α-1,4-Glucan phosphorylase forms from leaves of spinach (Spinacia oleracea L.) I. In situ localization by indirect immunofluorescence

Antisera were raised against two forms of -1,4-glucan phosphorylase (EC 2.4.1.1) which had been purified from leaves of Spinacia oleracea L. Immunoglobulin G preparations were isolated from the antisera, and their specificity was ensured by immunoplobulin G preparations were used for in situ localization of the two phosphorylase forms in spinach leaf thin sections by indirect immuno-fluorescence. Both enzyme forms were present in the palisade and spongy parenchyma and in the guard cells, but their intracellular distribution was complementary. One phosphorylase form (designated as the chloroplastic form) was restricted to the stromal space of chloroplasts whereas the other (the non-chloroplastic form) was present only in the cytoplasm of chloroplast-containing cells. Thus, the phosphorylases represent two distinct compartment-specific enzyme forms which reside within the same photosynthetically active mesophyll cell.Abbreviations DBM diazobenzyloxymethyl - FITC fluorescein isothiocyanate - IgG immunoglobulin G - PMSF phenylmethylsulfonyl fluoride     

A rapid method for isolation of purified,physiologically active chloroplasts,used to study the intracellular distribution of amino acids in pea leaves

A procedure is described for the rapid (<5 min) isolation of purified, physiologically active chloroplasts from Pisum sativum L. Mitochondrial and microbody contamination is substantially reduced and broken chloroplasts are excluded by washing through a layer containing a treated silica sol. On average the preparations contain 93% intact chloroplasts and show high rates of ¹⁴CO₂ fixation and CO₂-dependent O₂ evolution (over 100 mol/mg chlorophyll(chl)/h); they are also able to carry out light-driven incorporation of leucine into protein (4 nmol/mg chl/h). The amino-acid contents of chloroplasts prepared from leaves and from leaf protoplasts have been determined. Asparagine is the most abundant amino acid in the pea chloroplast (>240 nmol/mg chl), even thought it is proportionately lower in the chloroplast relative to the rest of the cell. The chloroplasts contain about 20% of many of the amino acids of the cell, but for individual amino acids the percentage in the chloroplast ranges from 8 to 40% of the cell total. Glutamic acid, glutamine and aspartic acid are enriched in the chloroplasts, while asparagine, homoserine and -(isoxazolin-5-one-2-yl)-alanine are relatively lower. Leakage of amino acids from the chloroplast during preparation or repeated washing was ca. 20%. Some differences exist between the amino-acid composition of chloroplasts isolated from intact leaves and from protoplasts. In particular, -aminobutyric acid accumulates to high levels, while homoserine and glutamic acid decrease, during protoplast formation and breakage.     

Purification of a non-chloroplastic α-glucan phosphorylase from spinach leaves

The non-chloroplastic -glucan phosphorylase (EC 2.4.1.1) from spinach leaves has been purified to homogeneity as revealed by dodecylsulfate gel electrophoresis. Both purification and separation from the chloroplastic phosphorylase were achieved by chromatography on Sepharose-bound dextrin. The chloroplastic phosphorylase did not bind to Sepharose-dextrin and was removed from the column by washing with buffer, as verified by polyacrylamide gel electrophoresis of the buffer eluate and by chromatography of a preparation from isolated intact chloroplasts. The non-chloroplastic phosphorylase did bind to a high extent to Sepharose-dextrin and could be eluted by a dextrin gradient. Based on dodecylsulfate gel electrophoresis and pyridoxal phosphate determination, a molecular weight of about 90,000 was found for the monomer. Molecular-weight determination by porosity density gradient electrophoresis and gel filtration on Sephadex G-200 suggested that the native enzyme is a dimer, as are other phosphorylases.Abbreviations DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetic acid - G1P glucose 1-phosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - PMSF phenylmethyl sulphonyl fluoride - SDS sodium dodecylsulfate - Tris Tris (hydroxymethyl)aminomethane Dedicated to Professor Dr. A. Pirson on the occasion of his 70th birthday     

Isolation and partial characterization of clathrin-coated vesicles from pea (Pisum sativum L.) cotyledons

Summary Clathrin-coated vesicles have been isolated from cotyledons of both developing and germinating pea seeds using differential centrifugation, ribonuclease treatment, discontinuous sucrose gradients, and isopycnic centrifugation on a linear D₂O-Ficoll gradient. The yield of coated vesicles from developing pea cotyledons was exceptional, being 1.6 × higher than the yield from hog and bovine brain, 5.3 × higher than the yield from carrot suspension cultures, and 13 × the yield from cotyledons of germinating pea seeds. The pea coated vesicles are similar to other plant coated vesicles in size (approximately 80 nm in diameter) and in having a clathrin heavy chain of 190,000 M_r. The lipid phosphorus to protein ratio, 190–250 nmol P per mg protein, of the coated vesicles from plants is comparable to that reported for highly purified coated vesicles from animals. The nondenatured pea clathrin reacted weakly with an antiserum to bovine brain clathrin, but pea clathrin denatured by sodium dodecyl sulfate did not.Abbreviations CLC Clathrin light chain - CHC clathrin heavy chain - CV coated vesicle - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TBS Tris buffered saline     

α-1,4-glucan phosphorylase forms from leaves of spinach (Spinacia oleracea L.)

Peptide patterns and immunological properties of the cytoplasmic and chloroplastic -1,4-glucan phosphorylase (EC 2.4.1.1) from spinach leaves have been studied and were compared with those of phosphorylases from other sources. The two spinach leaf phosphorylases were immunologically different; a limited cross-reactivity was observed only at high antigen or antibody concentrations. Peptide mapping of the two enzymes resulted in complex patterns composed of more than 20 fragments; but no peptide was electrophoretically identical in both proteins. Approximately 13 to 15 of the fragments exhibited antigeneity but no cross-reactivity of any peptide was observed. Therefore, the two compartment-specific phosphorylase forms from spinach leaves represent isoenzymes possessing different primary structures. Peptide patterns of potato tuber and rabbit muscle phosphorylase were different from those of the two spinach leaf enzymes. Although the potato tuber phosphorylase resides in the plastidic compartment and is kinetically closely related to the chloroplastic spinach enzyme, it reacted more strongly with the anti-cytoplasmic-phosphorylase immunoglobulin G. Similar results were obtained with rabbit muscle phosphorylase. These observations support the assumption that the chloroplast-specific phosphorylase isoenzyme has a higher structural diversity than does the cytoplasmic counterpart.Abbreviations EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PEG polyethylene glycol (approx. MW 8000) I=Schächtele and Steup 1986     

Positional Specificity and Fatty Acid Selectivity of Purified sn-Glycerol 3-Phosphate Acyltransferases from Chloroplasts

Soluble acyl-CoA:sn-glycerol 3-phosphate acyltransferases (EC 2.3.1.15) which are localized in chloroplasts were purified from leaves of Pisum sativum and Spinacia oleracea and obtained free from interfering activities. The purification raised the specific activities by factors of about 1,000 for pea and 200 for spinach preparations. In pea chloroplasts, acyltransferase activity occurs in two soluble forms with apparent isoelectric points of 6.3 and 6.6. For both forms, the same molecular weight of about 42,000 was determined. The enzyme from spinach chloroplasts showed a slightly higher molecular weight and a lower isoelectric point of 5.2.     

In-vitro degradation of starch granules isolated from spinach chloroplasts

The initial reactions of transitory starch degradation in Spinacia oleracea L. were investigated using an in-vitro system composed of native chloroplast starch granules, purified chloroplast and non-chloroplast forms of phosphorylase (EC 2.4.1.1) from spinach leaves, and -amylase (EC 3.2.1.1) isolated from Bacillus subtilis. Starch degradation was followed by measuring the release of soluble glucans, by determining phosphorylase activity, and by an electron-microscopic evaluation following deep-etching of the starch granules. Starch granules were readily degraded by -amylase but were not a substrate for the chloroplast phosphorylase. Phosphorolysis and glucan synthesis by this enzyme form were strictly dependent upon a preceding amylolytic attack on the starch granules. In contrast, the non-chloroplast phosphorylase was capable of using starch-granule preparations as substrate. Hydrolytic degradation of the starch granules was initiated at the entire particle surface, independently of its size. As a result of amylolysis, soluble glucans were released with a low degree of polymerization. When assayed with these glucans as substrate, the chloroplast phosphorylase form exhibited a higher apparent affinity and a higher reaction velocity compared with the non-chloroplast phosphorylase form. It is proposed that transitory starch degradation in vivo is initiated by hydrolysis; phosphorolysis is most likely restricted to a pool of soluble glucan intermediates.Abbreviations Glc1P Glucose 1-phosphate - Mes 2(N-morpholino)ethanesulfonic acid - Pi Orthophosphate     

Mode of glucan degradation by purified phosphorylase forms from spinach leaves

The glucan specifity of the purified chloroplast and non-chloroplast forms of -1,4-glucan phosphorylase (EC 2.4.1.1) from spinach leaves (Steup and E. Latzko (1979), Planta 145, 69–75) was investigated. Phosphorolysis by the two enzymes was studied using a series of linear maltodextrins (degree of polymerization 11), amylose, amylopectin, starch, and glycogen as substrates. For all unbranched glucans (amylose and maltodextrins G₅–G₁₁), the chloroplast phosphorylase had a 7–10-fold higher apparent affinity (determined by initial velocity measurements) than the non-chloroplast phosphorylase form. For both enzyme forms, the minimum chain length required for a significant rate of phosphorolysis was five glucose units. Likewise, phosphorolysis ceased when the maltodextrin was converted to maltotetraose. With the chloroplast phosphorylase, maltotetraose was a linear competitive inhibitor with respect to amylose or starch (K _i-0.1 mmol 1^-1); the inhibition by maltotetraose was less pronounced with the non-chloroplast enzyme. In contrast to unbranched glucans, the non-chloroplast phosphorylase exhibited a 40-, 50-, and 300-fold higher apparent affinity for amylopectin, starch, and glycogen, respectively, than the chloroplast enzyme. With respect to these kinetic properties the chloroplast phosphorylase resembled the type of maltodextrin phosphorylase.Abbreviations G1P Glucose 1-phosphate - MES 2(N-morpholino)ethane sulphonic acid - P_i orthophosphate - Tris Tris(hydroxymethyl)aminomethane     

Intracellular localization of serine acetyltransferase in spinach leaves

Intact chloroplasts isolated from spinach leaves by a combination of differential and Percoll density gradient centrifugation and free of mitochondrial and peroxisomal contamination contained about 35% of the total leaf serine acetyltransferase (EC 2.3.1.30) activity. No appreciable activity of the enzyme could be detected in the gradient fractions containing broken chloroplasts, mitochondria, and peroxisomes. L-cysteine added to the incubation mixture at 1 mM almost completely inhibited serine acetyltransferase activity, both of leaf and chloroplast extracts. D-cysteine was much less inhibitory. L-cystine up to 5 mM and O-acetyl-L-serine up to 10 mM had no effect on the enzyme activity. When measured at pH 8.4, the enzyme extracted from the leaves had a K _m for L-serine of 2.4, the enzyme from the chloroplasts a K _m of 2.8 mM.Abbreviations NAS N-acetyl-L-serine - NADP-GPD NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - OAS O-acetyl-L-serine - OASSase O-acetyl-L-serine sulfhydrylase - 3-PGA D-3-phosphoglycerate - SATase serine acetyltransferase     

Soluble and microsomal glutatione S-transferase activities in pea seedlings (Pisum sativum L.)

Epicotyl and primary leaves of pea seedlings (Pisum sativum L., var. Alaska) were found to contain soluble and microsomal enzymes catalyzing the addition of glutathione to the olefinic double bond of cinnamic acid. Glutathione S-cinnamoyl transfer was also obtained with enzyme preparations from potato slices and cell suspension cultures of parsley and soybean.The pea transferases had pH-optima between pH 7.4 and 7.8 K_m-values were 0.1–0.4 mM and 1–4 mM for cinnamic acid and glutathione, respectively. V-values were between 2–15 nmol mg^-1 protein x min.Chromatography on Sephacryl S-200 indicated that the soluble pea glutathione S-cinnamoyl transferase activity existed in molecular weight forms of 37,000, 75,000, and 150,000. The glutathione-dependent cleavage of the herbicide fluorodifen was catalyzed by a different soluble enzyme activity which eluted in molecular weight positions of 47,000 and/or 82,000.The microsomal fraction from pea primary leaves also catalyzed the conjugation of the carcinogen benzo[]pyrene with glutathione.Abbreviations GSH glutathione - DDE 1,1-Dichloro-2,2-bis-(4-chlorophenyl)-ethylene - DDMU l-Chloro-2,2-bis-(4-chlorophenyl)-ethylene     

Bacteriorhodopsin in a bloom of halobacteria in the Dead Sea

A dense bloom of red halobacteria developed in the Dead Sea in the summer 1980, bacterial densities of up to 1.9 x10⁷ cells ml^-1 were observed. The population consisted of two types: pleomorphic, cup-shaped cells and rod-shaped cells. A high content of bacteriorhodopsin was found in the bloom (up to 0.4 nmol per mg protein). The rod-shaped Halobacterium was isolated and was shown to contain bacteriorhodopsin.Abbreviations 20 specific gravity at 20°C - Tris Tris-(hydroxymethyl)-aminomethane     

α-1,4-Glucan phosphorylase forms in the green alga Eremosphaera viridis

In extracts of the unicellular green alga Eremosphaera viridis DeBary (Chlorococcales, Chlorophyceae) the average specific activity of α-1,4-glucan phosphorylase (E.C. 2.4.1.1) was 200 nmol glucose 1-phosphate formed per min and mg protein. Using continuous and discontinuous electrophoresis on polyacrylamide gels, three phosphorylase forms were found. When the log of the relative mobility of the three enzyme forms was plotted versus the acrylamide gel concentration (Ferguson plot) parallel lines were obtained, indicating that the three enzymes were indiscernible with respect to molecular weight. Electrophoresis on density gradient gels resulted in three activity zones lying close to each other. The relative molecular mass ( M _r) of the three enzymes was estimated to be around 180,000 with a difference of less than 7,000 between the small and the large forms.     

Resolution of two molecular forms of sucrose-phosphate synthase from maize,soybean and spinach leaves

Two forms of sucrose-phosphate synthase (EC 2.4.1.14) were resolved from leaves of three species, maize (Zea mays L. cv. Pioneer 3184), soybean (Glycine max (L.) Merr., cv. Ransom) and spinach (Spinacia oleracea L. cv. Resistoflay) by hydroxyapatite Ultrogel chromatography, using a 75-mM (designated peak 1) and 250-mM (peak 2) K-phosphate discontinuous-gradient elution. Rechromatography of the two forms showed that they were not readily interconvertible. The distribution of activity between the two forms differed among species and changed during purification of the enzyme. Recovery of peak-1 activity was specifically lowered when maize leaf extracts were prepared in the absence of magnesium, indicating that the two forms may differ in stability. In addition, the forms of the enzyme from maize differed in the extent of glucose-6-phosphate activation. These results provide evidence for the existence of multiple forms of sucrose-phosphate synthase in leaves of different species and that the forms differ in regulatory properties.Abbreviations Fru6P fructose 6-phosphate - Glc6P glucose 6-phosphate - HAU hydroxyapatite Ultrogel - Pi inorganic phosphate - SPS sucrose-phosphate synthase - UDP uridine 5-diphosphate - UDPG uridinediphosphate glucose Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh. Paper No. 10511 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh. Supported in part by USDA Competitive Research Grant No. 85-CRCR-1-1568     

Comparison of the kinetic properties,inhibition and labelling of the phosphate translocators from maize and spinach mesophyll chloroplasts

The kinetic properties of the phosphate translocator from maize (Zea mays L.) mesophyll chloroplasts have been determined. We have used a double silicone-oil-layer centrifugation system in order to obtain true initial uptake rates in forward-reaction experiments. In addition, it was possible to perform back-exchange experiments and to study the effects of illumination and of preloading the chloroplasts with different substrates on transport. It is shown that the phosphate translocator from mesophyll chloroplasts of maize, a C₄ plant, transports inorganic phosphate and phosphorylated C₃ compounds in which the phosphate group is linked to the C₃ atom (e.g. 3-phosphoglycerate and triose phosphate). The affinities of the transported metabolites towards the translocator protein are about one order of magnitude higher than in mesophyll chloroplasts from the C₃ plant, spinach. In contrast to the phosphate translocator from C₃-mesophyll chloroplasts, that of C₄-mesophyll chloroplasts catalyzes in addition the transport of C₃ compounds where the phosphate group is attached to the C₂ atom (e.g. 2-phosphoglycerate, phosphoenolpyruvate). The phosphate translocator from both chloroplast types is strongly inhibited by pyridoxal-5-phosphate (PLP), 2,4,6-trinitrobenzenesulfonic acid and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). In the case of the spinach translocator protein these inhibitors were shown to react with the same amino-acid residue at the substrate binding site, and one molecule of either DIDS or PLP is obviously required per substrate binding site for the inactivation of the translocation process. In the functionally active dimeric translocator protein only one substrate-binding site appears to be accessible at a particular time, indicating that the site might be exposed to each side of the membrane in turn. Using [³H]-H₂DIDS for the labelling of maize mesophyll envelopes the radioactivity was found to be associated with two polypeptides of about 29 and 30 kDa. Since Western-blot analysis showed that only the 30 kDa polypeptide reacted with an antiserum directed against the spinach phosphate translocator protein it is suggested that this polypeptide presumably represents the phosphate translocator from maize mesophyll chloroplasts.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - PEP phosphoenolpyruvate - 2-,3-PGA 2-,3-phosphoglycerate - PLP pyridoxal-5-phosphate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TNBS 2,4,6-trinitrobenzenesulfonic acid - triose P triose phosphate This work was supported by the Deutsche Forschungsgemeinschaft     

Light activation and molecular-mass changes of NAD(P)-glyceraldehyde 3-phosphate dehydrogenase of spinach and maize leaves

Light modulation of chloroplast glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) has been investigated. Complete activation of NADPH-dependent activity is achieved at 25 W.m^–2 photosynthetically active radiation in spinach (Spinacia oleracea L.) and 100 W.m^–2 in maize (Zea mays L.) leaves. Light activation is stronger in spinach (fivefold on average) than in maize (twofold), which shows higher dark activity. The NADH dependent activity does not change appreciably. Several substrate activators can simulate in vitro the light effect with recovery of latent NADPH-dependent activity of spinach enzyme, but they are almost inactive with maize enzyme. A mixture of activators has been devised to fully activate the spinach enzyme under most conditions. The NAD(P)-GAPDH protein can be resolved by rapid gel filtration (fast protein liquid chromatography) into three conformers which have different molecular masses according to the light conditions. Enzyme from darkened leaves or chloroplasts, or dichlorophenyl-1,1-dimethylurea-treated chloroplasts is mainly a 600-kDa regulatory form with low NADPH-dependent activity relative to NADH-activity. Enzyme from spinach leaves or chloroplasts during photosynthesis is mainly a 300-kDa oligomer, which along with the 600-kDa form also occurs in leaves of darkened maize. The conformer of illuminated maize leaves is mainly a 160-kDa species. Results are consistent with a model of NAD(P)-GAPDH freely interconvertible between protomers of the 160-kDa (or 300-kDa intermediate) form with high NADPH-activity, produced in the light by the action of thioredoxin and activating metabolites (spinach only), and a regulatory 600-kDa conformer with lower NADPH-activity produced in darkness or when photosynthesis is inhibited. This behavior is reminiscent of the in-vitro properties of purified enzyme; therefore, it seems unlikely that NAD(P)-GAPDH in the chloroplast is part of a stable multienzyme complex or is bound to membranes.Abbreviations AEM activator equilibrium mixture - Chl chlorophyll - DCMU dichlorophenyl 1,1-dimethylurea - DTT dithiothreitol - FPLC fast protein liquid chromatography - NAD(P)-GAPDH glyceraldehyde 3-phosphate dehydrogenase, NAD(P)-dependent - PAR photosynthetic active radiation - PGK phosphoglycerate kinase - Tricine N-tris(hydroxy-methyl) methyl-glycine This work was supported by grants from the Ministero dell'Università e della Ricerca Seientifica e Tecnologica (40%, years 1990 and 1991).     

One carbon metabolism in methanogenic bacteria

Two strains of Methanosarcina (M. Barkeri strain MS, isolated from sewage sludge, and strain UBS, isolated from lake sediments) were found to have similar cellular properties and to have DNA base compositions of 44 mol percent guanosine plus cytosine. Strain MS was selected for further studies of its one-carbon metabolism. M. barkeri grew autotrophically via H₂ oxidation/CO₂ reduction. The optimum temperature for growth and methanogenesis was 37°C. H₂ oxidation proceeded via an F₄₂₀-dependent NADP⁺-linked hydrogenase. A maximum specific activity of hydrogenase in cell-free extracts, using methyl viologen as electron acceptor, was 6.0 mol min · mg protein at 37°C and the optimum pH (9.0). M. barkeri also fermented methanol andmethylamine as sole energy sources for growth. Cell yields during growth on H₂/CO₂ and on methanol were 6.4 and 7.2 mg cell dry weight per mmol CH₄ formed, respectively. During mixotrophic growth on H₂/CO₂ plus methanol, most methane was derived from methanol rather than from CO₂. Similar activities of hydrogenase were observed in cell-free extracts from H₂/CO₂-grown and methanol-grown cells. Methanol oxidation apparently proceeded via carrierbound intermediates, as no methylotrophy-type of methanol dehydrogenase activity was observed in cell-free extracts. During growth on methanol/CO₂, up to 48% of the cell carbon was derived from methanol indicating that equivalent amounts of cell carbon were derived from CO₂ and from an organic intermediate more reduced than CO₂. Cell-free extracts lacked activity for key cell carbon synthesis enzymes of the Calvin cycle, serine path, or hexulose path.Abbreviations CAPS cycloaminopropane sulfonic acid - CH₃-SCoM methyl coenzyme M - DCPIP 2,6-dichlorophenolindophenol - DEAE diethylaminoethyl - dimethyl POPOP 1,4-bis-2-(4-mothyl-5-phenyloxazolyl)-benzene - DNA deoxyribonucleic acid - dpm dismtegrations per min - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - F₄₂₀ factor 420 - G+C guanosine plus cytosine - NAD⁺ nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - PBBW phosphate buffered basal Weimer - PMS phenazine methosulfate - PPO 2,5-diphenyloxazole - rRNA ribosomal ribonucleic acid - RuBP ribulose-1,5-bisphosphate - Tris tris-hydroxymethyl-aminomethane - max maximum specific growth rate     

Subcellular localization of the common shikimate-pathway enzymes in Pisum sativum L.

5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19), 3-dehydroquinate dehydratase (EC 4.2.1.10) and shikimate: NADP⁺ oxidoreductase (EC 1.1.1.25) were present in intact chloroplasts and root plastids isolated from pea seedling extracts by sucrose and modified-silica density gradient centrifugation. In young (approx. 10-d-old) seedling shoots the enzymes were predominantly chloroplastic; high-performance anion-exchange chromatography resolved minor isoenzymic activities not observed in density-gradientpurified chloroplasts. The initial enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was also associated with intact density-gradient-purified chloroplasts. 3-Dehydroquinate synthase (EC 4.6.1.3) and shikimate kinase (EC 2.7.1.71) were detected together with the other pathway enzymes in stromal preparations from washed chloroplasts. Plastidic EPSP synthase was inhibited by micromolar concentrations of the herbicide glyphosate.Abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - DEAE diethylaminoethyl - DHQase 3-dehydroquinate dehydratase - DTT dithiothreitol - EPSP 5-enolpyruvylshikimate 3-phosphate - SORase shikimate:NADP⁺ oxidoreductase     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

-Isopropylmalate (IPM) synthase, the first enzyme in the biosynthesis of l-leucine, was purified to a specific activity of 12 mole/min x mg protein from the valine-isoleucine double auxotrophic mutant A-81 of the hydrogen bacterium Alcaligenes eutrophus H 16. The activity in crude extracts of derepressed cells was 0.106 moles of isopropylmalate formed per min and per mg protein. Gel electrophoresis and regel electrophoresis of the isolated main band resulted in several distinct bands, which were not altered by the additions of substrate -ketoisovalerate, feedback inhibitor leucine or other effectors.The isoelectric points of the enzyme protein was between 3.9 and 4.0. The molecular weight was 114500 daltons and 100000 respectively in the absence and presence of the feedback inhibitor leucine. The enzyme activity depended strongly on the pH, the optimum is at pH 8.2. The enzyme was could labile and exhibits temperature anomalies.Abbreviations Used CoA Coenzyme A - Tris Tris(hydroxymethyl)aminomethane hydrochloride - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - IPM -Isopropylmalate - KIV -Ketoisovalerate Prepared from doctoral thesis of the University of Göttingen 1973     

Sucrose-phosphate synthase is regulated via metabolites and protein phosphorylation in potato tubers,in a manner analogous to the enzyme in leaves

(i) Sucrose-phosphate synthase (SPS) was purified 40-fold from stored potato (Solanum tuberosum L.) tubers to a final specific activity of 33–70 nkat·(mg protein)^–1 via batch elution from diethylaminoethyl (DEAE)-sephacel, polyethylene glycol (PEG) precipitation and Mono Q anion-exchange chromatography. (ii) Immunoblotting revealed a major and a minor band with molecular weights of 124.8 kDa and 133.5 kDa, respectively. Both bands were also present in extracts prepared in boiling SDS to exclude proteolysis. No smaller polypeptides were seen, except when the preparations were incubated before application on a polyacrylamide gel. (iii) The enzyme preparation was activated by glucose-6-phosphate and inhibited by inorganic phosphate. Both effectors had a large effect on the K _m (fructose-6-phosphate) and the K _m (uridine-5-diphosphoglucose) with phosphate acting antagonistically to glucose-6-phosphate. (iv) Preincubation of potato slices with low concentrations of okadaic acid or microcystin resulted in a three- to fourfold decrease in the activity of SPS when the tissue was subsequently extracted and assayed. The decrease was especially marked when the assay contained low concentrations of substrates and glucose-6-phosphate, and inorganic phosphate was included. Preincubation with mannose or in high osmoticum resulted in an increase of SPS activity. (v) Analogous changes were observed in germinating Ricinus communis L. seedlings. After preincubation of the cotyledons in glucose, high SPS activity could be measured, whereas okadaic acid, omission of glucose, or addition of phosphate or sucrose led to a large decrease of SPS activity in the selective assay. (vi) It is argued that SPS from non-photosynthetic tissues is regulated by metabolites and by protein phosphorylation in an analogous manner to the leaf enzyme.Abbreviations Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - Pi inorganic phosphate - PGI phosphoglucose isomerase - PP2A phosphoprotein phosphatase 2A - PEG polyethyleneglycol - SPS sucrose-phosphate synthase - UDPGlc uridine-5-diphosphoglucose This work was supported by the Deutsche Forschungsgemeinschaft, the BMFT and Sandoz AG, Basel, Switzerland. We are grateful to Prof. E. Beck (Pflanzenphysiologie, Bayreuth, Germany) for providing us with laboratory facilities, and to Dr. U. Sonnewald (Institut für Genbiologische Forschung, Berlin, Germany) for many discussions and providing us with unpublished data.     

On the control of long-chain-fatty acid synthesis in isolated intact spinach (Spinacia oleracea) chloroplasts

1. Chloroplasts isolated from spinach leaves by using the low-ionic-strength buffers of Nakatani & Barber [(1977) Biochim. Biophys. Acta.461, 510-512] had higher rates of HCO(3) (-)-dependent oxygen evolution (up to 369mumol/h per mg of chlorophyll) and higher rates of [1-(14)C]acetate incorporation into long-chain fatty acids (up to 1500nmol/h per mg of chlorophyll) than chloroplasts isolated by using alternative procedures. 2. Acetate appeared to be the preferred substrate for fatty acid synthesis by isolated chloroplasts, although high rates of synthesis were also measured from H(14)CO(3) (-) in assays permitting high rats of photosynthesis. Incorporation of H(14)CO(3) (-) into fatty acids was decreased by relatively low concentrations of unlabelled acetate. Acetyl-CoA synthetase activity was present 3-4 times in excess of that required to account for rates of [1-(14)C]acetate incorporation into fatty acids, but pyruvate dehydrogenase was either absent or present in very low activity in spinach chloroplasts. 3. Rates of long-chain-fatty acid synthesis from [1-(14)C]acetate in the highly active chloroplast preparations, compared with those used previously, were less dependent on added cofactors, but showed a greater response to light. The effects of added CoA plus ATP, Triton X-100 and sn-glycerol 3-phosphate on the products of [1-(14)C]acetate incorporation were similar to those reported for less active chloroplast preparations. 4. Endogenous [(14)C]acetyl-CoA plus [(14)C]malonyl-CoA was maintained at a constant low level even when fatty acid synthesis was limited by low HCO(3) (-) concentrations. Endogenous [(14)C]acyl-(acyl-carrier protein) concentrations increased with increasing HCO(3) (-) concentration and higher rates of fatty acid synthesis, but were slightly lower in the presence of Triton X-100. It is proposed that rates of long-chain-fatty acid synthesis in isolated chloroplasts at saturating [1-(14)C]acetate concentrations and optimal HCO(3) (-) concentrations may be primarily controlled by rates of removal of the products of the fatty acid synthetase.