P. Wang et al., AFFINITY PURIFICATION OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE LARGE SUBUNIT N-EPSILON-METHYLTRANSFERASE/, Protein expression and purification, 6(4), 1995, pp. 528-536
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.