Choi Kyung Min, Shin Kyung Su, Yun Soon Kyu, Ki Mi Ran, Hwang Se Young
Department of Bioinformatics and Graduate School of Biotechnology, Korea University, Chochiwon, Chungnam 339-800, Republic of Korea.
Anal Biochem. 2007 Aug 15;367(2):167-72. doi: 10.1016/j.ab.2007.05.021. Epub 2007 May 26.
A spectrophotometric assay to determine peptide transport has been developed. Using two chromogenic peptide mimetics, L-phenylalanyl-L-2-sulfanilylglycine (PSG) and L-phenylalanyl-L-3-thiaphenylalanine (PSP), the peptide transport patterns in individual cell species can be evaluated effectively. After the addition of PSG to a HeLa cell suspension, sulfanilic acid accumulated progressively inside, but not outside, the cells, demonstrating that PSG was transported wholly intact. The addition of PSP to the same cell suspension was followed immediately by extracellular thiophenol production. Measurement of the rate of thiophenol release thereby provided direct determination of PSP transport. The thiophenol release was consistent with Michaelis-Menten kinetics, with a K(m) of 0.016 mM and a V(max) of 5.07 nmol/min (1 x 10(6) cells/ml, pH 7.4, 37 degrees C). The resulting kinetic constants estimated were in agreement with values determined by single-substrate enzyme kinetics. Using PSP, transport kinetics of various dipeptides was examined by competitive spectrophotometry. As a result, dipeptides tested could be ranked in order of kinetic power for their transport.
