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C S Andreo

Publications and source records attributed to C S Andreo.

At least 37 records · Page 2Linked to original sources
PubMed1990

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves. Kinetic properties of different oligomeric structures.

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves was inhibited by increasing the ionic strength in the assay medium. The inhibitory effect was higher at pH 7.0 than 8.0, with median inhibitory concentrations (IC50) of 89 mM and 160 mM respectively, for inhibition by NaCl. Gel-filtration experiments indicated that the enzyme dissociated into dimers and monomers when exposed to high ionic strength (0.3 M NaCl). By using the enzyme-dilution approach in the absence and presence of 0.3 M NaCl, the kinetic properties of each oligomeric species of the protein was determined at pH 7.0 and 8.0. Tetrameric, dimeric and monomeric structures were shown to be active but with different V and Km values. The catalytic efficiency of the oligomers was tetramer greater than dimer greater than monomer, and each quaternary structure exhibited higher activity at pH 8.0 than 7.0. Dissociation constants for the equilibria between the different oligomeric forms of the enzyme were determined. It was established that Kd values were affected by pH and Mg2+ levels in the medium. Results suggest that the distinct catalytic properties of the different oligomeric forms of NADP-dependent malate dehydrogenase and changes in their equilibrium could be the molecular basis for an efficient physiological regulation of the decarboxylation step of C4 metabolism.

Chromatography, Gel↗
PubMed1990

Kinetic and Structural Properties of NADP-Malic Enzyme from Sugarcane Leaves.

Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP(+), and Mg(2+) were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (K(m) = 58 micromolar), but also inhibition by high substrate (K(i) = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (K(m) = 120 micromolar). At both pH conditions, activity response to NADP(+) exhibited Michaelis-Menten behavior with K(m) values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg(2+) suggested the presence of at least two Mg(2+) - binding sites with different affinity. The K(a) values for Mg(2+) obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg(2+) levels could be important for the physiological regulation of NADP-malic enzyme.

Journal Article↗
PubMed1989

Protection of Pyruvate,Pi Dikinase from Maize against Cold Lability by Compatible Solutes.

Most C(4) species are chilling sensitive and certain enzymes like pyruvate,Pi dikinase of the C(4) pathway are also cold labile. The ability of cations and compatible solutes to protect maize (Zea mays) dikinase against cold lability was examined. The enzyme in desalted extracts at pH 8 from preilluminated leaves could be protected against cold lability (at 0 degrees C) by the divalent cations Mn(2+), Mg(2+), and Ca(2+). There was substantial protection by sulfate based salts but little protection by chloride based salts of potassium or ammonium (concentration 250 millimolar). The degree of protection against cold lability under limiting MgCl(2) (5 millimolar) was pH sensitive (maximum protection at pH 8), but independent of ionic strength (up to 250 millimolar by addition of KCl). In catalysis Mg(2+) is required and Mn(2+) could not substitute as a cofactor. Several compatible solutes reduced or prevented the cold inactivation of dikinase (in desalted extracts and the partially purified enzyme), including glycerol, proline, glycinebetaine and trimethylamine-N-oxide (TMAO). TMAO and Mg(2+) had an additive effect in protecting dikinase against cold inactivation. TMAO could largely substitute for the divalent cation and addition of TMAO during cold treatment prevented further inactivation. Cold inactivation was partially reversed by incubation at room temperature; with addition of TMAO reversal was complete. The temperature dependence of inactivation at pH 8 and 3 millimolar MgCl(2) was evaluated by incubation at 2 to 17 degrees C for 45 minutes, followed by assay at room temperature. At preincubation temperatures below 11 degrees C there was a progressive inactivation which could be prevented by TMAO (450 millimolar). The results are discussed relative to possible effects of the solutes on the quaternary structure of this enzyme, which is known to dissociate at low temperatures.

Journal Article↗
PubMed1989

Maize leaf phosphoenolpyruvate carboxylase : oligomeric state and activity in the presence of glycerol.

Maize (Zea mays L.) leaf phosphoenopyruvate (PEP) carboxylase activity at subsaturating levels of PEP was increased by the inclusion of glycerol (20%, v/v) in the assay medium. The extent of activation was dependent on H(+) concentration, being more marked at pH 7 (with activities 100% higher than in aqueous medium) than at pH 8 (20% activation). The determination of the substrate concentration necessary to achieve half-maximal enzyme activity (S(0.5)) (PEP) and maximal velocity (V) between pH 6.9 and 8.2 showed a uniform decrease in S(0.5) in the presence of glycerol over the entire pH range tested, and only a slight decrease in V at pH values near 8. Including NaCl (100 millimolar) in the glycerol containing assay medium resulted in additional activation, mainly due to an increase in V over the entire range of pH. Glucose-6-phosphate (5 millimolar) activated both the native and the glycerol-treated enzyme almost to the same extent, at pH 7 and 1 millimolar PEP. Inhibition by 5 millimolar malate at pH 7 and subsaturating PEP was considerably lower in the presence of glycerol than in an aqueous medium (8% against 25%, respectively). Size-exclusion high performance liquid chromatography in aqueous buffer revealed the existence of an equilibrium between the tetrameric and dimeric enzyme forms, which is displaced to the tetramer as the pH was increased from 7 to 8. In the presence of glycerol, only the 400 kilodalton tetrameric form was observed at pH 7 or 8. However, dissociation into dimers by NaCl could not be prevented by the polyol. We conclude that the control of the aggregation state by the metabolic status of the cell could be one regulatory mechanism of PEP carboxylase.

Journal Article↗
PubMed1988

Carboxylation and dephosphorylation of phosphoenol-3-fluoropyruvate by maize leaf phosphoenolpyruvate carboxylase.

The analogue (Z)-phosphoenol-3-fluoropyruvate [(Z)-3-fluoro-2-(phosphono-oxy)propenoic acid] was tested as substrate of maize leaf phosphoenolpyruvate carboxylase. Studies with NaH14CO3 indicate that the analogue is carboxylated by the enzyme. However, this reaction accounts for only one-tenth of the activity measured by Pi liberation. The rest of the analogue is merely dephosphorylated. This is the first analogue for which both carboxylation and dephosphorylation have been observed.

Bicarbonates↗
PubMed1988

Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase. A resonance energy transfer study.

Phosphoenolpyruvate carboxylase, purified from maize leaves, is rapidly inactivated by the fluorescence probe dansyl chloride. The loss of activity can be ascribed to the covalent modification of an R-NH2 group, presumably the epsilon-NH2 group of lysine. Analysis of the data by the statistical method of Tsou [Sci. Sin. 11, 1535-1558 (1962)] provides clear evidence that a pH 8 eight R-NH2 groups can be modified in the tetrameric form of the enzyme, four of which are essential for catalytic activity. Essential groups are modified about five times more rapidly than the non-essential ones. The enzyme was completely protected against inactivation by Mg2+ plus phosphoenolpyruvate and consequently binding of the modifier to the essential groups is completely abolished. Hence the four essential groups seemed to be located at or near the active site(s). One of the four essential groups was modified with dansyl chloride and the other three progressively with eosin isothiocyanate. In the doubly labeled protein non-radiative single-singlet energy transfer between dansyl chloride (donor) and eosin isothiocyanate (acceptor) was observed. The low variance (+/- 5%) in the efficiency of energy transfer obtained at a particular acceptor stoichiometry (0.8-1.1, 1.9-2.1, 2.9-3.1) in triplicate samples provided confidence that the measured transfer efficiency may be interpreted as transfer between specific sites. The distances calculated from the efficiency of resonance energy transfer revealed two acceptor sites, equally separated, 4.8-5.1 nm from the donor site and third site being 6.4 nm apart from the donor. Under conditions where the tetrameric enzyme dissociates into the monomers, no transfer of resonance energy between the protein-bound dansyl chloride and eosin isothiocyanate was observed. Most likely the four essential lysyl residues in the tetrameric enzyme are located in different subunits of the enzyme, hence each of the subunits would contain a substrate-binding site with one lysyl residue crucial for activity.

Affinity Labels↗
PubMed1988

Stereoselectivity of the interaction of E- and Z-2-phosphoenolbutyrate with maize leaf phosphoenolpyruvate carboxylase.

The aim of this work was to investigate the stereoselectivity of maize leaf phosphoenolpyruvate carboxylase with E- and Z-2-phosphoenolbutyrate as inhibitors and substrates. In addition, a procedure is presented for the separation of the isomers of 2-phosphoenolbutyrate. The method is based on the different interaction of those compounds with a strong anion-exchange high-pressure liquid chromatography column using 50 mM potassium phosphate (pH 3) as elution buffer, and allows the obtention of pure E- and Z-P-enolbutyrate with high yield. The same system was used to identify Z-P-enolbutyrate as the product of the phosphorylation of 2-oxobutyrate by rabbit muscle pyruvate kinase. In the presence of 5 mM Mg2+, both isomers of P-enolbutyrate inhibited C4-plant P-enolpyruvate carboxylase; the values of Ki were 15-20 microM and 100-110 microM for Z- and E-P-enolbutyrate, respectively. With 0.5 mM Mn2+, the Z isomer was also effective as inhibitor (Ki = 35-40 microM), while the E isomer produced activation of the carboxylase probably due to its binding at an allosteric site. Both compounds were substrates of the enzyme with similar V/Km values; however, V and Km for the two isomers were significantly different (i.e. Km = 110 microM for Z-P-enolbutyrate and 220 microM for E-P-enolbutyrate). The results indicate the existence of stereoselectivity for the binding of P-enolbutyrate to the active site of P-enolpyruvate carboxylase. However, this fact does not affect the use of the isomers as substrates by the plant carboxylase.

Animals↗
PubMed1987

Changes in the quaternary structure of phosphoenolpyruvate carboxylase induced by ionic strength affect its catalytic activity.

Phosphoenolpyruvate carboxylase from maize leaves dissociated into dimers and/or monomers when exposed to increasing ionic strength (e.g. 200-400 mM NaCl) as indicated by gel filtration experiments. Changes in the oligomerization state were dependent on pH, time of preincubation with salt and protein concentration. A dissociation into dimers and monomers was observed at pH 8, while at pH 7 dissociation into the dimeric form only was observed. Exposure of the enzyme to higher ionic strength decreased the activity in a time-dependent manner. Turnover conditions and glucose 6-phosphate protected the carboxylase from the decay in activity, which was faster at pH 7 than at pH 8. The results suggest that changes in activity of the enzyme, following exposure to high ionic strength, are the consequence of dissociation. Tetrameric and dimeric forms of the phosphoenolpyruvate carboxylase seemingly reveal different catalytic properties. We suggest that the distinct catalytic properties of the different oligomeric species of phosphoenolpyruvate carboxylase and changes in the equilibrium between them could be the molecular basis for an effective regulation of metabolite levels by this key enzyme of C4 plants.

Carboxy-Lyases↗
PubMed1987

Interaction of acetyl phosphate and carbamyl phosphate with plant phosphoenolpyruvate carboxylase.

Acetyl phosphate produced an increase in the maximum velocity (Vmax. for the carboxylation of phosphoenolpyruvate catalysed by phosphoenolpyruvate carboxylase. The limiting Vmax. was 22.2 mumol X min-1 X mg-1 (185% of the value without acetyl phosphate). This compound also decreased the Km for phosphoenolpyruvate to 0.18 mM. The apparent activation constants for acetyl phosphate were 1.6 mM and 0.62 mM in the presence of 0.5 and 4 mM-phosphoenolpyruvate respectively. Carbamyl phosphate produced an increase in Vmax. and Km for phosphoenolpyruvate. The variation of Vmax./Km with carbamyl phosphate concentration could be described by a model in which this compound interacts with the carboxylase at two different types of sites: an allosteric activator site(s) and the substrate-binding site(s). Carbamyl phosphate was hydrolysed by the action of phosphoenolpyruvate carboxylase. The hydrolysis produced Pi and NH4+ in a 1:1 relationship. Values of Vmax. and Km were 0.11 +/- 0.01 mumol of Pi X min-1 X mg-1 and 1.4 +/- 0.1 mM, respectively, in the presence of 10 mM-NaHCO3. If HCO3- was not added, these values were 0.075 +/- 0.014 mumol of Pi X min-1 X mg-1 and 0.76 +/- 0.06 mM. Vmax./Km showed no variation between pH 6.5 and 8.5. The reaction required Mg2+; the activation constants were 0.77 and 0.31 mM at pH 6.5 and 8.5 respectively. Presumably, carbamyl phosphate is hydrolysed by phosphoenolpyruvate carboxylase by a reaction the mechanism of which is related to that of the carboxylation of phosphoenolpyruvate.

Bicarbonates↗
PubMed1986

Modification of an essential amino group of phosphoenolpyruvate carboxylase from maize leaves by pyridoxal phosphate and by pyridoxal phosphate-sensitized photooxidation.

Phosphoenolpyruvate carboxylase from maize leaves was inactivated by pyridoxal 5'-phosphate in the dark and in the light. A two-step reversible mechanism is proposed for inactivation in the dark, which involves the formation of a noncovalent complex prior to a Schiff base with amino groups of the enzyme. Spectral analysis of pyridoxal 5'-phosphate-modified phosphoenolpyruvate carboxylase showed absorption maxima at 432 and 327 nm, before and after reduction with NaBH4, respectively, suggesting that epsilon-amino groups of lysine residues are the reactive groups in the enzyme. A correlation between spectral data and the maximal inactivation obtained with several concentrations of inhibitor allowed us to establish that the incorporation of 4 mol of pyridoxal 5'-phosphate per mole of holoenzyme accounts for total inactivation. The absence of modifier bound to phosphoenolpyruvate carboxylase when the modification was carried out in the presence of phosphoenolpyruvate and MgCl2 suggests the existence of an essential lysine residue at the catalytic site of the enzyme. Modification of phosphoenolpyruvate carboxylase in the light under an oxygen atmosphere resulted in an irreversible inactivation, which was completely protected by phosphoenolpyruvate and MgCl2. Spectral analysis of the photomodified enzyme showed an absorption peak of 320 nm, suggesting light-mediated addition of a nucleophilic residue (probably an imidazole group) to the pyridoxal 5'-phosphate-lysine azomethine bond.

Binding Sites↗
PubMed1986

Active-site-directed inhibition of phosphoenolpyruvate carboxylase from maize leaves by bromopyruvate.

Bromopyruvate is a competitive inhibitor of maize leaf phosphoenolpyruvate carboxylase with respect to phosphoenolpyruvate (Ki: 2.3 mM at pH 8). Relatively low concentrations of this compound completely and irreversibly inactivated the enzyme. The inactivation followed pseudo-first-order kinetics. The haloacid combines first with the carboxylase to give a reversible enzyme-bromopyruvate complex and then alkylates the enzyme. The maximum inactivation rate constant was 0.27 min-1 at pH 7.2 and 30 degrees C and the concentration of bromopyruvate giving half-maximum rate of inactivation was 1.8 mM. The inactivation was prevented by the substrate phosphoenolpyruvate, in the absence or presence of MgCl2, and by the competitive inhibitor P-glycolate. Malate afforded protection at pH 7 but not at pH 8. MgCl2 enhanced the inactivation when it was carried out at pH 7; its effect was mainly due to a decrease in the dissociation constant of the complex between bromopyruvate and the enzyme from 2 to 1.4 mM. This behavior was not observed at pH 8. Analysis of the inactivation at different pH suggests that a group of pKa near 7.5 is important for the binding of the reagent to the carboxylase. Determination of the number of sulfhydryl groups of the native and modified enzyme with [3H]-N-ethylmaleimide suggests that the inactivation correlates with the modification of thiol groups in the enzyme. The substrate prevented the modification of these groups. The results suggest that the alkylating reagent modifies cysteinyl residues at the phosphoenolpyruvate binding site of the carboxylase.

Binding Sites↗
PubMed1985

Quaternary structure of chloroplast F1-ATPase in solution. Conformational changes in spatial arrangement of subunits upon activation.

The hydrodynamic properties of isolated ATPases were studied via their rotational diffusion in buffer solution. Chloroplast F1-ATPase (CF1) and Escherichia coli F1-ATPase (EF1) were covalently labeled with eosinisothiocyanate and then investigated by polarized laser spectroscopy. The rotational correlation time in aqueous buffer of latent (five-subunit) CF1 was 390 ns. Four-subunit (delta-deficient) CF1 showed the same correlation time, however, for three-subunit (delta, epsilon-deficient) CF1 the rotational correlation time was more than eight times larger (3200 ns). The rotational correlation time of activated CF1 was three times larger than the one of latent CF1. These large changes in the rotational correlation times are directly related to changes in the quaternary structure of CF1 upon activation. EF1 was found to behave essentially as activated CF1. Based on the observed rotational correlation times we concluded that the mass distributions of latent CF1 and of delta-deficient CF1 resemble a dimeric arrangement. The structure of delta, epsilon-deficient CF1 more likely resembles a hexagon, the mass centers of the six main subunits lie in one plane. The structure of the activated forms of CF1 can be described best as an intermediate between the dimeric arrangement of latent CF1 and an octahedron. The large changes in the quaternary structure of isolated CF1 are reversed when the activation of the enzyme is reversed.

Chemical Phenomena↗
PubMed1984

On the molecular mechanism of maize phosphoenolpyruvate carboxylase activation by thiol compounds.

Incubation of purified phosphoenolpyruvate carboxylase from Zea mays L. leaves with dithiothreitol resulted in an almost 2-fold increase in the enzymic activity. The activated enzyme showed the same affinity for its substrates and the same sensitivity with respect to malate and oxalacetate inhibition. The activation induced by dithiothreitol was reversed by diamide, an oxidant of vicinal dithiols, suggesting that the redox state of disulfide bonds of the enzyme may be important in the expression of the maximal catalytic activity.Titration of thiol groups before and after activation of maize phosphoenolpyruvate carboxylase by dithiothreitol shows an increase of the accessible groups from 8 to 12 suggesting that the reduction of two disulfide bonds accompanied the activation. The thiols exposed by the treatment with dithiothreitol were available to reagents in nondenatured enzyme and two of them were reoxidized to a disulfide bond by diamide. It is concluded that the mechanism of phosphoenolpyruvate carboxylase activation by dithiothreitol involves the net reduction of two disulfide bonds in the enzyme.

Journal Article↗
PubMed1981

Effects of Hydroxamic Acids Isolated from Gramineae on Adenosine 5'-triphosphate Synthesis in Chloroplasts.

Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7-methoxy-1,4-(2H)-benzoxazin-3(4H)-one (DIMBOA) and the 2-O-beta-d-glucopyranoside of DIMBOA, inhibit photophosphorylation by spinach chloroplasts. Both cyclic and noncyclic photophosphorylations were inhibited to the same extent. The concentrations producing 50% inhibition for DIMBOA and its glucoside were about 1 and 4 millimolar, respectively. These compounds inhibit coupled electron transport but do not affect basal or uncoupled electron transport. Both acids inhibit the ATPase activities of membrane-bound coupling factor 1 (CF(1)) and of purified CF(1). On the basis of these results, it is concluded that DIMBOA and its glucoside act as energy transfer inhibitors of photophosphorylation.

Journal Article↗
PubMed1980

Sulfhydryl groups in photosynthetic energy conservation. VI. Subunit distribution of sulfhydryl groups and disulfide bonds in chloroplast coupling factor and ATPase activity.

The subunit distribution of sulfhydryl groups and disulfide bonds of spinach chloroplasts coupling factor I has been determined. Native coupling factor I with a latent ATPase activity has eight sulfhydryl groups distributed 4 : 2 : 0 : 0 : 2 in the alpha, beta, gamma, delta and epsilon subunits, respectively. Heat treatment of coupling factor I, in addition to the activation of its ATPase activity, induces a dithiol-disulfide interchange between the gamma and the alpha subunit, changing the sulfhydryl groups' distribution to 2 : 2 : 2 : 0 : 2. Reduction of disulfide bonds of coupling factor I by dithioerythritol during heat treatment gives a subunit distribution of 4 : 4 : 4 : 0 : 2, suggesting that native coupling factor I has three disulfide bonds, two in the gamma subunit and one in one of the beta subunits. The results suggest an asymmetric redox state of some of the subunits of coupling factor I and an asymmetric positioning of some of them in the molecular structure of coupling factor I.

Adenosine Triphosphatases↗
PubMed1979

Sulphydryl groups in photosynthetic energy conservation. V. Localization of the new disulfide bridges formed by o-iodosobenzoate in coupling factor of spinach chloroplasts.

1. Chemical modification by o-iodosobenzoate of soluble chloroplast coupling factor 1 (CF1) during heat activation resulted in inhibition of its Ca-ATPase activity and in the formation of two new intrapeptide disulfide bridges as suggested by: (a) the disappearance of three out of four accessible thiol groups, two from gamma and one from a beta subunit as a consequence of CF1 modification by o-iodosobenzoate; (b) the total free sulphydryl groups of CF1 were reduced from 8 to 4 after modification of CF1 by o-iodosobenzoate. Two groups disappeared from beta and two from gamma subunits; (c) a second heating step of CF1 in the presence of 10 mM dithioerythritol reversed the inhibition of the ATPase and reduced both the newly formed disulfide bridges and those present in native CF1. 2. Modification of chloroplasts in the light with o-iodosobenzoate resulted in the inhibition of photophosphorylation and ATPase. CF1 isolated and purified from these chloroplasts had its Ca-ATPase activity inhibited and two new disulfide bridges. The total number of free sulphydryl groups was reduced from 8 to 4 and three accessible groups disappeared from beta and gamma subunits.

Adenosine Triphosphatases↗