Kinetics of uptake, transport, and accumulation of liposome-associated fluorescent dolichol esters in human fibroblasts.
The anthroyl and n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino (NBD) hexanoyl esters of dolichol were synthesized and incorporated into phospholipid liposomes. Fluorescence spectrometric methods were used to estimate the kinetics and dynamics of the incorporation and turnover of these esters in normal human fibroblasts. For anthroyl dolichol a saturable uptake of 2.5 x 10(3) pmol/10(6) fibroblasts was obtained. Half-maximum uptake was seen at a labeling concentration of 19 microM. The time required for half-maximum uptake of fluorescence (t1/2) was about 10 h. Over 50% of the anthroyl dolichol taken up remained in fibroblasts 24 h after the labeling medium was removed. Uptake was higher for esters of 9 isoprenes than for those with 16-21. Dolichol labeled with the NBD fluorophore appeared to enter fibroblasts in higher concentration than the same dolichol labeled with anthracene. Uptake was not influenced by the presence of agents that disrupt lysosome function (leupeptin and chloroquine) prior to or during fluorescence labeling. The amount of fluorescent dolichol in the (i) lysosomes and endosomes and (ii) nuclei of labeled fibroblasts was determined after Percoll density gradient centrifugation and cell lysis in culture, respectively. Most of the fluorescent dolichyl ester (and most of the free alcohol form) taken up by fibroblasts was recovered in lysosomes.
Jiang
L
L
Department of Biochemistry, University of Western Ontario, London, Canada.
Rip
J W
JW
D001692:Biological Transport; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004286:Dolichols / Q000737:chemistry / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D004952:Esters / Q000138:chemical synthesis / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
D004286:Dolichols; D004952:Esters; D005456:Fluorescent Dyes; D008081:Liposomes; D010743:Phospholipids