Subunit Interactions of Glucose-6-Phosphate Dehydrogenase from Human Erythrocytes

Abstract

Glucose-6-phosphate dehydrogenase from human erythrocytes shows successive aggregationdissociation equilibria. The effects of salt concentration and pH show that a molecule of 210,000 molecular weight and ~2 0 ,~ of 9.0 S dissociated to a half-molecule of 105,000 molecular weight and S Z O ,~ of 5.6 S. Evidence for this being a discrete dissociation was provided by molecular weight determinations, relation between sedimentation coefficients and molecular weights, and the occurrence of skew patterns a t a particular degree of dissociation.Some aggregation of the molecule to give apparent molecular weights above 210,000 occurred a t low s d t concentrations in the pH range 6-7.Reaction with maleic anhydride gave a species of molecular weight 53,000 and ~2 0 ,~ S,indicating that in the native protein the predominant molecular species (210,000) was a tetramer.The nature of the dissociations indicate the dissimilarities between the subunit contacts in the tetrameric molecule. Optimum conditions for the enzyme assay suggest that the dimer contains the essential requirements for the catalytic function of the protein, and the implications of these findings are discussed.

Biochemical mechanisms of glucose-6-phosphate dehydrogenase deficiency.

“…The content of G6PD CRM was lower than estimated by the double-antibody radiommunoassay (8), probably because of the higher background of that procedure with respect to the solid-phase radioimmunoassay. The resulting specific activity in the eight normal subjects reveals a decided similarity to values observed for homogeneous preparations of the B-type enzyme (11,(23)(24)(25)(26).…”

Human 6-Phosphogluconate Dehydrogenase. Purification of the Erythrocyte Enzyme and the Influence of Ions and NADPH on its Activity

“…Further purification of glucose-6-phosphate dehydrogenase was achieved using DEAE-Sephadex a t pH 6.5 and CM-Sephadex a t pH 5.8 in phosphate, with the buffer conditions as previously described [ 5 ] . These steps were carried out in a single day using 12.5-cm Buchner funnels for the separation.…”

“…The procedure is described according to the steps involved with reference to 6-phosphogluconate dehydrogenase. Glucose-6-phosphate dehydrogenase was separated from 6-phosphogluconate dehydrogenase in step 4 and purified further according to the procedure of Cohen and Rosemeyer [5].…”

“…I n the present communication details of a bulk separation procedure are given for a 5000-fold purification yielding 50 mg of enzyme with a specific activity of 10 unitslmg, starting from 70 pints of human blood. The method allowed the concurrent purification of glucose-6-phosphate dehydrogenase, the procedure for which had previously been developed in this laboratory [5].…”

“…Scheme for purification of 6-phosphoglumte dehydrogenase. The precipitate marked * was used for the purification of glucose-6-phosphate dehydrogenase using the procedure previously reported [5] Eur. J.…”