into T. pyriformis cells is in agreement with the finding that no efflux of this amino acid takes place as well. The uptake of L-asparagine is pH and K+ dependent, whereas Na+ ions strongly inhibit this uptake. The Km and Vmax values of L-asparagine uptake is 1.43 mM and 0.7 nmol/min, respectively. The half life of L-asparagine "protein transport system" was 40 min, a value which is very close to the half life of the membrane-bound L-asparaginase of this microorganism. Ouabain and vanadate inhibit the uptake of L-asparagine by more than 80%, while ouabain or vanadate inhibit in vivo 5% or 95% the activity of L-asparaginase, respectively. This indicates the lack of interrelationship between the L-asparagine "protein transport system" and the L-asparaginase protein molecule.
Laboratory of Biochemistry, Faculty of Chemistry, School of Sciences, Aristotelian University of Thessaloniki, Greece.
I K
IK
Tsavdaridis
D J
DJ
Triantafillou
D A
DA
Kyriakidis
D000818:Animals; D001215:Asparaginase / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D001693:Biological Transport, Active; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D013769:Tetrahymena pyriformis / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D001224:Aspartic Acid; D014638:Vanadates; D010042:Ouabain; D001216:Asparagine; D001215:Asparaginase; D011188:Potassium