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

https://doi.org/10.1006/prep.1995.1070Get rights and content

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit ϵN-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.

References (0)

Cited by (23)

  • Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants

    2016, Molecular Plant
    Citation Excerpt :

    One member of this subclass is located in chloroplasts, and the enzyme from pea (Pisum sativum) was originally shown to trimethylate Lys14 of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme that catalyzes the CO2 fixation step during photosynthesis. The enzyme was named PsLSMT for Pisum sativum Large Subunit Rubisco Methyltransferase (Wang et al., 1995). PsLSMT was extensively characterized at the biochemical and structural levels (for reviews, see Dirk et al., 2006; Houtz et al., 2008).

  • Neuroprotective effects of hypothermia on synaptic actin cytoskeletal changes induced by perinatal asphyxia

    2014, Brain Research
    Citation Excerpt :

    Protein was quantified by Bradford’s method (Bradford 1976) [12] using bovine serum albumin as standard. Western blot analysis was carried out using PSD fractions separated on 10% SDS-PAGE and the transfer of separated proteins to polyvinylidene difluoride (PVDF) membrane were performed as described previously (Wang et al., 1995; Dunah et al., 1996; Luo et al., 1996). Samples containing 20 μg of protein from each groups were applied to each lane.

  • Polypeptide substrate specificity of PsLSMT: A set domain protein methyltransferase

    2007, Journal of Biological Chemistry
    Citation Excerpt :

    At least one study, capitalizing on high resolution ternary complexes formed between SET 7/9 and polypeptide substrates, used a consensus recognition sequence to predict other potential polypeptide substrates for SET7/9 and verified the substrate in vitro (18). Although LSMT has provided important structural and molecular information regarding the SET domain and the catalytic mechanism for protein lysyl methylation (20, 21, 35, 43), ternary complexes between LSMT and polypeptide substrates have not proven feasible as with other SET domain PKMTs. Small synthetic polypeptides do not bind with appreciable affinity to LSMT, and co-crystallization with Rubisco has been unsuccessful.

View all citing articles on Scopus
View full text