Plant and Cell Physiology Advance Access published online on September 27, 2005
Plant and Cell Physiology, doi:10.1093/pcp/pci203
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1 Department of Biology, Queen's University, Kingston, Ontario, K7L 3N6, Canada; Present address: Department of Botany, University of Toronto, Toronto, Ontario M5S 3B2, Canada
* To whom correspondence should be addressed. Two novel phosphoenolpyruvate carboxylase (PEPC) isoforms have been biochemically characterized from endosperm of developing castor oil seeds (COS) (Blonde and Plaxton, 2003 J Biol Chem 278: 11867-11873). The association of a 107 kDa PEPC subunit (p107) with an immunologically-unrelated bacterial PEPC-type 64 kDa polypeptide leads to marked physical and kinetic differences between the PEPC1 p107 homotetramer and PEPC2 p107/p64 heterooctamer. COS p107 is quite susceptible to limited proteolysis during PEPC purification. An endogenous asparaginyl endopeptidase appears to catalyze the in vitro cleavage of an approximate 120 amino acid polypeptide from the N-terminal end of p107 producing a truncated 98 kDa polypeptide (p98). Immunoblotting was used to estimate proteolytic activity by following the disappearance of p107 and concomitant appearance of p98 during incubation of clarified COS extracts at 4 °C. The in vitro proteolysis of p107 to p98 only occurred in the combined presence of 2 mM dithiothreitol and high salt concentrations (particularly SO42- and PO42- salts). Although p107 degrading activity was present throughout COS development, it was most pronounced in endosperm extracts from older beans. Several protease inhibitors, including two commercially available protease inhibitor cocktails, were tested for their ability to prevent p107 proteolysis. All of the inhibitors were ineffective except for 2,2'-dipyridyl disulfide (DPDS), a relatively inexpensive and underutilized active site inhibitor of plant thiol proteases. Asparaginyl endopeptidase activity of COS extracts was unaffected by 20% (NH4)2SO4 when determined in the presence or absence of 2 mM dithiothreitol using a spectrophotometric assay based upon the hydrolysis of benzoyl-L-Asn-p-nitroanilide. Thus, we propose that the combined presence of 2 mM dithiothreitol and 20% (NH4)2SO4 promotes a p107 conformational change that exposes the N-terminal region asparaginyl residue where p107 hydrolysis is believed to occur.
Received June 24, 2005
Accepted September 13, 2005
Regular Paper
In vitro Proteolysis of Phosphoenolpyruvate Carboxylase from Developing Castor Oil Seeds by An Endogenous Thiol Endopeptidase
2 Department of Biology, Queen's University, Kingston, Ontario, K7L 3N6, Canada
3 Department of Biology, Queen's University, Kingston, Ontario, K7L 3N6, Canada; Department of Biochemistry, Queen's University, Kingston, Ontario, K7L 3N6, Canada
William C. Plaxton, E-mail: plaxton{at}biology.queensu.ca
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