Peter Schürmann

Thioredoxin and enzyme regulation

Abstract Thioredoxin, a hydrogen carrier protein that functions in DNA synthesis and in the transformation of sulfur metabolites, has recently been found to serve as a regulatory protein in linking light to the activation of enzymes during photosynthesis.

Characterization of Tic110, a Channel-forming Protein at the Inner Envelope Membrane of Chloroplasts, Unveils a Response to Ca2+ and a Stromal Regulatory Disulfide Bridge

Tic110 has been proposed to be a channel-forming protein at the inner envelope of chloroplasts whose function is essential for the import of proteins synthesized in the cytosol. Sequence features and topology determination experiments presently summarized suggest that Tic110 consists of six transmembrane helices. Its topology has been mapped by limited proteolysis experiments in combination with mass spectrometric determinations and cysteine modification analysis. Two hydrophobic transmembrane helices located in the N terminus serve as a signal for the localization of the protein to the membrane as shown previously. The other amphipathic transmembrane helices are located in the region composed of residues 92–959 in the pea sequence. This results in two regions in the intermembrane space localized to form supercomplexes with the TOC machinery and to receive the transit peptide of preproteins. A large region also resides in the stroma for interaction with proteins such as molecular chaperones. In addition to characterizing the topology of Tic110, we show that Ca2+ has a dramatic effect on channel activity in vitro and that the protein has a redox-active disulfide with the potential to interact with stromal thioredoxin.

Studies on the regulatory properties of chloroplast fructose-1,6-bisphosphatase.

The regulatory properties of chloroplast fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, (EC 3.1.3.11) were examined with a homogeneous enzyme preparation isolated from spinach leaves. The activation of the enzyme, that was earlier shown to occur via reduced thioredoxin, was found to be accompanied by a structural change that took place more slowly than the rate of catalysis. The recently found deactivation of the thioredoxin-activated enzyme by physiological oxidants such as oxidized glutathione and dehydroascorbic acid was also slow relative to catalysis. Under the conditions used, the activated enzyme showed a pH optimum of about 8.0, whereas the corresponding value for the non-activated form was pH 8.8. The importance of the thioredoxin-linked mechanism of enzyme regulation that is effected through photoreduced ferredoxin and ferredoxin-thioredoxin reductase is discussed in relation to other light-controlled regulatory agents in chloroplasts.

Role of cyclic photophosphorylation in photosynthetic carbon dioxide assimilation by isolated chloroplasts

Although the physiological nature of cyclic photophosphorylation is now well documented (MACLACHLAN & PORTER 1959, FORTI & PARISI 1963, URBACH & SIMONIS 1964, WIESSNER & GAFFRON 1964, TANNER et al. 1965, 1966, WIESSNER 1965, NULTSCH 1966, 1967, JESCHKE 1967, RAVEN 1967, RAMIREZ et al. 1968, ZANETTI 1969, GIMMLER 1970, MIGINIAC-MASLOW 1971) there is a divergence of views concerning its involvement in CO2 assimilation. A need for cyclic photophosphorylation seemed to arise from the requirement of an excess of ATP over NADPH (in a ratio of 3 to 2) for the assimilation of CO2 to the level of carbohydrate (CALVIN & BASSHAM 1962) - a requirement that cannot be met by noncyclic photophosphorylation alone which produces ATP and NADPH in a ratio of 1 to 1 (ref. ARNON et al 1958, JAGENDORF 1958, AVRON & JAGENDORF 1959, STILLER & VENNESLAND 1962, TURNER et al 1962, DEL CAMPO et al 1968). Consistent with this conclusion were CO2 assimilation experiments with broken chloroplasts in which sugar phosphates were formed only when both cyclic and noncyclic photophosphorylation were operating in a proper balance (TREBST et al 1959).

Effect of a systemic virus infection on chloroplast function and structure

Abstract The effect of virus infection on chloroplasts was investigated with squash plants systemically infected with squash mosaic virus. Infected leaves showed (i) a shift in products from sugars to amino acids and organic acids; (ii) an increase in cytoplasmic ribosomes and proteins of the enzyme fraction; and (iii) fewer chloroplasts. By contrast, chloroplasts isolated from healthy and virus-infected leaves showed no difference with regard to: (i) products of photosynthetic 14CO2 assimilation; (ii) sensitivity of photosynthetic 14CO2 assimilation to the inhibitors antimycin A and 3-(3′,4′-dichlorophenyl)-1, 1-dimethylurea (DCMU); (iii) rate of cyclic and noncyclic photophosphorylation; (iv) ultracentrifugation profile; (v) activity of the enzymes ribulose-1,5-diphosphate carboxylase, phosphoenolpyruvate (PEP) carboxylase, and malate dehydrogenase; and (vi) chloroplast ultrastructure. It was concluded that squash mosaic virus infection influences reactions of leaf cytoplasm but has no direct effect on photosynthetic activity or ultrastructure of chloroplasts.

Appearance of sedoheptulose 1,7-diphosphatase activity on conversion of chloroplast fructose 1,6-diphosphatase from dimer form to monomer form.

Chloroplast fructose 1,6-diphosphatase isolated at pH 5.5 as the dimer dissociated to the monomer at pH 8.5. When the pH was adjusted from 8.5 back to 5.5, the newly formed monomer partly reassociated to form the dimer. The monomer lacked the fructose diphosphatase activity characteristic of the dimer (measured in the presence of a saturating concentration of Mg++) but retained ferredoxin-dependent activity (measured in the presence of Mg++ plus protein factor and either reduced ferredoxin or dithiothreitol). In addition, the monomer acquired sedoheptulose 1,7-diphosphatase activity that was dependent on either reduced ferredoxin or dithiothreitol and the protein factor.

Role of ferredoxin in the activation of sedoheptulose diphosphatase in isolated chloroplasts

Abstract Sedoheptulose 1,7-diphosphatase activity of isolated spinach chloroplasts shows a requirement for (i) reduced ferredoxin and (ii) a protein factor. Activation by ferredoxin, reduced photochemically by chloroplast fragments, was optimal at pH 7.8 and at a Mg 2+ concentration of 5 mM. The protein factor needed for activation appears to be the same as that required by the chloroplast fructose-1,6-diphosphatase that is activated by reduced ferredoxin. The results indicate that sedoheptulose-1,7-diphosphatase, like fructose-1,6-diphosphatase, is a regulatory enzyme whose activity in chloroplasts is controlled via ferredoxin by light.

Role of the reductive carboxylic acid cycle in a photosynthetic bacterium lacking ribulose 1,5-diphosphate carboxylase

Abstract Evidence is presented that a green photosynthetic bacterium ( Chlorobium thiosulfatophilum , Tassajara) lacks ribulose 1,5-diphosphate carboxylase, the key enzyme of the reductive pentose phosphate cycle—the photosynthetic carbon reduction mechanism characteristic of green plants. The bacterium appears to use exclusively the reductive carboxylic acid cycle (and its associated reactions) not only, as previously recognized, in the photosynthetic conversion of CO 2 to amino acids and organic acids but also to carbohydrates. This conclusion is based on: (i) the absence in cell-free preparations of ribulose 1,5-diphosphate carboxylase; (ii) the assimilation by cell suspensions of 14 CO 2 , [ 14 C]acetate, and [ 14 C]succinate to give the products expected from the operation of the reductive carboxylic acid cycle; (iii) the demonstration in cell-free preparations of all enzymes needed for the conversion of CO 2 and the primary product of the reductive carboxylic acid cycle, acetyl-CoA, to carbohydrate. The results also show that CO 2 concentration influences the products formed by the reductive carboxylic acid cycle, with a low level of CO 2 favoring the synthesis of carbohydrates.

A regulatory mechanism for CO2 assimilation in plant photosynthesis: Activation of ribulose‐1,5‐diphosphate carboxylase by fructose 6‐phosphate and deactivation by fructose 1,6‐diphosphate

Fig. 1. Effect of fructose 6-phosphate and fructose 1,6-d& phosphate on the activity of RuDP carboxylase. The reaction mixture contained the following (added in the order indicated): Tricine buffer, pH 7.5, 200 mM; EDTA, 0.06 mM; MgC12, 1 mM; reduced glutathione, 5 mM; RuDP carboxylase (purified to homogeneity by a procedure based on that of Paulsen and Lane [ 3]), 20 fig: fructose 6-phosphate and fructose l,bdiphosphate, concentrations as shown; NaHCt4C01 (5 X lo-’ cpm ctmole), 1 mM; and RuDP, 0.3 mM. Volume, 0.5 ml; temperature, 25”; reaction time, 10 min. The reaction, carried out in scintillation vials, was stopped with 0.05 ml 6 N HCI; samples were evaporated to dryness and the 14C-phosphoglycerate (PGA) formed was determined in a scintillation counter. The control treatment gave 690 cpm PGA formed. PGA was identified as the reaction product in each of the treatments by thin-layer electrophoresi+chromatography [ 1 l]

Ribulose 1,5-Diphosphate Carboxylase: A Regulatory Enzyme in the Photosynthetic Assimilation of Carbon Dioxide

Publisher Summary This chapter discusses ribulose 1,5-diphosphate (RuDP) carboxylase. One property of RuDP carboxylase that appears to be in conflict with its proposed role in photosynthesis is a requirement for a CO 2 concentration about 100-fold greater than that normally present in air. Since its initial isolation, RuDP carboxylase has continued to attract wide interest not only because of its key role in photosynthesis but also because of its unique features. The carboxylase is present at very high concentrations, accounting for up to 50% of the total soluble protein of leaves. On the basis of its carboxylase activity, RuDP carboxylase has been purified to homogeneity from algae and higher plants and was recently crystallized from tobacco leaves. The discovery that isolated chloroplasts photoassimilate bicarbonate to the level of carbohydrate extended more recently by findings that this reaction can be made to proceed at high rates, up to 60% those of leaves, permits an in vitro estimation of the concentration of bicarbonate needed to sustain a maximal rate of photosynthesis.