Open AccessAn Immunological Investigation of the Structure and Function of Ribulose 1,5‐Bisphosphate Carboxylase
Author(s) -
Gray John C.,
Kekwick Roy G. O.
Publication year - 1974
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1974.tb03506.x
Subject(s) - phaseolus , ribulose 1,5 bisphosphate , spinach , isoelectric focusing , biochemistry , chemistry , gel electrophoresis , rubisco , pyruvate carboxylase , polyacrylamide gel electrophoresis , enzyme , size exclusion chromatography , protein subunit , ribulose , isoelectric point , precipitin , chromatography , ultracentrifuge , antiserum , microbiology and biotechnology , biology , antibody , botany , gene , immunology
1 Ribulose bisphosphate carboxylase has been purified to homogeneity, as judged by poly‐acrylamide‐gel electrophoresis, isoelectric focusing and analytical ultracentrifugation, from the leaves of the French bean, Phaseolus vulgaris. The enzyme, of specific activity 1.52 units/mg protein, had a sedimentation coefficient of 17.9 S at 10 mg protein/ml. 2 The subunits of the enzyme were separated by gel filtration in the presence of sodium dodecylsulphate and the molecular weights of the isolated subunits were estimated to be 55000 and 15300 by sodium dodecylsulphate‐polyacrylamide gel electrophoresis. 3 Antibodies to the enzyme and its subunits were raised in rabbits and the reactions of the antisera were examined by gel diffusion and by quantitative precipitin analysis. Ribulose bis‐phosphate carboxylase activity was inhibited by the anti‐enzyme serum and by the anti‐large‐subunit serum, but not by the anti‐small‐subunit serum or by a control serum, suggesting that the large subunit of the enzyme contains the catalytic site. 4 Gel diffusion of the antisera against preparations of the enzyme and small subunits from bean and spinach‐beet, Beta vulgaris , revealed that the partial immunological identity of the bean and spinach‐beet enzymes was located in the large subunits.