Differential Subunit Aggregation of a Purified Protein from Cold-hardened and Unhardened Puma Rye

Abstract

Summary Purified ribulose-1,5-bisphosphate carboxylase-oxygenase from cold-hardened and unhardened Puma rye plants was osmotically concentrated by dialysis against buffer containing 25% (w/v) polyethylene-glycol for 16 h at 4°C in the presence or absence of β-mercaptoethanol. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of protein samples concentrated 100 to 200 fold in the absence of reducing agent indicated that the large subunit of the enzyme from unhardened rye plants aggregated to a greater extent than the large subunit of the enzyme from cold-hardened rye plants. This aggregation was reversed by the addition of β-mercaptoethanol to the concentrated enzyme samples implicating disulfide bond formation in the mechanism of aggregation. No structural alterations were observed by SDS polyacrylamide gel electrophoresis for the small subunits of the enzyme from either cold-hardened or unhardened rye plants. The relevance of these results to Levitt's sulfhydryl hypothesis is discussed.