Journal Article

In vitro Proteolysis of Phospho<i>enol</i>pyruvate Carboxylase from Developing Castor Oil Seeds by an Endogenous Thiol Endopeptidase

Valerie Crowley, Sam Gennidakis and William C. Plaxton

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 46, issue 11, pages 1855-1862
Published in print November 2005 | ISSN: 0032-0781
Published online November 2005 | e-ISSN: 1471-9053 | DOI:
In vitro Proteolysis of Phosphoenolpyruvate Carboxylase from Developing Castor Oil Seeds by an Endogenous Thiol Endopeptidase

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry


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Two novel phosphoenolpyruvate carboxylase (PEPC) isoforms have been biochemically characterized from endosperm of developing castor oil seeds (COS). 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 approximately 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 SO4 2– and PO4 2– 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.

Keywords: Asparaginyl endopeptidase; Castor oil seeds; Phosphoenolpyruvate carboxylase; Proteolysis; Ricinus communis; AS, ammonium sulfate; BZ-Asn-pNA, benzoyl-l-Asn-p-nitroanilide; COS, castor oil seed; DPDS, 2,2′-dipyridyl disulfide; DTT, dithiothreitol; E-64, l-(trans)-epoxysuccinyl-leucylamido 4-guanidino butane; FPLC, fast protein liquid chromatography; p98 and p107, 98 and 107 kDa polypeptides, respectively; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; PKp, leucoplast pyruvate kinase; PMSF, phenylmethylsulfonyl fluoride; pNA, p-nitroaniline

Journal Article.  5962 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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