Purification and characterization of the phosphate/hydroxyl ion antiport protein from rat liver mitochondria.
The phosphate transport protein was purified from rat liver mitochondria by extraction in an 8% (v/v) Triton X-100 buffer followed by adsorption chromatography on hydroxyapatite and Celite. SDS/polyacrylamide-gel electrophoresis (10%, w/v) demonstrated that the purified polypeptide was apparently homogeneous when stained with Coomassie Blue and had a subunit Mr of 34,000. However, lectin overlay analysis of this gel with 125I-labelled concanavalin A demonstrated the presence of several low- and high-Mr glycoprotein contaminants. To overcome this problem, mitochondria were pre-extracted with a 0.5% (v/v) Triton X-100 buffer as an additional step in the purification of phosphate transport protein. SDS/polyacrylamide gradient gel electrophoresis (14-20%, w/v) of the hydroxyapatite and Celite eluates revealed one major band of Mr 34,000 when stained with Coomassie Blue. The known thiol group sensitivity of the phosphate transporter was employed to characterize the isolated polypeptide further. Labelling studies with N-[2-3H]ethylmaleimide showed that only the 34,000-Mr band was labelled in both the hydroxyapatite and Celite fractions, when purified from rat liver mitochondria. Further confirmation of its identity has been provided with an antiserum directed against the 34,000-Mr protein. Specific partial inhibition of phosphate uptake, as measured by iso-osmotic swelling in the presence of (NH4)2HPO4, was achieved when mitoplasts (mitochondria minus outer membrane) were incubated with this antiserum. Finally, amino acid analysis of the rat liver mitochondrial phosphate/hydroxyl ion antiport protein indicates that it is similar in composition to the equivalent protein isolated from ox heart.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D017920:Antiporters; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003208:Concanavalin A; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D006878:Hydroxides; D017665:Hydroxyl Radical; D007106:Immune Sera; D008930:Mitochondria, Liver / Q000378:metabolism*; D028044:Phosphate-Binding Proteins; D051381:Rats; D011919:Rats, Inbred Strains
D000596:Amino Acids; D017920:Antiporters; D002352:Carrier Proteins; D006878:Hydroxides; D007106:Immune Sera; D028044:Phosphate-Binding Proteins; D003208:Concanavalin A; D017665:Hydroxyl Radical; D005033:Ethylmaleimide