Isomers in thioredoxins of spinach chloroplasts

We have developed a method for the concomitant purification of several components of the ferredoxin/thioredoxin system of spinach chloroplasts. By applying this method to spinach-leaf extract or spinach-chloroplast extract we separated and purified three thioredoxins indigenous to chloroplasts. The three thioredoxins, when reduced, will activate certain chloroplast enzymes such as fructose-1,6-bisphosphatase and NADP-dependent malate dehydrogenase. Fructose-1,6-bisphosphatase is activated by thioredoxin f exclusively. Malate dehydrogenase is activated by thioredoxin mb and thioredoxin mc in a similar way, and it is also activated by thioredoxin f but with different kinetics. All three thioredoxins have very similar relative molecular masses of about 12,000 but distinct isoelectric points of 6.1 (thioredoxin f), 5.2 (thioredoxin mb) and 5.0 (thioredoxin mc). The amino acid composition as well as the C-terminal and N-terminal sequences have been determined for each thioredoxin. Thioredoxin f exhibits clear differences in amino acid composition and terminal sequences when compared with the m-type thioredoxins. Thioredoxin mb and thioredoxin mc, however, are very similar, the only difference being an additional lysine residue at the N-terminus of thioredoxin mb. Amino acid analyses, terminal sequences, immunological tests and the activation properties of the thioredoxins support our conclusion that thioredoxins mb and mc are N-terminal redundant isomers coming from one gene whereas thioredoxin f is a different protein coded by a different gene.     

Induction by different thioredoxins of ATPase activity in coupling factor 1 from spinach chloroplasts

ATPase activity of the coupling factor 1, CF1, isolated from spinach chloroplasts, was enhanced by reduction with dithiothreitol. Reduced thioredoxins from spinach chloroplasts, Escherichia coli and human lymphocytes replaced dithiothreitol as reductant and activator of the ATPase. CF1 must be in an oxidized activated state to be further activated by reduced thioredoxin. This state was obtained either by heating CF1 or removing the inhibitory intrinsic epsilon subunit from CF1. Efficiency and primary structure of the different thioredoxins were compared. The progressive addition of KCl during ATPase activation by reduced thioredoxin increases then decreases this process. We proposed that three basic amino acids corresponding to arginine 73 and lysines 82 and 96 in Escherichia coli thioredoxin play an important role in the anchorage of the thioredoxin to the negatively charged surface of the CF1 and are involved in the dual effect of KCl. The variations in the screening effect of the negative charges of the CF1 surface by K+ ions can indeed explain the changes in the anchorage of these 3 basic amino acids with concomitant variation in ATPase activity. Human thioredoxin must be 10 times more concentrated than Escherichia coli or spinach chloroplast thioredoxin to exhibit the same activation effect on the ATPase. This fact was related to the properties of a sequence equivalent to the part from amino acid 59 to 72 in Escherichia coli thioredoxin. This part which joins the two lobes of the thioredoxin is more hydrophilic and more negatively charged in human thioredoxin than in Escherichia coli or spinach chloroplast thioredoxin. Although ATPase activation was obtained at a very low concentration of the reduced spinach chloroplast thioredoxin, the thioredoxin formed only a loose complex with CF1.     

Further characterization and amino acid sequence of m-type thioredoxins from spinach chloroplasts

The complete primary structure of m-type thioredoxin from spinach chloroplasts has been sequenced by conventional sequencing including fragmentation, Edman degradation and carboxypeptidase digestion. As already reported [Tsugita, A., Maeda, K. & Schürmann, P. (1983) Biochem. Biophys. Res. Commun. 115, 1-7] these thioredoxins contain the same active-site sequence as thioredoxins from other sources. Based on the amino acid sequence thioredoxin mc contains 103 residues, has a relative molecular mass of 11425 and a molar absorption coefficient at 280 nm of 19 300 M-1 cm-1. The spinach thioredoxin mc has an overall homology of 44% with the thioredoxin from Escherichia coli mainly due to differences in the N-terminal and C-terminal regions.     

Hydrogenation of geranylgeraniol : two pathways exist in spinach chloroplasts

The reduction of geranylgeranylpyrophosphate to phytylpyrophosphate in spinach chloroplasts is described for the first time. The reductase is localized in the chloroplast envelope. By contrast, the reduction of the geranylgeranyl moiety in Chl synthesis is catalyzed in the thylakoids (via Chl synthetase). NADPH functions as electron donor in both reactions. Chl synthetase is firmly bound to the thylakoid membranes, and very little activity is found in the stroma fraction. Chl synthetase in chloroplasts can use the pyrophosphate ester of either phytol, geranylgeraniol, or farnesol, phytylpyrophosphate being the preferred substrate. Exogenous Chlide exhibits no influence on Chl synthesis by chloroplast subfractions.     

Ferrochelatase of spinach chloroplasts

Spinach chloroplasts catalyse the incorporation of Fe(2+) into protoporphyrin, mesoporphyrin and deuteroporphyrin to form the corresponding haems. This ferrochelatase activity was detected by pyridine haemochrome formation with acetone-dried powders of chloroplasts, or from the formation of [(59)Fe]haems by intact chloroplasts. Decreasing the mitochondrial contamination of the chloroplasts by density-gradient centrifugation did not cause any loss of activity: spinach ferrochelatase appears to be principally a chloroplast enzyme. The characteristics of the enzyme were examined by using [(59)Fe]haem assay. The activity was pH-dependent: for both mesohaem and protohaem formation there were two pH maxima, a major peak at about pH7.8 and a smaller peak at about pH9.2. Lineweaver-Burk plots showed that the K(m) for Fe(2+) incorporation into protoporphyrin was 8mum and that for Fe(2+) incorporation into mesoporphyrin was 36mum. At non-saturating Fe(2+) concentrations the K(m) for protoporphyrin was 0.2mum and that for mesoporphyrin was 0.4mum. Ferrochelatase was not solubilized by treatment of chloroplasts with ultrasound but was solubilized by stirring in 1% (w/v) Tween 20 at pH10.4. Unlike the rat liver mitochondrial enzyme, chloroplast ferrochelatase was not stimulated by treatment with selected organic solvents. The spinach enzyme was inactive in aerobic conditions and it was shown by using an oxygen electrode that under such conditions the addition of Fe(2+) to buffer solutions caused a rapid uptake of dissolved oxygen, believed to be due to the oxidation of Fe(2+) to Fe(3+); Fe(3+) is not a substrate for ferrochelatase.     

At least two Toc34 protein import receptors with different specificities are also present in spinach chloroplasts

The receptor components of the chloroplast protein import machinery, Toc34 and Toc159, are both encoded by small gene families in Arabidopsis thaliana. Recent results suggest that each member of these families preferentially interacts with different groups of precursor proteins. Here we address the question, whether multiple homologous Toc receptors are unique to Arabidopsis or whether they are a general phenomenon in plants. Indeed, in spinach we could identify at least two Toc34 proteins with different substrate specificities as demonstrated by competition and antibody inhibition experiments. In addition, an analysis of the available genomic data revealed the presence of at least two Toc34 homologs in six other plant species.     

Fluorescence induction in intact spinach chloroplasts

Under conditions in which the Photosystem II quencher is rapidly reduced upon illumination, either after a preillumination or following treatment with dithionite, the fluorescence-induction curve of intact spinach chloroplasts (class I type) displays a pronounced dip. This dip is probably identical with that observed after prolonged anaerobic incubation of whole algal cells (“I-D dip”). It is inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea and occurs in the presence of dithionite, sufficient to reduce the plastoquinone pool. It is influenced by far red light, methylviologen, anaerobiosis and uncouplers in a manner consistent with the interpretation that it represents a photochemical quenching of fluorescence by an electron transport component situated between the Photosystem II quencher and plastoquinone. Glutaraldehyde inhibition may indicate that protein structural changes are involved.     

Starch degradation in isolated spinach chloroplasts

A method for loading isolated intact spinach (Spinacia oleracea L.) chloroplasts with ¹⁴C-starch is described. These intact chloroplasts were incubated aerobically in the dark for 30 minutes. Radioactivity in starch declined and glyceric acid 3-phosphate and maltose were the major radioactive products. It is proposed that starch is degraded within the chloroplast to glyceric acid 3-phosphate and to maltose.     

Glycolate formation in intact spinach chloroplasts

Photosynthetic ¹⁴CO₂ fixation and the accumulation of photosynthetic products and the response of each process to both 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) and ascorbate were investigated in the intact spinach chloroplast.     

Crystal structures of two Bacillus carboxylesterases with different enantioselectivities

Naproxen esterase (NP) from Bacillus subtilis Thai I-8 is a carboxylesterase that catalyzes the enantioselective hydrolysis of naproxenmethylester to produce S-naproxen (E > 200). It is a homolog of CesA (98% sequence identity) and CesB (64% identity), both produced by B. subtilis strain 168. CesB can be used for the enantioselective hydrolysis of 1,2-O-isopropylideneglycerol (solketal) esters (E > 200 for IPG-caprylate). Crystal structures of NP and CesB, determined to a resolution of 1.75 Å and 2.04 Å, respectively, showed that both proteins have a canonical α/β hydrolase fold with an extra N-terminal helix stabilizing the cap subdomain. The active site in both enzymes is located in a deep hydrophobic groove and includes the catalytic triad residues Ser130, His274, and Glu245. A product analog, presumably 2-(2-hydroxyethoxy)acetic acid, was bound in the NP active site. The enzymes have different enantioselectivities, which previously were shown to result from only a few amino acid substitutions in the cap domain. Modeling of a substrate in the active site of NP allowed explaining the different enantioselectivities. In addition, Ala156 may be a determinant of enantioselectivity as well, since its side chain appears to interfere with the binding of certain R-enantiomers in the active site of NP. However, the exchange route for substrate and product between the active site and the solvent is not obvious from the structures. Flexibility of the cap domain might facilitate such exchange. Interestingly, both carboxylesterases show higher structural similarity to meta-cleavage compound (MCP) hydrolases than to other α/β hydrolase fold esterases.     

EGTA, a calcium chelator, inhibits electron transport in photosystem II of spinach chloroplasts at two different sites

A fifteen minute incubation of spinach chloroplasts with the divalent Ca²⁺ chelator, EGTA, in concentrations 50–250 μM, inhibits electron transport through both photosystems. All photosystem II partial reactions, including indophenol, ferricyanide and the DCMU-insensitive silicomolybdate reduction are inhibited from 70–100%. The photosystem II donor reaction, diphenyl carbazide → indophenol, is also inhibited, indicating that the inhibition site comes after the Mn²⁺ site, and that the first Ca²⁺ effect noted (site II) is not on the water oxidation enzyme, as is commonly assumed, but between the Mn²⁺ site and plastoquinone A pool. The other photosystem II effect of EGTA (Ca²⁺ site I), occurs in the region between plastoquinone A and P700 in the electron transport chain of chloroplasts. About 50% inhibition of the reaction ascorbate + TMPD → methyl viologen is given by incubation with 200 μM EGTA for 15 min. Ca²⁺ site II activity can be restored with 20 mM CaCl₂. Ca²⁺ site I responds to Ca²⁺ and plastocyanin added jointly. More than 90% activity in the ascorbate + TMPD → methylviologen reaction can be restored. Various ways in which Ca²⁺ ions could affect chloroplast structure and function are discussed. Since EGTA is more likely to penetrate chloroplast membranes than EDTA, which is known to remove CF₁, the coupling factor, from chloroplast membranes, and since Mg²⁺ ions are ineffective in restoring activity, it is concluded that Ca²⁺ may function in the electron transport chain of chloroplasts in a hitherto unsuspected manner.