Affinity purification of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase.
Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase (Protein methylase III, Rubisco LSMT, EC 2.1.1.43) catalyzes methylation of the epsilon-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 approximately 60 kDa.
Department of Horticulture and Landscape Architecture, University of Kentucky, Lexington 40546-0091, USA.
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Houtz
D002846:Chromatography, Affinity; D004800:Enzymes, Immobilized; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008024:Ligands; D008567:Membranes, Artificial; D008745:Methylation; D008970:Molecular Weight; D018532:Pisum sativum / Q000201:enzymology; D011145:Polyvinyls; D012436:S-Adenosylmethionine; D018724:Spinacia oleracea / Q000201:enzymology; D013379:Substrate Specificity; D005465:Fluorocarbon Polymers
D004800:Enzymes, Immobilized; D011495:Histone-Lysine N-Methyltransferase; D008024:Ligands; D008567:Membranes, Artificial; D011145:Polyvinyls; D012436:S-Adenosylmethionine; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C097344:ribulose-1,5-bisphosphate carboxylase-oxygenase large subunit epsilonN-methyltransferase