title: Association of glyceraldehyde-3-phosphate dehydrogenase with the human red cell membrane. A kinetic analysis.
abstract: A rapid filtration technique was used to analyze the kinetics of the binding reaction between glyceraldehyde-3-P dehydrogenase and the band 3 protein of the human erythrocyte membrane. Saponin was used to eliminate the membrane as a rate-limiting barrier. The re-equilibration of the enzyme following dilution of membranes in buffer took only a few seconds. Dissociation rates were greatly stimulated and association rates were reduced by increasing ionic strength. A Brönsted-Bjerrum analysis suggested that two or three charges of opposite sign on each protein were involved in binding. NADH appeared to elute the enzyme by interaction with its catalytic site, but the band 3 binding site extended beyond the NADH site, permitting the formation of a ternary NADH . enzyme . membrane complex. The release of the enzyme from fresh erythrocytes immediately following saponin lysis showed kinetics similar to the release of enzyme from ghosts. The extrapolated zero time intercepts of these reactions suggested that two-thirds of cellular glyceraldehyde-3-P dehydrogenase was membrane bound prior to hemolysis. This value is similar to that calculated for the association of the enzyme with band 3 in the intact red cell when the high ionic strength and enzyme-eluting compounds in the cytoplasm are taken into account.
mesh_terms: D004910:Erythrocyte Membrane / Q000187:drug effects / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D005374:Filtration; D005634:Fructose-Bisphosphate Aldolase / Q000302:isolation & purification; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D009243:NAD / Q000494:pharmacology; D009994:Osmolar Concentration; D011485:Protein Binding / Q000187:drug effects; D012503:Saponins / Q000494:pharmacology
chemical_list: D012503:Saponins; D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005634:Fructose-Bisphosphate Aldolase