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Effect of hydrogen peroxide on spinach (Spinacia oleracea) chloroplast fructose bisphosphatase.

ABSTRACT: Thiol-treated spinach (Spinacia oleracea) chloroplast fructose bisphosphatase (EC 3.1.3.11) is severely inhibited by H2O2, whereas the freshly purified enzyme is little affected. Dithiothreitol reverses inhibition by H2O2, indicating that essential thiol groups are oxidized during H2O2 inactivation. A new role for the dithiol and thioredoxin systems that are operative in illuminated chloroplasts is proposed.

SUBMITTER: Charles SA

PROVIDER: S-EPMC1162009 | biostudies-other | 1980 Aug

REPOSITORIES: biostudies-other

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Spinach (Spinacia oleracea) chloroplast sedoheptulose-1,7-bisphosphatase. Activation and deactivation, and immunological relationship to fructose-1,6-bisphosphatase.

Project description:In this paper we study activation by dithiothreitol and reduced thioredoxins and deactivation by oxidized thioredoxins f of sedoheptulose-1,7-bisphosphatase. The behaviour of the enzyme when chromatographed on a thioredoxin-Sepharose column is also described. The enzyme is autoxidizable upon removal of reducing agents, and is activated when reduced by any of the thioredoxins. This mechanism may allow the regulation of the Calvin cycle upon light-dark and dark-light transitions. The formation of a stable complex between enzyme and thioredoxin could explain the inhibitory effect of high thioredoxin concentrations. The use of immunological techniques shows that sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase are poorly related immunologically.

| S-EPMC1149281 | biostudies-other

Properties of oxidized and reduced spinach (Spinacia oleracea) chloroplast fructose-1,6-bisphosphatase activated by various agents.

Project description:Fructose-1,6-bisphosphatase (FBPase) can be reduced and activated by either dithiothreitol or reduced thioredoxin. This activation is pH-dependent. An amino acid group with a pK value of 5.55 is involved in this process. Both enzyme forms can also be stimulated by agents such as fructose 1,6-bisphosphate, Mg2+, Ca2+ and Ca2+/fructose 1,6-bisphosphate. FBPase reduced by dithiothreitol is more strongly activated than the enzyme reduced by thioredoxin. The specificity constant (kcat./Km) is enhanced over 2.5-25-fold and 1.5-2-fold (depending on the agent used) for FBPase reduced by dithiothreitol and thioredoxin respectively. In both cases, no new kinetic properties appeared. The pH-activity profile of the stimulated enzyme is slightly shifted towards the acidic side with respect to the reduced enzyme. A lag phase is observed in the progress curve of both enzymic forms, treated or untreated. Each agent used to stimulate must induce a new conformation of the enzyme, more active than the initial one, characterized by a specificity constant and a relaxation time. This lag phase tends to disappear when the assay temperature is increased. Temperature has the same effect on the activity of oxidized, reduced and stimulated FBPase, but different effects on the stability of the different forms.

| S-EPMC1151415 | biostudies-other

Action of calcium ions on spinach (Spinacia oleracea) chloroplast fructose bisphosphatase and other enzymes of the Calvin cycle.

Project description:Thiol-treated spinach (Spinacia oleracea) chloroplast fructose bisphosphatase is powerfully inhibited by Ca2+ non-competitively with respect to its substrate, fructose 1,6-bisphosphate. 500 microM-Ca2+ causes virtually complete inhibition and the Ki is 40 microM. Severe inhibition of sedoheptulose bisphosphatase is also caused by Ca2+. A role for Ca2+ in regulation of the Calvin cycle in spinach chloroplasts is proposed.

| S-EPMC1161961 | biostudies-other

pH and kinetic studies of chloroplast sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea).

Project description:The aim of this paper is to study some steady-state kinetic properties of sedoheptulose-1,7-bisphosphatase, its pH-dependence and the effect of a substrate analogue, fructose 2,6-bisphosphate. Studies were carried out with sedoheptulose 1,7-bisphosphate and with fructose 1,6-bisphosphate, an alternative substrate. The pK values are identical for both substrates, and fructose 2,6-bisphosphate behaves like a competitive inhibitor. These results suggest that there exists a unique active site for either sedoheptulose 1,7-bisphosphate or fructose 1,6-bisphosphate on the enzyme molecule. Increasing Mg2+ concentrations shifted the optimum pH. As for fructose-1,6-bisphosphatase, we believe that this shift is due to the neutralization of negative charges near the active centre [Cadet, Meunier & Ferté (1987) Eur. J. Biochem. 162, 393-398]. The free species of sedoheptulose 1,7-bisphosphate and fructose 1,6-bisphosphate are not the usual substrates of enzyme, nor is Mg2+. But the kinetics relative to the (Mg2+-substrate4-)2- complex is not consistent with this complex being the substrate. An explanation of this discrepancy is proposed, involving both the negative charges near the active centre and the positive charges of Mg2+. The observed Vmax. of the reduced enzyme is 65% of the theoretical Vmax. for both substrates, but the observed Vmax. relative to sedoheptulose 1,7-bisphosphate is 3 times the one relative to fructose 1,6-bisphosphate. The specificity constant (kcat./Km), 1.62 x 10(6) M-1.s-1 with respect to sedoheptulose 1,7-bisphosphate compared with 5.5 x 10(4) M-1.s-1 with respect to fructose 1,6-bisphosphate, indicates that the enzyme specificity towards sedoheptulose 1,7-bisphosphate is high but not absolute.

| S-EPMC1149282 | biostudies-other

Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplasts. The effect of hydrogen peroxide and of Paraquat.

Project description:The stroma of spinach chloroplasts contains ascorbic acid and glutathione at millimolar concentrations. [Reduced glutathione]/[oxidized glutathione] and [ascorbate]/[dehydroascorbate] ratios are high under both light and dark conditions and no evidence for a role of oxidized glutathione or dehydroascorbate in the dark-deactivation of fructose bisphosphatase could be obtained. Addition of H2O2 to chloroplasts in the dark decreases the above ratios, an effect that is reversed on illumination. Addition of Paraquat to illuminated chloroplasts caused a rapid oxidation of reduced glutathione and ascorbate, and apparent loss of dehydroascorbate. Paraquat rapidly inactivated fructose bisphosphatase activity, as assayed under physiological conditions.

| S-EPMC1154305 | biostudies-other

Isolation and purification of chloroplastic spinach (Spinacia oleracea) sedoheptulose-1,7-bisphosphatase.

Project description:Higher-plant sedoheptulose-1,7-bisphosphatase was isolated and purified over 200-fold from spinach (Spinacia oleracea) chloroplast stromal extracts to apparent electrophoretic homogeneity by DEAE-Fractogel, molecular sieving on Sephadex G-200 and Blue B dye-matrix affinity chromatography. It is a protein of Mr 66,000, made up of two apparently identical subunits (Mr 35,000). The enzyme is activated by reduced thioredoxin fb in the presence of dithiothreitol. Its specificity towards sedoheptulose 1,7-bisphosphate versus fructose 1,6-bisphosphate is high, but not absolute.

| S-EPMC1147525 | biostudies-other

Properties of freshly purified and thiol-treated spinach chloroplast fructose bisphosphatase.

Project description:Freshly purified spinach chloroplast fructose bisphosphatase is powerfully inhibited by inorganic phosphate competitively with respect to its substrate fructose 1,6-bisphosphate. The concentrations of phosphate and substrate in the chloroplast stroma are such that the enzyme in this form could not operate at a significant rate in vivo. Incubation of the enzyme with dithiothreitol for 24 h decreases the Km for fructose 1,6-bisphosphate from 0.8 to 0.033 mM, decreases the Km for Mg2+ from 9 to 2 mM and substantially alleviates inhibition by inorganic phosphate. The physiological significance of thiol activation of the enzyme is discussed.

| S-EPMC1161446 | biostudies-other

Spinacia oleracea strain:Spinach

Project description:Spinacia oleracea strain:Spinach Raw sequence reads

| PRJNA576552 | ENA

The complete mitochondrial genome sequence of spinach, <i>Spinacia oleracea</i> L.

Project description:Spinach (Spinacia oleracea L.) is an economically important vegetable crop. Here we describe the complete mitochondrial DNA sequence of spinach, which has a length of 329,613 bp and a GC content of 43.4%. It is separated by a pair of directly repeated elements of 7286 bp, to form a large single copy region of 229,375 bp and a small single copy region of 85,666 bp. The genome contains 29 protein-coding genes, 4 pseudogenes, 24 tRNA genes, and 3 rRNA genes. A phylogenetic analysis revealed that spinach was closely related to Beta vulgaris (sugar beet), both belonging to the Amaranthaceae family.

| S-EPMC7799700 | biostudies-literature

Project description:Hyperthermophilic archaea have an unusual phosphatase that exhibits activity toward both inositol-1-phosphate and fructose-1,6-bisphosphate, activities carried out by separate gene products in eukaryotes and bacteria. The structures of phosphatases from Archaeoglobus fulgidus (AF2372) and Methanococcus jannaschii (MJ0109), both anaerobic organisms, resemble the dimeric unit of the tetrameric pig kidney fructose bisphosphatase (FBPase). A striking feature of AF2372, but not of MJ0109, is that the sulfhydryl groups of two cysteines, Cys150 and Cys186, are in close proximity (4 A). A similar arrangement of cysteines has been observed in chloroplast FBPases that are regulated by disulfide formation controlled by redox signaling pathways (ferredoxin/thioredoxin). This mode of regulation has not been detected in any other FBPase enzymes. Biochemical assays show that the AF2372 phosphatase activity can be abolished by incubation with O(2). Full activity is restored by incubation with thiol-containing compounds. Neither the C150S variant of AF2372 nor the equivalent phosphatase from M. jannaschii loses activity with oxidation. Oxidation experiments using Escherichia coli thioredoxin, in analogy with the chloroplast FBPase system, indicate an unexpected mode of regulation for AF2372, a key phosphatase in this anaerobic sulfate reducer.

| S-EPMC2323843 | biostudies-literature

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