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Suborganellar localization and molecular characterization of nonoproteolytic degraded leukoplast pyruvate kinase from developing castor oil seeds

Author:
Negm, F.B., Cornel, F.A., Plaxton, W.C.
Source:
Plant physiology 1995 v.109 no.4 pp. 1461-1469
ISSN:
0032-0889
Subject:
Ricinus communis, endosperm, pyruvate kinase, plastids, isolation, cell membranes, isozymes, chromosome mapping, polypeptides, amino acid sequences, molecular weight, genes, cell structures
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'-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 alpha3,beta3-heterohexamer. Comparison of the respective N-terminal sequences of the 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.
Agid:
664264