Affinity Purification of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit ϵN-Methyltransferase

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit ϵ N-methyltransferase (Protein methylase III, Rubisco LSMT, EC 2.1.1.43) catalyzes methylation of the ϵ-amino group of Lys-14 in the large subunit of Rubisco. In this paper, an affinity purification procedure for pea ( Pisum sativum L. cv Laxton′s Progress No. 9) Rubisco LSMT is described and characterized. Spinach ( Spinacia oleracea L. cv Melody) Rubisco, a substrate for pea Rubisco LSMT, was immobilized to polyvinylidene fluoride (PVDF) transfer membranes (Immobilon-P) and used as a ligand for the affinity purification of Rubisco LSMT from pea leaf extracts and chloroplast lysates. Pea Rubisco LSMT specifically bound to PVDF-immobilized spinach Rubisco but not to control PVDF membranes which contained immobilized BSA or pea Rubisco. Rubisco LSMT was not eluted by 1 M KCl but was specifically released by S-adenosyl-L-methionine (AdoMet) or spinach Rubisco. Elution of Rubisco LSMT by AdoMet was a result of catalytic methylation of the PVDF-immobilized spinach Rubisco, and was therefore more efficient than elution by the competitive Ligand spinach Rubisco, An increase in the specific activity of Rubisco LSMT of approximately 7000-fold was achieved in one step with this affinity purification technique. Rubisco LSMT is a monomeric protein with a molecular mass of ∼60 kDa.