A comparative study of the binding of aldolase and glyceraldehyde-3-phosphate dehydrogenase to the human erythrocyte membrane

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A high percentage (generally ~60%) of the aldolase-binding capacity of isolated human erythrocyte membranes was lost either during storage of the membranes at 4 °C in 10 m m imidazole-1 m m MgCl 2-10 m m 2-mercaptoethanol, pH 6.8, for 24–48 h, or during brief heating at 25–45 °C. In contrast, relatively little (generally ~25%) of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) binding capacity of the membranes was lost under these same conditions. Binding of both aldolase and G3PDH occurred in (at least) two kinetically distinguishable phases, with a rapidly equilibrating and a slowly equilibrating process being noted. The major portion of the aldolase binding was of a heat-labile rapidly equilibrating type, while the major portion of the G3PDH binding was of a heat-stable slowly equilibrating type. Binding of both aldolase and G3PDH was inhibited by P i in the presence of Mg 2+, with dianionic P i being the effective species. Elution of peripheral proteins, including erythrocyte actin (Band 5), did not cause a decrease in binding of either enzyme that could be attributed to the loss of peripheral proteins. Binding of G3PDH had a markedly detrimental effect on binding of aldolase, but the reverse was not true. Based on this lack of mutual competition as well as the differences in stability of the aldolase and G3PDH binding sites, it is concluded that these enzymes do not bind to a single molecular feature on the erythrocyte membrane.