Mild purification procedure and subunit structure of glucosephosphate isomerase from baker's yeast.

A mild procedure for the purification of glucosephosphate isomerase from baker's yeast (Saccharomyces cerevisiae) is reported. The purified enzyme was homogeneous and did not contain charge isomers as shown by polyacrylamide gel electrophoresis as well as DEAE-Sepharose column chromatography. Its molecular weight determined by gel filtration and sucrose density gradient centrifugation was approximately 120 000, which agreed with that of the enzyme in the crude extract as well as that of the renatured enzyme. Gel filtration in 6M guanidine/HCl as well as acrylamide gel electrophoresis of sodium dodecyl sulfate denatured glucosephosphate isomerase showed one single peak and gave a subunit molecular weight of 60 000. Cross-linking patterns obtained with yeast glucosephosphate isomerase after treatment with dimethyl suberimidate resulted in the appearance of dimers as the largest-linked product of the enzyme subunit. After dissociation the enzyme can readily be reassociated and renatured with a yield of maximum 73% and a pseudo first order rate constant of 0.12 min-1 at 25 degrees C.