Inhibition of transport system b0,+ in blastocysts by inorganic and organic cations yields insight into the structure of its amino acid receptor site.
The most conspicuous, Na(+)-independent amino acid transport process in preimplantation mouse blastocysts was provisionally designated system b0,+ because it accepts some cationic and zwitterionic amino acids about equally well as substrates. Although system b0,+ is not Na(+)-stimulated, it has not been determined if it is inhibited by Na+, or if its activity is affected by most other ions. Therefore, we measured uptake of amino acids by blastocysts in isotonic solutions of different ionic and nonionic osmolites. Na(+)-independent L-leucine uptake was unaffected by the ion concentration, but L-lysine transport was several-fold faster in isotonic solutions of non-electrolytes than in similar solutions of inorganic and organic salts or zwitterions. The Km value for 'Na(+)-independent' L-lysine transport was about 10-fold higher in isotonic salt solutions than in solutions of nonionic osmolites, whereas the Km value for L-leucine transport was about the same in either type of solution. Therefore, inorganic and organic cations and the cationic portions of zwitterions appear to compete with cationic but not zwitterionic amino acids for system b0,+ receptor sites. The cation, harmaline, was a particularly strong competitive inhibitor of 'Na(+)-independent' L-lysine uptake by system b0,+, even in isotonic salt solutions, whereas it inhibited L-leucine uptake noncompetitively. Moreover, harmaline appeared to compete with inorganic cations for the lysine receptor sites of system b0,+. Harmaline also has been found by other investigators to competitively inhibit L-lysine uptake by the Na(+)-independent system asc1 in horse erythrocytes, whereas it noncompetitively inhibits alanine uptake by the same system. Similarly, harmaline noncompetitively inhibits L-alanine uptake by the Na(+)-dependent system ASC in human erythrocytes, but it appears to compete for binding with L-alanine's cosubstrate, Na+. In addition, others have found that the positively-charged side chains of cationic amino acids seem to take the place of Na+ needed near side chains in order for zwitterionic amino acids to be transported by systems ASC and y+. We conclude that system b0,+ may be similar to systems asc1, ASC and y+, and that each of these systems may be a variant of the same ancestral transport process. We speculate that since it appears to accept a broader scope of substrates and to interact with a wider variety of cations than do systems asc1, ASC or y+, system b0,+ may more closely resemble the proposed ancestral transport process than any of the other contemporary systems.
Van Winkle
L J
LJ
Department of Biochemistry, Chicago College of Osteopathic Medicine, Downers Grove, IL 60515.
Campione
A L
AL
Gorman
J M
JM
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001755:Blastocyst / Q000378:metabolism*; D002412:Cations; D006246:Harmaline / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D012964:Sodium / Q000502:physiology
D000596:Amino Acids; D002412:Cations; D012964:Sodium; D006246:Harmaline; D007930:Leucine; D008239:Lysine