Use of glyceraldehyde-3-phosphate dehydrogenase-depleted human erythrocyte ghosts as specific high affinity adsorbents for the purification of glyceraldehyde-3-phosphate dehydrogenase from various tissues.
Human erythrocyte ghosts depleted of glyceraldehyde-3-phosphate dehydrogenase are used as specific high-affinity adsorbents for the purification of glyceraldehyde-3-phosphate dehydrogenase from mouse muscle, liver, kidney and brain. On incubation with the crude tissue homogenates, the depleted ghosts bind glyceraldehyde-3-phosphate dehydrogenase, aldolase, and a few other proteins. Washing the incubated ghosts several times with 5 mM phosphate buffer(pH 8.0) removed several of the non specifically bound proteins. Aldolase can be eliminated from the membrane by incubating the ghosts for 30 min in 5 mM phosphate buffer (pH 8.0)/2mM fructose 1,6-biphosphate, and then washing with the same solution. Glyceraldehyde-3-phosphate dehydrogenase can then be specifically eluted from the ghosts by incubating them with 2 mM NADH in 5mM phosphate buffer (pH 8.0). Although the enzyme from brain appears to bind less strongly to the ghosts it was possible, using this procedure, to purify glyceraldehyde-3-phosphate dehydrogenase from all the tissues investigated. The purified enzyme exhibits high specific activity and migrates as a single band (during SDS polyacrylamide gel electrophoresis) which corresponds to a protomer molecular weight of 37 000.
D000818:Animals; D001921:Brain / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods; D004910:Erythrocyte Membrane / Q000201:enzymology; D004912:Erythrocytes / Q000201:enzymology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D009132:Muscles / Q000201:enzymology
D005987:Glyceraldehyde-3-Phosphate Dehydrogenases