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Articles

Purification and Partial Characterization of Ribulose Bisphosphate Carboxylase Holoenzyme and Its Subunits from Chlorella sorokiniana and Use of Its Antigen Affinity-Purified Antibodies in Specific Immunoprecipitation and Immunoadsorption Procedures ¹

Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611, Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061

Chlorella sorokiniana ribulose-1,5-bisphosphate carboxylase (RuBPCase) was purified to homogeneity with yields of 35 to 40%. Molecular weights of the holoenzyme and its large subunit (LS) and small subunit (SS) were estimated to be 562,000, 55,000, and 15,800, respectively. Amino acid compositions of LS from C. sorokiniana and spinach were similar, whereas the compositions of their SS were very different. Antisera prepared against holoenzyme, LS, and SS were purified by antigen-affinity column chromatography. Purified anti-holoenzyme immunoglobulin G (IgG) and anti-LS IgG cross-reacted with holoenzyme and LS but not with SS. Anti-SS IgG reacted neither with holoenzyme nor with LS. Because purified anti-holoenzyme IgG or the anti-LS IgG inhibited RuBPCase activity, antibody preparations were titered by the amount of ³⁵S-labeled RuBPCase immunoprecipitated. Approximately 40% of the total RuBPCase activity in cell homogenates was tightly particulate-bound and was solubilized with 0.5% Nonidet P-40 without inhibition of enzyme activity. Direct-immunoprecipitation and indirect-immunoadsorption procedures, with affinity-purified anti-holoenzyme IgG, gave specific and quantitative recovery of ³⁵S-labeled RuBPCase from cell extracts containing Nonidet P-40. Affinity-purified anti-LS IgG and anti-SS IgG were used to immunoprecipitate either the LS or SS antigens synthesized in vitro in a mRNA-dependent in vitro translation assay system. Rocket immunoelectrophoresis was used to quantify as little as 50 nanograms of RuBPCase antigen in cell extracts.

³ New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915.

⁴ Present address: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611.

¹ Supported by the Science and Education Administration of the United States Department of Agriculture under grant 5901-0410-8-0097-0 and 59-2121-0-1-097-0 from the Competitive Research Grants Office. Florida Agriculture Experiment Stations Journal Series, No. 6358.