PLANT PHYSIOLOGY , Vol 109, Issue 4 1461-1469, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Suborganellar Localization and Molecular Characterization of Nonproteolytic Degraded Leukoplast Pyruvate Kinase from Developing Castor Oil Seeds
F. B. Negm, F. A. Cornel and W. C. Plaxton
Departments of Biology and Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6
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.
