AFFINITY PURIFICATION OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE LARGE SUBUNIT N-EPSILON-METHYLTRANSFERASE/

Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-epsilo n-methyltransferase (Protein methylase III, Rubisco LSMT, EC 2.1.1.43) catalyzes methylation of the epsilon-amino group of Lys-14 in the lar ge subunit of Rubisco. In this paper, an affinity purification procedu re for pea (Pisum sativum L. cv Laxton's Progress No. 9) Rubisco LSMT is described and characterized. Spinach (Spinacia oleracea L. cv Melod y) Rubisco, a substrate for pea Rubisco LSMT, was immobilized to polyv inylidene 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 e luted by 1 M KCl but was specifically released by S-adenosyl-L-methion ine (AdoMet) or spinach Rubisco. Elution of Rubisco LSMT by AdoMet was a result of catalytic methylation of the PVDF-immobilized spinach Rub isco, and was therefore more efficient than elution by the competitive Ligand spinach Rubisco, An increase in the specific activity of Rubis co LSMT of approximately 7000-fold was achieved in one step with this affinity purification technique. Rubisco LSMT is a monomeric protein w ith a molecular mass of similar to 60 kDa. (C) 1995 Academic Press, In c.