Formation of Disulphide Cross-Linked Aggregates of Large Subunit from Higher Plant Ribulose-1, 5-Bisphosphate Carboxylase-Oxygenase

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Abstract
The properties of the large and small subunit polypeptides of purified wheat ribulose-1, 5-bisphosphate carboxylase-oxygenase (Rubisco, E.C. 4.1.1.39) were studied. The protein was dissociated into subunits by extreme pH or detergent treatment. The separated subunits were unable to reassemble into functional holoprotein when the starting conditions were restored. Some of the separated small subunit polypeptides retained their ability to form functional heterologous Rubisco when mixed with large subunits from a cyanobacterial Rubisco. The separated large subunits of wheat Rubisco formed non-functional, high-molecular-weight aggregates. Treatment with both sodium dodecyl sulphate and thiol reductant was necessary to disrupt the aggregated structures, which indicates that the large subunits had been cross-linked by disulphide bridges. Since added thiol reductant did not prevent aggregation of the separated subunits during attempted reconstitution, oxidation of the sulphydryl groups apparently took place on contact faces sheltered by the secondary and tertiary structures of the polypeptides. High concentration of large subunits or freezing and thawing of the solution stimulated the formation of disulphide cross-links between the large subunits. The presence of small subunits did not prevent aggregation of large subunits. The results suggest that large subunits have a tendency to cross-link with disulphide bridges thus preventing proper assembly with small subunits.

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