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. 1995 Dec;109(4):1461–1469. doi: 10.1104/pp.109.4.1461

Suborganellar Localization and Molecular Characterization of Nonproteolytic Degraded Leukoplast Pyruvate Kinase from Developing Castor Oil Seeds.

F B Negm 1, F A Cornel 1, W C Plaxton 1
PMCID: PMC157682  PMID: 12228683

Abstract

Plastid pyruvate kinase (PKp) activity and anti-(castor oil seed [COS] PKp) immunoglobulin G immunoreactive polypeptides were recovered in the stroma but not from envelope membranes of purified COS leukoplasts that had been subfractionated by sucrose density gradient centrifugation. The PKp was highly purified from isolated leukoplasts using anion-exchange and ADP-agarose chromatographies. Proteolysis of PKp was almost entirely eliminated by including 2,2[prime]-dipyridyl disulfide in purification buffers. The final preparation contained 63.5-kD ([alpha] subunit) and 54-kD ([beta] subunit) polypeptides that stained for protein and cross-reacted with anti-(COS PKp) immunoglobulin G with similar intensities. These two polypeptides co-eluted following gel-filtration chromatography and co-migrated during nondenaturing isoelectric focusing-polyacrylamide gel electrophoresis. The enzyme's native Mr was estimated to be 334,000. This PKp thus appears to exist as an [alpha]3[beta]3-heterohexamer. Comparison of the respective N-terminal sequences of the [alpha] and [beta] subunits with the deduced amino acid sequences for several PKp cDNAs indicated that (a) the [alpha] and [beta] subunits are encoded by COS genes previously designated as PKpA and PKpG, respectively, and (b) respective transit peptides of 4.8- and 5.5-kD are cleaved from the [alpha] and [beta] subunit preproteins following their translocation into the leukoplast.

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Selected References

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