Purified Spinach Chloroplasts Research Articles

Leucine synthesis in chloroplasts: Leucine/isoleucine aminotransferase and valine aminotransferase are different enzymes in spinach chloroplasts

Summary This work demonstrates that the complete synthesis of leucine is localized in chloroplasts. Isolated, purified spinach chloroplasts, supplied with 2-oxoisovalerate (3 mmol L −1 ), incorporated [2 −14 C] acetate (2 mmol L −1 ) into leucine as the only amino acid labelled. A leucine/isoleucine aminotransferase and a valine aminotransferase as terminal enzymes of the branched-chain amino acid synthesis (both enzymes belonging to the EC 2.6.1 class) were isolated from chloroplast stroma and purified about 110-fold. The enzymes differed strongly in their substrate specificities. The apparent K m -values for 2-oxoisocaproate and 2-oxo-3-methylvalerate of the leucine/isoleucine aminotransferase and for 2-oxoisovalerate of the valine aminotransferase were 0.12; 0.089 and 1.13 mmol L −1 , respectively. The optimum pH of the aminotransferase reaction was 8. 9. Both enzymes were competitively inhibited by higher concentrations of leucine, isoleucine and valine. However, the efficient regulation of leucine synthesis takes place by a sensitive feedback control of 2-isopropylmalate synthase by leucine at a micromolar level (Hagelstein and Schultz, 1993). Concerning the integration of leucine synthesis into chloroplast metabolism, the problem of pathways competing for the same introductory substrates, acetyl-CoA and 2-oxoisovalerate, is discussed.

Reductive Modification and Nonreductive Activation of Purified Spinach Chloroplast NADP-Dependent Glyceraldehyde-3-phosphate Dehydrogenase

Spinach chloroplast NAD(P)-glyceraldehyde-3-phosphate dehydrogenase (NAD(P)-GAPDH; EC, 1.2.1.13) was purified as the 600-kDa oligomer of low specific activity. Incubation of the enzyme with either a reductant or a 1,3-bisphosphoglycerate (1,3bisPGA) generating system, but most effectively with both, resulted in an increase of the apparent NADPH-dependent activity. Only the 1,3bisPGA treatment caused dissociation and yielded the 150-kDa heterotetramer (A2B2). The higher activity of the tetramer is largely due to a decreasedKMvalue for the substrate 1,3bisPGA. Reductive treatment alone does not dissociate the enzyme. Reduction was equally effective with glutathione as with dithiothreitol or with reduced thioredoxin f. The concentration of 1,3bisPGA required to obtain 50% activity (Ka) was 19.5 ± 4.1 μMfor the untreated enzyme and 2.0 ± 1.4 μMfor the thiol-pretreated enzyme. Thus,in vitro1,3bisPGA, alone or—at much lower concentrations—together with a reductant can activate (and dissociate) NAD(P)-GAPDH. The enzyme exhibits similarKavalues in its reduced and its oxidized form for ATP (1–2 mM), NADP (50–200 μM), and NADPH (0.3–0.5 mM) as positive effectors, but these effectors do not lead to any activation when present together with 0.14 mMNAD. Only 1,3bisPGA retained its characteristic effect in the presence of NAD. The dissociated enzyme reaggregates upon removal of the positive effectors. From these results it is concluded (i) that the role of the reduction of the NAD(P)-GAPDHin vivois to increase its sensitivity toward the activator 1,3bisPGA and (ii) that the actual activation (and aggregation) state of the enzyme in chloroplasts in the light is regulated by the concentration of 1,3bisPGA as activator in the stroma and its actual activity by the availability of 1,3bisPGA as substrate.

Abortive and productive elongation catalysed by purified spinach chloroplast RNA polymerase.

Experimental conditions are reported under which purified spinach chloroplast RNA polymerase catalyses the abortive elongation reaction on a synthetic poly[d(A-T)] template. The reaction only occurs under very stringent conditions and absolutely requires Mn2+ as the metal activator. No reaction can be detected in the presence of Mg2+. Furthermore, the rate of abortive elongation with the chloroplast enzyme is extremely sensitive to the presence of added salts, such as KCl or (NH4)2SO4, in the reaction assays. In the combined presence of Mn2+ and Mg2+, a marked inhibition of abortive elongation is associated with an activation of productive elongation and an increased length of RNA chains. Thus, whereas Mn2+ is more active than Mg2+ for phosphodiester bond formation, it appears that Mg2+ favors the stabilization of the ternary transcription complexes. These results are compared with those obtained under similar conditions for wheat germ RNA polymerase II and Escherichia coli RNA polymerase.

Biosynthesis of aromatic amino acids by highly purified spinach chloroplasts – Compartmentation and regulation of the reactions

The ability of chloroplasts to synthesize aromatic amino acids from CO2 was investigated using highly purified, intact spinach (Spinacia oleracea L. cv. Viking II) chloroplasts and 14CO2. Incorporation of 14C into aromatic amino acids was very low, however, and this was assumed to be due to lack of phosphoenolpyruvate (PEP), one of the substrates for the shikimate/arogenate pathway leading to aromatic amino acids in chloroplasts. Therefore, the glycolytic enzymes phosphoglycerate mutase (EC 2.7.5.3) and enolase (EC 4.2.1.11) were added to the 14CO2 fixation medium in order to convert labelled 3‐phosphoglycerate exported from the intact chloroplasts to 2‐phosphoglycerate and PEP. In this way a part of the glycolytic pathway was reconstituted outside the chloroplasts to substitute for the cytoplasm lost on isolation. The presence of both enzymes in the medium increased incorporation of 14C into Tyr and Phe more than ten‐fold and incorporation into Trp about two‐fold, while total 13CO2 fixation rates were not affected. Our results suggest that chloroplasts do not contain phosphoglycerate mutase or enolase, and that, in vivo, PEP is synthesized in the cytoplasm and imported to the chloroplast stroma for the biosynthesis of aromatic amino acids. The biosynthesis of all three aromatic amino acids was under feedback control. Using expected physiological concentrations (below 100 μM), each of the aromatic amino acids exerted a strict feedback inhibition of its own biosynthesis only.

Amino acid sequence similarity between spinach chloroplast and mammalian gluconeogenic fructose-1,6-bisphosphatase

Chloroplast fructose-1,6-bisphosphatase is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars and the properties of this enzyme are clearly distinct from cytosolic gluconeogenic fructose-1,6-bisphosphatase. Light-dependent activation via a ferredoxin/thioredoxin system and insensitivity to inhibition by AMP are unique characteristics of the chloroplast enzyme. In the present study, purified spinach chloroplast fructose-1,6-bisphosphatase was reduced, S-carboxymethylated with iodoacetic acid, and cleaved with either cyanogen bromide or trypsin. The resulting peptides were purified by reversed-phase high performance liquid chromatography. Automated Edman degradation of some of the purified peptides showed amino acid sequences highly homologous to residues 72–86, 180–199, and 277–319 of pig kidney fructose-1,6-bisphosphatase. These findings suggest a common evolutionary origin for mammalian gluconeogenic and chloroplast fructose-1,6-bisphosphatase, enzymes catalyzing the same reaction but having different functions and modes of regulation.

Identification and mutational analysis of the promoter for a spinach chloroplast transfer RNA gene.

A transcription extract from purified spinach chloroplast was used to test chloroplast DNA sequences for their function as promoter elements. Chloroplast tRNA genes are correctly transcribed in the extract by a soluble RNA polymerase, and precursor molecules are processed into mature tRNAs. Transcription of the spinach chloroplast tRNA2Met gene (trnM2) in vitro requires 5' upstream DNA sequences. Deletion of 5' DNA sequences with exonuclease Bal31 was used to establish the 5' boundary of the promoter region. This boundary is part of a DNA sequence with partial homology to the prokaryotic -35 region. Seventeen base pairs downstream from this sequence a DNA sequence occurs which is homologous to the prokaryotic -10 region. We used synthetic oligonucleotides fused to trnM2 5' deletion mutants to create insertions, deletions and base substitutions in these regions. Internal deletion mutants demonstrated that the -10 promoter element is also required for transcription in vitro. The arrangement of DNA sequences recognised by the chloroplast RNA polymerase resembles the prokaryotic promoter organization.

Presence of adenine phosphoribosyltransferase and adenosine kinase in chloroplasts of spinach leaves

1. 1. Activity of adenine phosphoribosyltransferase and adenosine kinase was detected in purified spinach chloroplasts by using differential centrifugation and discontinuous Percoll density gradients. 2. 2. This is the first report of purine salvage enymes being located in chloroplasts. The rôle of adenine and adenosine salvage in chloroplasts is discussed.

Synthesis of prenyl lipids in cells of spinach leaf. Compartmentation of enzymes for formation of isopentenyl diphosphate.

Purified spinach chloroplasts incorporate [1-14C]isopentenyl diphosphate into prenyl lipids in high yields. The immediate biosynthetic precursors of isopentenyl diphosphate (hydroxymethylglutaryl-CoA, mevalonate, mevalonate-5-phosphate, mevalonate-5-diphosphate), on the other hand, are not accepted as substrates and the corresponding enzymes hydroxymethylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase are not present in the organelles. These enzymes can only be detected in a membrane-bound form at the endoplasmic reticulum (hydroxymethylglutaryl-CoA reductase) and as soluble activities in the cytoplasm. The concept is developed that isopentenyl diphosphate is formed in the cytoplasm as a 'central intermediate' and is distributed then to other cellular compartments (endoplasmic reticulum, plastids, mitochondria) for further biosynthetic utilization.

Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. I. Electrophoretic and immunochemical analyses.

We have developed a fast and reliable method for the separation of two membrane fractions respectively enriched in outer and inner envelope membranes from isolated, intact, purified spinach chloroplasts kept in a hypertonic medium (0.6 M mannitol). This separation was achieved by osmotically shrinking the inner envelope membrane, thus widening the intermembrane space, and then subsequently removing the "loosened" outer envelope membrane by applying low pressure to the shrunken chloroplasts and slowly extruding them through the small aperture of a Yeda press under controlled conditions. By centrifugation of the mixture obtained through a discontinuous sucrose gradient, we were able to separate two membrane fractions having different densities (fraction 2 or light fraction, d = 1.08 g/cm3, and fraction 3 or heavy fraction, d = 1.13 g/cm3). The recent characterization of polypeptides localized on the outer envelope membrane from spinach chloroplasts, E10 and E24 (Joyard, J., Billecocq, A., Bartlett, S. G., Block, M. A., Chua, N.-H., and Douce, R. J. Biol. Chem., 258, 10000-10006) enabled us to characterize our two membrane fractions. Analyses of the polypeptides by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and immunoblotting have shown that fraction 2 (light fraction) was completely devoid of polypeptide E30, which is involved in the transport of phosphate across the inner envelope membrane, but was enriched in polypeptides E10 and E24. The reverse was true for fraction 3 (heavy fraction). Under these conditions, it is clear that fraction 2 is strongly enriched in outer envelope membrane whereas fraction 3 consisted mostly of inner envelope membrane. Indeed, by immunoelectrophoresis, we were able to demonstrate that, on a protein basis, fraction 2 contained about 90% of outer membrane, whereas fraction 3 contained about 80% of inner membrane. Further characterization of the outer envelope membrane was achieved by using thermolysin, a nonpenetrant protease.

On the compartmentation of isopentenyl diphosphate synthesis and utilization in plant cells

Purified spinach chloroplast and daffodil chromoplast preparations do not use mevalonate, phosphomevalonate, and diphosphomevalonate for the synthesis of isopentenyl diphosphate. Isopentenyl diphosphate, on the other hand, is incorporated into plastidal polyprenoids in large amounts. In the presence of a cytoplasmic supernatant, however, mevalonate and the phosphomevalonates were incorporated into the plastidal polyprenoids in equally large amounts, which demonstrates that the enzymes mevalonate kinase (EC 2.7.1.36), phosphomevalonate kinase (EC 2.7.4.2), and diphosphomevalonate decarboxylase (EC 4.1.1.33) are soluble cytoplasmic enzymes and that they apparently do not occur as isoenzymes within the plastids. The concept is developed that isopentenyl diphosphate is a central intermediate in plant polyprenoid formation which is channeled into several compartment for different biosynthetic pathways.

Control of Shikimate Pathway in Spinach Chloroplasts by Exogenous Substrates

Summary In isolated, purified spinach chloroplasts, tryptophan formation by shikimate pathway is strongly enhanced by adding phosphoenylpyruvate, glutamine and serine as substrates. No other substrate is needed, however, added intermediates (especially erythrose-4-phosphate and chorismate) interact with the endogenous pathway intermediates.

Tocopherol and plastoquinone synthesis in spinach chloroplasts subfractions

Subfractions isolated from intact purified spinach chloroplasts are able to prenylate the aromatic moiety of α-tocopherol and plastoquinone-9 precursors. The biosynthesis of α-tocopherol and plastoquinone-9 is a compartmentalized process. The chloroplast envelope membranes are the only site of the enzymatic prenylation in α-tocopherol synthesis whereas the thylakoid membrane is also involved in the prenylation and methylation sequence of plastoquinone-9 biosynthesis. A very active kinase which forms phytyl- PP is localized in the stroma. Phytol but not geranylgeraniol is the polyprenol precursor of the side chain of α-tocopherol in spinach chloroplasts.

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

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.

Transcription activity of a DNA-protein complex isolated from spinach plastids.

A DNA . protein complex of about 150 S is isolated from purified spinach chloroplasts by Sepharose 4B gel filtration. A DNA-dependent RNA polymerase activity is found associated with the complex. This DNA protein complex is able to initiate RNA chains in vitro. The RNA synthesis is more dependent on CTP than other nucleoside triphosphates. 50% of the activity is still present with 0.6 M KCl. The temperature optimum occurs between 30 degrees C and 35 degrees C. Rifampicin and rifamycin SV have no inhibitory effect. TNA products have been characterized by gel filtration and by hybridization with chloroplast DNA (ctDNA). At the beginning of transcription DNA products are linked to the transcription complex and are later detached. The molecular weight of the product ranges between 0.07 X 10(6) and 2 X 10(6). A part of the product (3--4%) has a molecular weight higher than 2 X 10(6). No endogenous RNase activity was present during the molecular weight determinations experiments. Hybridization experiments show that at least 75% of the RNA products are hybridizable with ctDNA and that 40% of these products are composed of chloroplast ribosomal RNA, showing that rDNA is preferentially transcribed.

An essential arginyl residue in the soluble chloroplast ATPase.

The photosynthetic formation of ATP in chloroplasts requires a coupling factor wtih latent ATPase activity, which has been purified to homogeneity [ 1,2] , has a molecular weight of 325 000 [ 1 ] and is made up of five different subunits [3] . Studies intended to characterize the active site(s) of the coupling factor have been carried out by chemical modification with Nethylmaleimide [4,5] , 7-chloro-4~nitrobenzo-2-oxa-I ,3diazole [6] , o-iodosobenzoate [7] ,2,2’-dithio-bis(5nitropyridine) [8,9] and trypsin treatment [6] . Inhibition of the ATPase activity of the chloroplast coupling factor by 7-chloro4-nitrobenzo-2-oxa-1,3diazole suggests the involvement of tyrosine residues in the catalytic site [6] . The participation of arginyl residues in the binding of anionic substrates or ligands in several enzymes have been shown by chemical modification with cu-dicarbonyl reagents [lo-141 . Recently, Borders et al. [lo] suggested that the adenine nucleotide binding site of creatine kinase contains an essential arginyl residue and Marcus et al. [ 1 l] found that modification of an arginyl residue of the mitochondrial ATPase, probably at the hydrolytic site, resulted in enzyme inactivation. The present paper reports the effects of an arginine-specific reagent, 2,3-butanedione in borate buffer, on the ATPase activity of the purified spinach chloroplast coupling factor. The results obtained suggest that there is one essential arginine per active site in the chloroplast ATPase. 2. Experimental

Physical Properties of Spinach Chloroplast Lamellar Proteins

The chlorophyll-containing lamellae within higher plant chloroplasts are the site of the light reactions and associated electron transport reactions of the photosynthetic process (12, 19). Investigations on the chemical composition of purified spinach chloroplast lamellae have shown that they are about 50 % protein and 50 % lipid (13). Of the 2 fractions, the lipid has been studied the most extensively owing to the considerable interest in the photosynthetic pigments. and the majority of the fraction can be accounted for by known compounds (8, 11). The protein fraction contains the 2 cytochromes, f anid b,; and the transitioln metals iron (nonheme), manigalnese and copper (13). This fraction has received very little direct attention, presumably owing to the extreme insolubility of the material at phvsiological pH and the lack of appropriate enzymological tools to cope with the problem. Davenport and Hill (3) purified cytochromle f from an ammoniacal ethanolic extract of fresh parsley leaves by ammonium sulfate fractionation and calcium phosphate gel adsorption. The pure protein was shown to be MW = 110,000 and contained 2 hemes. Indirect evidence concerning the properties of the protein fraction comes from work primarily designed to investigate the nature of the chlorophyll-protein link in attempts to elucidate the state of chlorophyll in vivo (14,15). For this purpose a wide variety of detergents and organic solvents have been used to solubilize the membranes of chloroplasts. It was decided that a similar use of detergents for the removal of lipid from the lamelflae and solubilizationi of the protein would be a fruitful approach to the studv of the chloroplast protein fraction. These methods have also been shown to be of great value in the study of the mitochonidrion where similar problems of protein insolubility prevail (5). This report describes the preparation and some physical properties of the protein fraction of chlloroplast lamellae in an attempt to derive informiiationl pertinent to the molecular architecture of the photosynthetic membrane.

The deoxyribonucleic acid content of purified spinach chloroplasts

Chloroplasts isolated from spinach leaves in aqueous media and purified by a variety of techniques were found to contain DNA. Identification of the DNA was based on its chemical properties and on the absorption spectra of the reaction products of the material with diphenylamine, cysteine-sulfuric acid, and indole. Values of 1.3–7.1 μg. of DNA per milligram of protein were found; however, values of 3–4 μg. DNA per milligram protein were typical.