Kinetics of PAPS translocase: evidence for an antiport mechanism.
In order to gain an understanding of the mechanisms involved in the transfer of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) from the cytosol where it is synthesized to the Golgi lumen where it serves as the universal sulfate donor for sulfate ester formation in higher organisms, we have undertaken a kinetic characterization of the PAPS translocase from rat liver Golgi. Analyzing the PAS translocase activity in both intact Golgi vesicles and in a reconstituted liposome system, we have determined a number of physical and kinetic parameters. Strong competitive inhibition in zero-trans uptake experiments only with beta-methylene PAPS and adenosine 3',5'-biphosphate (PAP) suggest the transporter is highly specific for the 3'-phosphate. The demonstration of trans acceleration as observed by stimulation of transport activity under exchange conditions suggests that the translocase is a carrier with distinct binding sites accessible from both faces of the membrane. The behavior of the PAPS translocase in the presence of equilibrium concentrations of PAP supports the function of an antiport mechanism. Thus the translocase is characterized by its kinetic properties as a specific transporter of PAPS which acts through an antiport mechanism with PAP as the returning ligand. This characterization of the transport activity has proved instrumental in the identification of an approximate 230 kDa Golgi membrane protein as the PAPS translocase protein [Ozeran, J.D., Westley, J., & Schwartz, N.B. (1996) Biochemistry 35, 3695-3703 (accompanying paper)].
Ozeran
J D
JD
Department of Pediatrics, University of Chicago, Chicago, Illinois 60637, USA.
Westley
J
J
Schwartz
N B
NB
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D017920:Antiporters / Q000378:metabolism*; D001278:Atractyloside / Q000031:analogs & derivatives / Q000494:pharmacology; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology* / Q000378:metabolism; D017136:Ion Transport / Q000502:physiology*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D010171:Palmitoyl Coenzyme A / Q000494:pharmacology; D010724:Phosphoadenosine Phosphosulfate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D013431:Sulfates / Q000378:metabolism
D017920:Antiporters; D008081:Liposomes; D008565:Membrane Proteins; D009711:Nucleotides; C099313:PAPS translocase protein, rat; D013431:Sulfates; C099314:beta-methylene PAPS; D010171:Palmitoyl Coenzyme A; D001278:Atractyloside; D010724:Phosphoadenosine Phosphosulfate; D000244:Adenosine Diphosphate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; C003853:carboxyatractyloside