Reconstitution of lysosomal sulfate transport in proteoliposomes.
As part of a strategy to purify the lysosomal sulfate transporter, we developed a method for reconstitution of transport in artificial membrane vesicles. Lysosomal membranes were prepared from Percoll density gradient purified rat liver lysosomes and membrane proteins were solubilized using the non-ionic detergent, Triton X-100. The solubilized proteins were mixed with liposomes prepared by sonication of egg yolk lecithin and the detergent was removed by passage of the mixture over Bio-beads XAD2. The resulting proteoliposomes exhibited saturable sulfate transport with characteristics that were very similar to those observed in lysosomal membranes. Transport in proteoliposomes had a Km of 155 microM, exhibited pH dependence and was sensitive to inhibition by DIDS. Reconstitution of transport in proteoliposomes may be useful as an assay for purification of the lysosomal sulfate carrier.
Koetters
P J
PJ
Department of Pediatrics, Harbor-UCLA Medical Center, Torrance 90502, USA.
Chou
H F
HF
Jonas
A J
AJ
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002499:Centrifugation, Density Gradient; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D008099:Liver / Q000648:ultrastructure; D008247:Lysosomes / Q000378:metabolism*; D017830:Octoxynol; D051381:Rats; D017207:Rats, Sprague-Dawley; D013431:Sulfates / Q000378:metabolism*
D008081:Liposomes; D013431:Sulfates; D017830:Octoxynol; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid