Transport of L-methionine in human diploid fibroblast strain WI38.
The transport of L-methionine in human diploid fibroblast strain WI38 was investigated. The uptake of L-methionine was measured in sparse cell cultures in a simple balanced salt solution buffered with either Tris.HCl of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Similar results were obtained with these two buffers. Cultures were allowed to equilibrate with the buffered saline before transport was measured. The presence of glucose in the buffered saline results in a slight reduction in the initial rate of transport for the first 2 h of equilibration in buffered saline. L-Methionine is actively transported in WI38 by saturable, chemicallly specific mechanisms which are temperature, pH and, in part Na+ dependent, and are reactive with both L- and D-stereoisomers. Kinetic analysis of initial rates of transport at substrate concentrations from 0.0005 to 100 mM indicated the presence of two saturable transport systems. System 1 has an apparent KM of 21.7 micrometer and an apparent V of 3.57 nmol/mg per min. System 2 has an apparent KM of 547 micrometer and an apparent V of 22.6 nmol/mg per min. Kinetic analysis of initial rates of transport in Na+-free media or after treatment with ouabain suggested that system 1 is Na+ independent and that system 2 is Na+ dependent. Preloading of cells with unlabeled L-methionine greatly increases the initial rate of uptake. Efflux of transported methionine is temperature dependent, and is greatly increased in the presence of unlabeled L- or D-methionine or L-phenylalanine, but not in the presence of L-arginine. L-Methionine transport is strongly inhibited by other neutral amino acids, and is very weakly inhibited by dibasic amino acids, dicarboxylic amino acids, proline or glycine.
D000596:Amino Acids / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D004171:Diploidy; D005347:Fibroblasts / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D013237:Stereoisomerism
D000596:Amino Acids; D003470:Culture Media; D010042:Ouabain; D012964:Sodium; D008715:Methionine