Relationship between the Subunits of Leucoplast Pyruvate Kinase from <i>Ricinus communis</i> and a Comparison with the Enzyme from Other Sources

Blakeley, Stephen D.; Plaxton, William C.; Dennis, David T.

doi:10.1104/pp.96.4.1283

Cited by 29 publications

(39 citation statements)

References 13 publications

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“…Although the evidence presented here and elsewhere Italicized and underlined letters represent the N-terrninal amino acid sequence previously determined by Blakeley et al (1991) for the 57.5-kD COS PK p a subunit that had been proteolyzed during the enzyme's purification from a developing COS endosperm homogenate . The arrow indicates the processing site for the 44-amino acid transit peptide that is cleaved during import of the a subunit preprotein into the leukoplast.…”

Section: Discussionmentioning

confidence: 96%

“…The generation of a 57.5-kD degradation product from the 63.5-kD a subunit arises from the specific action of an endogenous asparaginyl endopeptidase, since the site of cleavage is on the carboxy-terminal side of a unique sequence of four consecutive Asn residues (Blakeley et al, 1991). The asparaginyl endopeptidase of developing COS was shown to be a Cys protease that displays characteristics consistent with its putative involvement in the turnover and/or elimination of PK, during COS maturation (Plaxton, 1991;Cornel and Plaxton, 1994).…”

mentioning

confidence: 99%

“…PK,A, PK,B, and PK,G) from a developing COS cDNA library. The deduced sequence of PK,A encodes eight amino acid residues that have been identified as the N terminus of the 57.5-kD a subunit of the proteolyzed COS PK, (Blakeley et al, 1991). In vitro import assays utilizing isolated COS leukoplasts, radiolabeled translation products of the PK,A and PK,G cDNAs, and specific antibodies against the overexpressed C termini of PK,A and PKpG initially led to the hypothesis that leukoplasts of developing COS may contain distinct PK, isozymes in the leukoplast envelope (i.e.…”

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confidence: 99%

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

1995

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Plastid pyruvate kinase (PK,) activity and anti-(castor oil seed [COSI PK, ) immunoglobulin C 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 PK, was highly purified from isolated leukoplasts using anion-exchange and ADP-agarose chromatographies. Proteolysis of PK, was almost entirely eliminated by including 2,2'-dipyridyl disulfide in purification buffers. The final preparation contained 63.5-kD ( a subunit) and 54-kD ( p subunit) polypeptides that stained for protein and cross-reacted with anti-(COS PK, ) immunoglobulin C 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 M, was estimated t o be 334,000. This PK, thus appears to exist as an a,p,-heterohexamer. Comparison of the respective N-terminal sequences of the a and subunits with the deduced amino acid sequences for severa1 PK, cDNAs indicated that (a) the a and p subunits are encoded by COS genes previously designated as PKpA and PKpC, respectively, and (b) respective transit peptides of 4.8-and 5.5-kD are cleaved from the a and p subunit preproteins following their translocation into the leukoplast.

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Section: Discussionmentioning

confidence: 96%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

1995

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“…The physical, immunological, and/or kinetic properties of several purified plant cytosolic and plastid PKs (PKc and PKp, respectively) have been studied in detail (1, 9, 11, 13-15, 17-19, 21). As well, cDNAs encoding higher plant cytosolic and plastid PKs have recently been cloned and sequenced (2)(3).…”

Section: Vl [1990] Plant Physiol 94: 1528-1534) By Contrast Immuno-mentioning

confidence: 99%

Leucoplast Pyruvate Kinase from Developing Castor Oil Seeds

Plaxton

1991

Plant Physiol.

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Leucoplast pyruvate kinase (PKp; EC 2.7.1.40) from endosperm of developing castor oil seeds (Ricinus communis L. cv Baker 296) appears to be highly susceptible to limited degradation by a cysteine endopeptidase during the purification of the enzyme or incubation of clarified homogenates at 40C. Purified (1, 9, 11, 13-15, 17-19, 21). As well, cDNAs encoding higher plant cytosolic and plastid PKs have recently been cloned and sequenced (2-3).Like most nonplant PKs, the cytosolic enzyme from developing endosperm and leaftissue of the castor oil plant appear to be 230 to 240 kD homotetramers (18). By contrast, PK, from germinating COS endosperm is a heterotetramer composed of two types of closely related, but nonidentical, 57 and 56 kD subunits (17-18), whereas chloroplast PK from the green alga Selenastrum minutum was shown to be a 210 kD monomer (13).The subunit composition for leucoplast PK from developing oil seeds has not yet been resolved. Developing COS endosperm leucoplast PK was recently purified to near homogeneity, and had a native molecular mass of about 305 kD (19). SDS-PAGE and western blot analysis of the final preparation revealed two major protein staining bands of 57.5 and 44 kD, that were immunologically related and occurred in an approximate 2:1 ratio, respectively. It was tentatively proposed that either there was proteolytic degradation ofthe 57.5 kD subunit yielding the 44 kD subunit, or that the enzyme might be a heterohexamer composed oftwo different types of related subunits ( 19). The present study was initiated to verify the in vivo subunit structure ofdeveloping COS PK,. Evidence is presented that demonstrates that: (a) throughout the course of endosperm development, PK, is composed in vivo of equal amounts of homologous 63.5 and 54 kD subunits; and (b) both subunits are highly susceptible to limited in vitro degradation by a nonleucoplast localized cysteine endopeptidase.

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“…PK, and PKp differ significantly in their molecular and kinetic characteristics and are immunologically unrelated proteins (12,15,16,(23)(24)(25)(26). DNA sequence analyses have shown PK, and PKp to be distinct isozymes (1,2). PKc from plants is similar to PK found in other eukaryotes, with approximately 50% of the amino acid residues identical to the nonplant enzyme and with all amino acids essential for substrate binding at identical locations (1).…”

mentioning

confidence: 99%

Normal Growth of Transgenic Tobacco Plants in the Absence of Cytosolic Pyruvate Kinase

Gottlob-McHugh

Sangwan²,

Blakeley³

et al. 1992

Plant Physiol.

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The coding sequence of the cytosolic isozyme of potato tuber pyruvate kinase (PK) was attached to the transit peptide of the small subunit of pea ribulose-1,5-bisphosphate carboxylase oxygenase and placed under the control of the cauliflower mosaic virus 35S promoter. This construct was transformed into Nicotiana tabacum. Unexpectedly, two primary transformants were recovered in which PK activity in leaves was greatly reduced. The reduction in PK activity appeared to result from the complete absence of the cytosolic form of the enzyme (PKl

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Relationship between the Subunits of Leucoplast Pyruvate Kinase from Ricinus communis and a Comparison with the Enzyme from Other Sources

Cited by 29 publications

References 13 publications

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

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

Leucoplast Pyruvate Kinase from Developing Castor Oil Seeds

Normal Growth of Transgenic Tobacco Plants in the Absence of Cytosolic Pyruvate Kinase

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