Activation of spinach chloroplast glyceraldehyde 3-phosphate dehydrogenase: effect of glycerate 1,3-bisphosphate

Spinach (Spinacia oleracea L.) chloroplast NAD(P)-dependent glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) was purified. The association state of the protein was monitored by fast protein liquid chromatography-Superose 12 gel filtration. Protein chromatographed in the presence of NADP⁺ and dithiothreitol consisted of highly NADPH-active protomers of 160 kDa; otherwise, it always consisted of a 600-kDa oligomer (regulatory form) favoured by the addition of NAD⁺ in buffers and with low NADPH-dependent activity (ratio of activities with NADPH versus NADH of 0.2–0.4). Glycerate 1,3-bisphosphate (BPGA) was prepared enzymatically using rabbit-muscle NAD-GAPDH, and purified. Among known modulators of spinach NAD(P)-GAPDH, BPGA is the most effective on a molar basis in stimulating NADPH-activity of dark chloroplast extracts and purified NAD(P)-GAPDH (activation constant, K _a= 12 M). It also causes the enzyme to dissociate into 160-kDa protomers. The K _m of BPGA both with NADPH or NADH as coenzyme is 4–7 M. NAD⁺ and NADH are inhibitory to the activation process induced by BPGA. This compound, together with NADP(H) and ATP belongs to a group of substrate-modifiers of the NADPH-activity and conformational state of spinach NAD(P)-GAPDH, all characterized by K _a values three- to tenfold higher than the K _m. Since NADP(H) is largely converted to NAD(H) in darkened chloroplasts Heineke et al. 1991, Plant Physiol. 95, 1131–1137, it is proposed that NAD⁺ promotes NAD(P)-GAPDH association into a regulatory conformer with low NADPH-activity during dark deactivation. The process is reversed in the light by BPGA and other substrate-modifiers whose concentration increases during photosynthesis, in addition to reduced thioredoxin.Abbreviations BPGA glycerate 1,3-bisphosphate - Chl chlorophyll - DTT dithiothreitol - FPLC fast protein liquid chromatography - NAD(P)-GAPDH glyceraldehyde 3-phosphate dehydrogenase, NAD(P)-dependent - 3-PGA glyerate 3-phosphate - PGK phosphoglycerate kinase - Prt protein - Tricine N-tris (hydroxymethyl) methyl-glycine This work was supported by grants from the Ministero dell'Università e della Ricerca Scientifica e Technologica in years 1990–1991. We are grateful to Dr. G. Branlant (Laboratoire d'Enzymologie et de Génie Génétique, Vandoeuvre les Nancy, France) for introducing us to the BPGA purification procedure.     

Dark modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase in the chloroplast

The properties of the system which reverses light modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase activity in pea chloroplasts were examined. A factor catalyzing dark modulation of these enzymes was found. This factor cochromatographed with thioredoxin in all systems used (Sephacryl S-200, Sephadex G-75, DEAE-cellulose). Inhibition of dithiothreitol-dependent modulation and of dark reversal by antibody against Escherichia coli thioredoxin further suggest that the dark factor is in fact thioredoxin. It appears that the reaction is the reverse of the previously described dithiothreitol-dependent thioredoxin-catalyzed modulation of enzymes. The limiting step in vitro seems to be the oxidation of thioredoxin during the dark period.     

Increase in chloroplastic thiol groups by SO2 and its effect on light modulation of NADP-dependent glyceraldehyde 3-phosphate dehydrogenase

In broken spinach chloroplasts the total amount of thiol groups is about 3.7 mol mg^-1 chlorophyll. Two thirds are represented by the masked form (which is only titratable after unfolding of the protein). Of the free groups, those reacting with NBD·Cl (1.2–2.0 mol mg^-1 chlorophyll) seem to be undergoing oxidation more readily than those reacting with DTNB (1.0 mol mg^-1 chlorophyll). SO₂ application causes a maximal increase of 25% in free thiols, and doubles the amount of the masked thiols. The light triggered increase in SH, which starts at an elevated level, runs parallel to that of the controls. SO₂ application of 1.8 mg m^-3 (=28 nmol l^-1) for 1 h does not affect the dark level of NADP-GPD but enhances the light modulation by increasing the ratio of activation. This enhancement is explained by an increase in masked thiol groups during the preceding fumigation period.Abbreviations DTNB 5,5 dithiobis-2-nitrobenzene-2-oxa-1,3 diazole - NBD·Cl 7-chloro-4-nitrobenzene-2-oxa-1,3 diazole - PCMB p-chloromercuribenzoate - SDS sodium dodecylsulfate - NADP-GPD NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2[N-Morpholino]ethanesulfonic acid - PGA 3-phosphoglyceric acid     

Clinical strains of Candida albicans express the surface antigen glyceraldehyde 3-phosphate dehydrogenase in vitro and in infected tissues

We have previously described the presence of an enzymatically active form of glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) in the cell surface of Candida albicans ATCC 26555 which is also a fibronectin and laminin binding protein. Immunohistochemical analysis of tissue sections from patients with disseminated candidiasis with a polyclonal antiserum to GAPDH from C. albicans (PAb anti-CA-GAPDH) revealed that the enzyme is expressed at the surface of fungal cells in infected tissues. The same PAb detected the presence of GAPDH species, with a molecular mass of approximately 33 kDa, in cell wall extracts obtained from clinical isolates of the fungus. These cell surface-bound GAPDH moieties exhibited a dose-dependent dehydrogenase activity. These results indicate that this cell surface-bound GAPDH plays a role during infection probably contributing to the attachment of fungal cells to host tissues.     

Structural characterization of transglutaminase-catalyzed cross-linking between glyceraldehyde 3-phosphate dehydrogenase and polyglutamine repeats

The accumulation of abnormal polyglutamine-containing protein aggregates within the cytosol and nuclei of affected neurons is a hallmark of the progressive neurodegenerative disorders caused by an elongated (CAG)(n) repeat in the genome. The polyglutamine domains are excellent substrates for the enzyme transglutaminase type 2 (tissue), resulting in the formation of cross-links with polypeptides containing lysyl groups. Enzymatic activity toward the Q(n) domains increases greatly upon lengthening of such Q(n) stretches (n > 40). Among the possible amine donors, the glycolytic enzyme glyceraldehyde-3-phosphate-dehydrogenase was shown to tightly bind several proteins involved in polyglutamine expansion diseases. Recently, the authors have shown that K191, K268, and K331, out of the 26 lysines present in glyceraldehyde-3-phosphate-dehydrogenase, are the reactive amine-donor sites forming cross-links with substance P, which bears the simplest Q(n) domain (n = 2). The present study reports that synthetic peptides of both pathological and nonpathological length (n = 43 and 17, respectively) form cross-links with the same K residues located in the C-terminal region of glyceraldehyde-3-phosphate-dehydrogenase. In addition, it is shown that extra K residues present in the C termini of glyceraldehyde-3-phosphate-dehydrogenase are susceptible to cross-linking in the presence of transglutaminase. The present results indicate a possible modulating effect of Q(n) stretches on tissue transglutaminase substrate specificity and mechanism of recognition.     

Cloning and characterisation of the glyceraldehyde 3-phosphate dehydrogenase gene of Candida bombicola and use of its promoter

The glyceraldehyde-3-phosphate dehydrogenase gene (GPD) of the sophorolipid producing yeast Candida bombicola was isolated using degenerated PCR and genome walking. The obtained 3,740 bp contain the 1,008 bases of the coding sequence and 1,613 and 783 bp of the upstream and downstream regions, respectively. The corresponding protein shows high homology to the other known GPD genes and is 74% identical to the gyceraldehyde-3-phosphate dehydrogenase of Yarrowia lipolytica. The particular interest in the C. bombicola GPD gene sequence originates from the potential use of its promoter for high and constitutive expression of homologous and heterologous genes. Southern blot analysis did not give any indication for the presence of multiple GPD genes and it can therefore be expected that the promoter can be used for efficient and high expression. This hypothesis was further confirmed by the biased codon usage in the GPD gene. GDP promoter fragments of different lengths were used to construct hygromycin resistance cassettes. The constructs were used for the transformation of C. bombicola and all of them, even the ones with only 190 bp of the GPD promoter, were able to render the cells resistant to hygromycin. The efficacy of a short GPD promoter can be a convenient characteristic for the construction of compact expression cassettes or vectors for C. bombicola. The GenBank accession number of the sequence described in this article is EU315245.     

Purification and some properties of glyceraldehyde 3-phosphate dehydrogenase fromSynechococcus sp.

Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was purified 386 fold to apparent homogeneity from the thermophilic cyanobacteriumSynechococcus sp. grown at optimum light intensities in batch cultures. The molecular mass of the tetrameric form of the enzyme was 160 kDa as determined by gel filtration and sucrose gradient centrifugation in a phosphate buffer containing DTT. The pH optimum for the oxidation of NADPH was broad (6–8) and the enzyme had a pI of 4.5. The turnover number was 36,000 min^–1 at 40° C. The activation energy was 12.4 Kcal for t>29° C and 20.6 Kcal for t<29° C. The specific absorption coefficient, A _{280 mm} ^{1% 1cm} of the pure enzyme in phosphate buffer at pH 6.8 was 15.2.By SDS gel electrophoresis molecular masses of 78 kDa and 39 kDa were found, indicating that the purified enzyme is a tetramer, probably a homotetramer.When Tris was used as buffer in the homogenization and phosphate and DTT were omitted, a high molecular form with a molecular mass above 500 kDa was found. This form was less active than the purified tetrameric form. Acetone and other organic solvents stimulated the native enzyme several fold.     

Disruption of NAD~+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

AIM:To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase(GAPDH),and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS:We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters(diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching(FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding.RESULTS:Using MALDI-TOF analysis,we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94,S98,and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH,we demonstrated accumulation of phospho-T99-GAPDH inthe nuclear fractions of A549,HCT116,and SW48 cancer cel s after cytotoxic stress. We performed site-mutagenesis,and estimated enzymatic properties,intranuclear distribution,and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+(Km = 741 ± 257 μmol/L in T99 I vs 57 ± 11.1 μmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP(fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION:Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners.     

Glucose-6-phosphate dehydrogenase in barley roots: kinetic properties and localisation of the isoforms

Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP⁺/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, K _m for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg²⁺ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots. Received: 21 June 2000 / Accepted: 28 July 2000     

Somatic and sperm-specific isoenzymes of glyceraldehyde-3-phosphate dehydrogenase: Comparative analysis of primary structures and functional features

The elucidation of the interdependence between structural features and functions of somatic and sperm-specific isoenzymes of glyceraldehyde-3-phosphate dehydrogenase (GAPD and GAPDS, respectively) was the goal of comparative analysis of their primary structures. GAPDS was shown to lack the sequence similar to the atypical nuclear export signal motif (NES) of the somatic isoenzyme GAPD. This finding is confirmed by experimental data on the absence of interaction between GAPDS and antibodies 6C5 recognizing the NES motif in the sequence of GAPD. The lack of NES correlates with functional peculiarities of the sperm-specific enzyme that is tightly bound to the fibrous sheath of the sperm flagellum. The sequences of the two isoenzymes were examined for the short motifs that might participate in apoptosis, endocytosis, and DNA repair. Sites of phosphorylation by different protein kinases have been revealed in both isoenzymes, and their characteristic features are discussed. These observations can serve as the basis for subsequent search for new ways of regulating the two isoenzymes.     

Transgenic potato plants with altered expression levels of chloroplast NADP-malate dehydrogenase: interactions between photosynthetic electron transport and malate metabolism in leaves and in isolated intact chloroplasts

The contribution of the malate valve in the regulation of steady-state photosynthesis was studied in transgenic potato (Solanum tuberosum L. cv Désirée) plants with altered expression of plastidic NADP-dependent malate dehydrogenase (NADP-MDH; EC 1.1.1.82). Mutant plants were obtained after transformation with the homologous Nmdh gene in antisense orientation, or with the Nmdh gene from pea (Pisum sativum L.) in sense orientation. A total number of nine stable sense and antisense lines with 10% or 30%, and 400% of wild-type NADP-MDH capacity were selected. Intact chloroplasts were isolated from leaves of wild-type and mutant plants. In chloroplasts from sense transformants the increased enzyme amount was activated as in wild-type chloroplasts, but increased rates of oxaloacetate-dependent malate formation were only measured upon partial uncoupling. In contrast, chloroplasts from antisense transformants produced only little malate upon oxaloacetate addition. Measurements with intact leaves during steady-state photosynthesis yielded no differences in gas-exchange parameters and chlorophyll fluorescence. The leaf malate content was unchanged in NADP-MDH underexpressors, but twice as high in overexpressing plants. The altered NADP-MDH expression clearly influences the redox state of ferredoxin, especially in low light. Furthermore, the malate valve can successfully compete for electrons with cyclic electron flow, but the conditions under which this occurs are quite artificial. Received: 14 February 1998 / Accepted: 12 May 1998     

Cloning, gene expression and characterization of a novel bacterial NAD-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Neisseria meningitidis strain Z2491

Alignment of the amino acid sequence of some archaeal, bacterial and eukaryotic non-phosphorylating glyceraldehydes-3-phosphate dehydrogenases (GAPNs) and aldehyde dehydrogenases (ALDHs) with the sequence of a putative GAPN present in the genome of the Gram-negative bacterium Neisseria meningitidis strain Z2491 demonstrated the conservation of residues involved in the catalytic activity. The predicted coding sequence of the N. meningitidis gapN gene was cloned in Escherichia coli XL1-blue under the expression of an inducible promoter. The IPTG-induced GAPN was purified ca. 48-fold from E. coli cells using a procedure that sequentially employed conventional ammonium sulfate fractionation as well as anion-exchange and affinity chromatography. The purified recombinant enzyme was thoroughly characterized. The protein is a homotetramer with a 50-kDa subunit, exhibiting absolute specificity for NAD and a broad spectrum of aldehyde substrates. Isoelectric focusing analysis with the purified fraction showed the presence of an acidic polypeptide with an isoelectric point of 6.3. The optimum pH of the purified enzyme was between 9 and 10. Studies on the effect of increasing temperatures on the enzyme activity revealed an optimal value ca. 64 °C. Molecular phylogenetic data suggest that N. meningitidis GAPN has a closer relationship with archaeal GAPNs and glyceraldehyde dehydrogenases than with the typical NADP-specific GAPNs from Gram-positive bacteria and photosynthetic eukaryotes.     

Differential synthesis of glyceraldehyde-3-phosphate dehydrogenase polypeptides in stressed yeast cells

Abstract Three unlinked genes, TDH1, TDH2 and TDH3 , encode the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (triose-phosphate dehydrogenase; TDK) in the yeast Saccharomyces cerevisiae . We demonstrate that the synthesis of the three encoded TDK polypeptides (TDHa, TDHb and TDHc, respectively) is not co-ordinately regulated and that TDHa is only synthesised as cells enter stationary phase, due to glucose starvation, or in heat-shocked cells. Furthermore, the synthesis of TDHb, but not TDHc, is strongly repressed by a heat shock. Hence, the TDHa enzyme may play a cellular role, distinct from glycolysis, that is required by stressed cells.     

Kinetic properties of N-(2-carboxyethyl)-NAD(H) and poly(ethylene glycol)-bound NAD(H) for alcohol, lactate, malate and glyceraldehyde-3-phosphate dehydrogenase from different organisms

The steady-state kinetics of alcohol dehydrogenases (alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1 and alcohol:NADP⁺ oxidoreductase, EC 1.1.1.2), lactate dehydrogenases (l-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27 and d-lactate:NAD⁺ oxidoreductase, EC 1.1.1.28), malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37), and glyceraldehyde-3-phosphate dehydrogenases [d-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase (phosphorylating), EC 1.2.1.12] from different sources (prokaryote and eukaryote, mesophilic and thermophilic organisms) have been studied using NAD(H), N⁶-(2-carboxyethyl)-NAD(H), and poly(ethylene glycol)-bound NAD(H) as coenzymes. The kinetic constants for NAD(H) were changed by carboxyethylation of the 6-amino group of the adenine ring and by conversion to macromolecular form. Enzymes from thermophilic bacteria showed especially high activities for the derivatives. The relative values of the maximum velocity (NAD = 1) of Thermus thermophilus malate dehydrogenase for N⁶-(2-carboxyethyl)-NAD and poly(ethylene glycol)-bound NAD were 5.7 and 1.9, respectively, and that of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase for poly(ethylene glycol)-bound NAD was 1.9.     

Action of sulphite on the substrate kinetics of chloroplastic NADP-dependent glyceraldehyde-3-phosphate dehydrogenase

The kinetics of NADP-GPD from spinach chloroplasts are biphasic vs NADPH and PGA. Thus, two maximum velocities exist with an intermediary plateau and two K_m values. Activation by NADPH + DTT increases V_max of both sections, but does not change the substrate affinities. Sulphite reduces the maximum activities of both sections vs NADPH, however, it causes normal substrate kinetics vs PGA; even V_max is reduced. Sulphite, present only during the activation process, suppresses the enzyme form with the higher V_max. The kinetics vs NADH are also biphasic; the activity is strongly reduced by preincubation of the chloroplasts with NADH + DTT or at NADH concentrations > 0.4mM. Using NADH as cofactor, inverted peaks in the kinetics vs PGA occur; sulphite is active in a similar way as when NADPH is used as cofactor. The biphasic kinetics are discussed with respect to additional potential for regulation of enzyme activity according to illumination and NADPH concentrations respectively.     

A survey for isoenzymes of glucosephosphate isomerase,phosphoglucomutase, glucose-6-phosphate dehydrogenase and 6-Phosphogluconate dehydrogenase in C3-, C4-and crassulacean-acid-metabolism plants,and green algae

Two isoenzymes each of glucosephosphate isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.43) were separated by (NH₄)₂SO₄ gradient solubilization and DEAE-cellulose ion-exchange chromatography from green leaves of the C₃-plants spinach (Spinacia oleracea L.), tobacco (Nicotiana tabacum L.) and wheat (Triticum aestivum L.), of the Crassulacean-acid-metabolism plants Crassula lycopodioides Lam., Bryophyllum calycinum Salisb. and Sedum rubrotinctum R.T. Clausen, and from the green algae Chlorella vulgaris and Chlamydomonas reinhardii. After isolation of cell organelles from spinach leaves by isopyenic centrifugation in sucrose gradients one of two isoenzymes of each of the four enzymes was found to be associated with whole chloroplasts while the other was restricted to the soluble cell fraction, implying the same intracellular distribution of these isoenzymes also in the other species.Among C₄-plants, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were found in only one form in corn (Zea mays L.), sugar cane (Saccharum officinarum L.) and Coix lacrymajobi L., but as two isoenzymes in Atriplex spongiosa L. and Portulaca oleracea L. In corn, the two dehydrogenases were mainly associated with isolated mesophyll protoplasts while in Atriplex spongiosa they were of similar specific activity in both mesophyll protoplasts and bundle-sheath strands. In all five C₄-plants three isoenzymes of glucosephosphate isomerase and phosphoglucomutase were found. In corn two were localized in the bundle-sheath strands and the third one in the mesophyll protoplasts. The amount of activity of the enzymes was similar in each of the two cell fractions. Apparently, C₄ plants have isoenzymes not only in two cell compartments, but also in physiologically closely linked cell types such as mesophyll and bundle-sheath cells. New address: Institut für Pflanzenphyiologie und Zellbiologie, Freie Universität Berlin, Königin-Luise-Straße 12-16a, D-1000 Berlin 33     

Light activation and molecular-mass changes of NAD(P)-glyceraldehyde 3-phosphate dehydrogenase of spinach and maize leaves

Light modulation of chloroplast glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) has been investigated. Complete activation of NADPH-dependent activity is achieved at 25 W.m^–2 photosynthetically active radiation in spinach (Spinacia oleracea L.) and 100 W.m^–2 in maize (Zea mays L.) leaves. Light activation is stronger in spinach (fivefold on average) than in maize (twofold), which shows higher dark activity. The NADH dependent activity does not change appreciably. Several substrate activators can simulate in vitro the light effect with recovery of latent NADPH-dependent activity of spinach enzyme, but they are almost inactive with maize enzyme. A mixture of activators has been devised to fully activate the spinach enzyme under most conditions. The NAD(P)-GAPDH protein can be resolved by rapid gel filtration (fast protein liquid chromatography) into three conformers which have different molecular masses according to the light conditions. Enzyme from darkened leaves or chloroplasts, or dichlorophenyl-1,1-dimethylurea-treated chloroplasts is mainly a 600-kDa regulatory form with low NADPH-dependent activity relative to NADH-activity. Enzyme from spinach leaves or chloroplasts during photosynthesis is mainly a 300-kDa oligomer, which along with the 600-kDa form also occurs in leaves of darkened maize. The conformer of illuminated maize leaves is mainly a 160-kDa species. Results are consistent with a model of NAD(P)-GAPDH freely interconvertible between protomers of the 160-kDa (or 300-kDa intermediate) form with high NADPH-activity, produced in the light by the action of thioredoxin and activating metabolites (spinach only), and a regulatory 600-kDa conformer with lower NADPH-activity produced in darkness or when photosynthesis is inhibited. This behavior is reminiscent of the in-vitro properties of purified enzyme; therefore, it seems unlikely that NAD(P)-GAPDH in the chloroplast is part of a stable multienzyme complex or is bound to membranes.Abbreviations AEM activator equilibrium mixture - Chl chlorophyll - DCMU dichlorophenyl 1,1-dimethylurea - DTT dithiothreitol - FPLC fast protein liquid chromatography - NAD(P)-GAPDH glyceraldehyde 3-phosphate dehydrogenase, NAD(P)-dependent - PAR photosynthetic active radiation - PGK phosphoglycerate kinase - Tricine N-tris(hydroxy-methyl) methyl-glycine This work was supported by grants from the Ministero dell'Università e della Ricerca Seientifica e Tecnologica (40%, years 1990 and 1991).     

Half-of-the sites reactivity and negative co-operativity: the case of yeast glyceraldehyde 3-phosphate dehydrogenase

Covalent modification of two of the four cysteine-149 residues of yeast glyceraldehyde 3-phosphate dehydrogenase, at pH 8.5, decreases the reactivity of the remaining two cysteine-149 residues and essentially inactivates the protein. The structure of the modifying reagent has only a secondary influence on this half-of-the-sites effect. Reactivity studies, together with the existing X-ray and sequence studies, suggest that the four active sites are initially functionally identical both in activity and in cysteine reactivity. The half-of-the-sites effect therefore arises in part or in whole from ligand-induced negatively co-operative conformational changes. A detailed kinetic study with iodoacetamide gives relative values of two rapidly reacting groups, a third more slowly reacting, and a fourth very slowly reacting group. These data, added to the existing data on cytidine triphosphate synthetase and alkaline phosphatase, suggest that the half-of-the-sites phenomena in many enzymes may be explained by ligand-induced negative co-operativity triggered by binding or covalent bond formation or both.     

Identification of potential redox-sensitive cysteines in cytosolic forms of fructosebisphosphatase and glyceraldehyde-3-phosphate dehydrogenase

Tertiary-structure modeling suggests the occurrence of disulfide bonds in the cytosolic form of fructosebisphosphatase (EC 3.1.3.11) in spinach (Spinacia oleracea L.), sugarbeet (Beta vulgaris L.) and potato (Solanum tuberosum L.). Redox modulation could then control the AMP sensitivity of fructosebisphosphatase in the cytosol, as suggested by the experiments of E. Khayat et al. (1993, Plant Physiol. 101, 57–64). Modeling also reveals two cysteine residues correctly positioned to form a disulfide bond and hence potentially redox-sensitive in the cytosolic glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from the facultative crassulacean metabolism plant Mesembryanthemum crystallinum L.Abbreviations Cys cysteine - DTT dithiothreitol This work was supported by NSF DCB-9018265 and US Department of Energy, Office of Health and Environmental Research, under contract W-31-109-ENG-38, and by laboratory-directed research and development funding from Argonne National Laboratory. We thank Hans J. Bohnert, University of Arizona, for providing the ice plant seeds, Xiaomu Niu and Paul M. Hasegawa, Purdue University, for providing the salt bush roots, Larry Sykora and staff at the UIC Greenhouse for cultivating the ice plants, and Christie Aljets for assistance with some of the activity determinations.