Elucidation of an A and L system for amino acid transport in the human lymphoblast using a membrane filtration technique.
Optimum conditions have been established for the measurement of amino acid transport by human lymphoblastoid cell lines using a membrane-filtration technique. The parameters we found to be important for the reproducibility of the method are: the types and combination of filters, the strength of the vacuum applied to the filters and the density of the cultures at the time of harvesting and during uptake and filtration. We found that bovine serum albumin added to phosphate buffered saline (PBS) glucose in which the cells are washed, resuspended and assayed is essential for the maintenance of viability, the prevention of clumping and the retention of the accumulated amino acid. Using this procedure we have characterized two transport systems for the neutral amino acids; an A and an L system, which are similar but not identical to the A and L systems characterized in rodent cell lines. These A and L systems have characteristically lower Km's and Vm's for alanine and phenylalanine, when compared to rodent cell lines. In addition, we find alpha-AIB to be a poor competitor of alanine and phenylalanine uptake.
D000409:Alanine / Q000378:metabolism*; D000596:Amino Acids / Q000494:pharmacology; D000621:Aminoisobutyric Acids / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D003584:Cytological Techniques; D005374:Filtration; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism*; D012710:Serum Albumin, Bovine
D000596:Amino Acids; D000621:Aminoisobutyric Acids; D012710:Serum Albumin, Bovine; D010649:Phenylalanine; D003513:Cycloheximide; D000409:Alanine