Further Studies on the Quaternary Structure of d-Ribulose-1,5-bisphosphate Carboxylase from Alcaligenes eutrophus

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Abstract
Homogeneous D-ribulose-1,5-bisphosphate carboxylase [EC 4.1.1.39], isolated from the hydrogen bacterium A. eutrophus, was studied by analytical ultracentrifugation. Sedimentation equilibrium experiments showed the enzyme to have a MW .**GRAPHIC**. [c = protein concentration and r = distance of sample from axis of rotation] = 534,000. The sedimentation coefficient was .**GRAPHIC**. = 14.1 S. The 2 types of subunits constituting the ribulosebisphosphate carboxylase were separated by gel filtration in the presence of sodium dodecyl sulfate and the amino acid compositions of the isolated large and small subunits were determined. Rabbit antibodies were developed against the ribulosebisphosphate carboxylase and its isolated subunits. The specific reactivity of the respective antibodies with their homologous antigens was proven by double immunodiffusion and quantitative immunoprecipitation analyses. Antibodies elicited against the whole enzyme also reacted with both the isolated large and small subunits as did the subunit-specific antibodies with the whole enzyme. There was no immunological correspondence between the large and the small subunits. The specific inhibition of the enzyme activity by antibodies directed against sites on the large subunit suggests that the catalytic function resides in the large subunit. EM examination of antibody .cntdot. carboxylase complexes formed upon mixing of the specific immunoglobulin G with the enzyme was used to verify the arrangement of the large and small subunits in the recently proposed structural model of the enzyme molecule. Apparently, the large subunits are located in the central 2 layers of the 4-layered enzyme molecule, whereas the 2 outer layers consist of small subunits. The observations are discussed with respect to an alternative model for the quaternary structure of ribulosebisphosphate carboxylase from tobacco.