Three Enzymes of Carbon Metabolism or their Antigenic Analogs in Pea Leaf Nuclei

Anderson, Louise E.; Wang, Xin; Gibbons, Jonathan

doi:10.1104/pp.108.2.659

Cited by 44 publications

(25 citation statements)

References 20 publications

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“…The average NADPHto NADH-linked activity ratios were 4:l for the B-subunit enzyme and 5.7:l for the BA334-subunit enzyme. The activity ratio we found for the pea chloroplastic enzyme was 2.6:l (Anderson et al, 199513). Ratios of NADPH-to NADH-linked activity ranging from 0.1:l to 3.5:l have been reported for the purified chloroplastic enzymes from pea (Schulman and Gibbs, 1968;Melandri et al, 1970;McGowan and Gibbs, 1974;Pupillo and Faggiani, 1979), spinach (Yonuschot et al, 1970;Pupillo and GiulainiPiccarri, 1975;Ferri et al, 1990;Trost et al, 1993), white mustard (Cerff, 1978), beet, buttercup, and Arum italicum (Pupillo and Faggiani, 1979).…”

Section: Dual Nadp-/nad-linked Activitysupporting

confidence: 60%

Expression and Characterization of Pea Chloroplastic Glyceraldehyde-3-Phosphate Dehydrogenase Composed of Only the B-Subunit

Anderson

1997

Plant Physiology

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A cDNA fragment coding for the pea (Pisum sativum 1.) chloroplastic glyceraldehyde-3-P dehydrogenase (EC 1.2.1.1 3) B-subunit and a truncated form corresponding in length to the A-subunit have been cloned into an expression vector, expressed in the absence of the A-subunit in a gap-Escherichia coli strain, purified, and studied. Like the isolated enzyme from higher plant chloroplasts, the recombinant enzymes have dual specificity for NADPH and NADH. l h e recombinant glyceraldehyde-3-P dehydrogenases have the same optimal pH as the enzyme isolated from pea chloroplasts. Like the native chloroplast enzyme, the recombinant B-subunit has a marked tendency to form large aggregates, whereas the truncated B-subunit exists as the tetramer. The recombinant 8-subunit glyceraldehyde 3-P dehydrogenase is more sensitive to dithiothreitol than its truncated form. It seems likely that a different pair of cysteines is responsible for the redox sensitivity of the activity of the enzyme composed of B-subunits than the cysteine residues implicated in the modulation of the activity of the enzyme composed of A-subunits by previous work in this laboratory.In chloroplasts G-3-P dehydrogenase (EC 1.2.1.13) catalyzes the reversible reduction and dephosphorylation of 1,3-bisphosphoglycerate to G-3-P using NADPH generated by PSI in the light. The enzyme is light-and DTT-activated (Buchanan, 1980; Anderson, 1986). It can use either NADP(H) or NAD(H) as the pyridine nucleotide substrate. In angiosperm chloroplasts there are two G-3-P dehydrogenase isozymes: one is composed of two A-subunits and two B-subunits (A,B,, isozyme I) and has a marked tendency to aggregate; the other is composed of four identical A-subunits (A4, isozyme 11) and does not aggregate Chambers, 1978, 1979;Ferri et al., 1978Ferri et al., , 1990Cerff, 1979;Pupillo and Faggiani, 1979;Scagliarini et al., 1993).The A-and B-subunits are very similar, except that the B-subunit has a highly negatively charged C-terminal extension that contains two Cys groups and is not found in other G-3-P dehydrogenases (Shih et al., 1986(Shih et al., , 1991Brinkmann et al., 1989;Liaud et al., 1990;Baalmann et al., 1996) This work was supported by National Science Foundation grant no. MCB-9513498.Present address: Department of Genetics, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1x8.* Corresponding author; e-mail louise@uic.edu; fax 1-312-413-2435. (Fig. 1). Both subunits are encoded in the nucleus (Cerff and Kloppstech, 1982;Shih et al., 1986). The existence of a B, tetramer in vivo has not been suggested. Baalmann et al. (1996) have developed an expression system for the A-and B-subunits of spinach (Spinacia oleracea) chloroplastic G-3-P dehydrogenase. Here we describe an expression system for the pea (Pisum sativum) chloroplastic G-3-P dehydrogenase B-subunit and for a truncated form corresponding in length to the A-subunit, and the characterization of the recombinant enzymes.

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Section: Dual Nadp-/nad-linked Activitysupporting

confidence: 60%

Expression and Characterization of Pea Chloroplastic Glyceraldehyde-3-Phosphate Dehydrogenase Composed of Only the B-Subunit

Anderson

1997

Plant Physiology

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“…Aldolase also has been reported to interact with V-type ATPases, leading to the suggestion that glycolysis may be localized to regions of high ATP demand (Lu et al, 2001). In plants, phosphoglycerate kinase, glyceraldehyde-3-P dehydrogenase, and aldolase have been shown to be present in the nuclei of pea leaf cells (Anderson et al, 1995), and glyceraldehyde-3-P dehydrogenase and aldolase also were shown to associate with the cytoskeleton in developing maize endosperm (Azama et al, 2003). Very recently, glycolytic enzymes were identified in a proteomic survey of human mitochondria (Taylor et al, 2003), although the functional significance of this observation was not investigated further.…”

Section: Discussionmentioning

confidence: 99%

Enzymes of Glycolysis Are Functionally Associated with the Mitochondrion in Arabidopsis Cells

et al. 2003

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Mitochondria fulfill a wide range of metabolic functions in addition to the synthesis of ATP and contain a diverse array of proteins to perform these functions. Here, we present the unexpected discovery of the presence of the enzymes of glycolysis in a mitochondrial fraction of Arabidopsis cells. Proteomic analyses of this mitochondrial fraction revealed the presence of 7 of the 10 enzymes that constitute the glycolytic pathway. Four of these enzymes (glyceraldehyde-3-P dehydrogenase, aldolase, phosphoglycerate mutase, and enolase) were also identified in an intermembrane space/outer mitochondrial membrane fraction. Enzyme activity assays confirmed that the entire glycolytic pathway was present in preparations of isolated Arabidopsis mitochondria, and the sensitivity of these activities to protease treatments indicated that the glycolytic enzymes are present on the outside of the mitochondrion. The association of glycolytic enzymes with mitochondria was confirmed in vivo by the expression of enolase-and aldolase-yellow fluorescent protein fusions in Arabidopsis protoplasts. The yellow fluorescent protein fluorescence signal showed that these two fusion proteins are present throughout the cytosol but are also concentrated in punctate regions that colocalized with the mitochondrion-specific probe Mitotracker Red. Furthermore, when supplied with appropriate cofactors, isolated, intact mitochondria were capable of the metabolism of 13 C-glucose to 13 C-labeled intermediates of the trichloroacetic acid cycle, suggesting that the complete glycolytic sequence is present and active in this subcellular fraction. On the basis of these data, we propose that the entire glycolytic pathway is associated with plant mitochondria by attachment to the cytosolic face of the outer mitochondrial membrane and that this microcompartmentation of glycolysis allows pyruvate to be provided directly to the mitochondrion, where it is used as a respiratory substrate.

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“…Our results strongly suggest that the NAD-dependent GAPDH is localized in the nucleus as well as in the cytosol. Interestingly, it was shown recently that an anti-NADP-linked GAPDH antibody reacts with protein(s) localized in chloroplasts, cytoplasm, and nucleus of leaf mesophyll cells of Pisum sativum (Anderson et al 1995). These results raised interesting possibilities that either NAPD-or NAD-linked GAPDH is also nuclear localized.…”

Section: Nuclear Localization Of Nad-dependent Gapdhmentioning

confidence: 93%

Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase inArabidopsis thaliana

et al. 1997

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Summary. NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (NAD-dependent GAPDH) was purified to homogeneity and injected into a rabbit to induce a polyclonal antibody. The antibody was judged to be of high specificity and high affinity. This antibody was used to probe sections of Arabidopsis leaf, stein or roots which were fixed using either paraformaldehyde or a high-pressure freezing method. Our results show that the NAD-dependent GAPDH localizes in the nucleus as well as in the cytosol. In phloem tissue, the NAD-dependent GAPDH was found in companion cells but not in the sieve element.

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Three Enzymes of Carbon Metabolism or their Antigenic Analogs in Pea Leaf Nuclei

Cited by 44 publications

References 20 publications

Expression and Characterization of Pea Chloroplastic Glyceraldehyde-3-Phosphate Dehydrogenase Composed of Only the B-Subunit

Expression and Characterization of Pea Chloroplastic Glyceraldehyde-3-Phosphate Dehydrogenase Composed of Only the B-Subunit

Enzymes of Glycolysis Are Functionally Associated with the Mitochondrion in Arabidopsis Cells

Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase inArabidopsis thaliana

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