Localization of ATP Sulfurylase and O-Acetylserine(thiol)lyase in Spinach Leaves

The intracellular compartmentation of ATP sulfurylase and O-acetylserine(thiol)lyase in spinach (Spinacia oleracea L.) leaves has been investigated by isolation of organelles and fractionation of protoplasts. ATP sulfurylase is located predominantly in the chloroplasts, but is also present in the cytosol. No evidence was found for ATP sulfurylase activity in the mitochondria. Two forms of ATP sulfurylase were separated by anion-exchange chromatography. The more abundant form is present in the chloroplasts, the second is cytosolic. O-Acetylserine(thiol)lyase activity is located primarily in the chloroplasts and cytosol, but is also present in the mitochondria. Three forms of O-acetylserine(thiol)lyase were separated by anion-exchange chromatography, and each was found to be specific to one intracellular compartment. The cytosolic ATP sulfurylase may not be active in vivo due to the unfavorable equilibrium constant of the reaction, and the presence of micromolar concentrations of inorganic pyrophosphate in the cytosol, therefore its role remains unknown. It is suggested that the plant cell may be unable to transport cysteine between the different compartments, so that the cysteine required for protein synthesis must be synthesized in situ, hence the presence of O-acetylserine(thiol)lyase in the three compartments where proteins are synthesized.     

The Second Serine Acetyltransferase, Bacterial-type O-Acetylserine (thiol) Lyase and Eukaryotic-type O-Acetylserine (thiol) Lyase from the Primitive Red Alga Cyanidioschyzon merolae

O -acetylserine (thiol) lyase (OASTL) genes were isolated by screening a genomic library of the primitive red alga Cyanidioschyzon merolae and named cmSAT2, and cmOASTL1 and cmOASTL2, respectively. cmSAT2 encoded a polypeptide of 406 amino acids. cmSAT2 was encoded on chromosome IX. cmOASTL1 and cmOASTL2 encoded for polypeptides of 389 and 390 amino acids, respectively. A molecular phylogenic tree of the amino acid sequences suggested that cmOASTL1 belongs to the group of eukaryotic plant OASTL while cmOASTL2 belongs to the bacterial type OASTL. cmOASTL1 and cmOASTL2 were encoded on chromosomes XVII and VIII, respectively. In Northern blot analyses, the probes for cmOASTL1 and cmOASTL2 hybridized with 1.4 kb and 1.3 kb bands, respectively. The identity of cmOASTL1 and cmOASTL2 was confirmed by genetic complementation in an OASTL- deficient mutant of Escherichia coli NK3 using sulfate or sulfide as a sole source of sulfur. Received 19 March 2001/ Accepted in revised form 7 May 2001     

Purification and Some Properties of Plant Serine Acetyltransferase

Serine acetyltransferase (SATase) that catalyzes the conversionof L-serine to O-acetyl-L-serine (OAS) in the presence of acetyl-CoAwas highly purified from rape leaf extract, using a coupledassay system in which OAS is converted to cysteine by enzymaticaction of exogenous cysteine synthase. Through purificationprocedures including heat treatment, ammonium sulfate fractionationand successive column chromatographies on DEAE-Toypearl, Blue-Toyopearland Toyopearl HW-55, the specific activity was raised to about4,800-fold over that in the crude extract. The molecular weightof rape enzyme was estimated to be about 350,000 by gel filtrationcolumn chromatography. The cysteine-forming activity of thefinal preparation was completely dependent on L-serine, acetyl-CoAand sulfide. However, this preparation had low activity forL-cysteine synthesis from L-serine even in the absence of exogenouscysteine synthase, suggesting that plant SATase exists as ahigh-molecular weight enzyme complexed with cysteine synthase. (Received November 6, 1987; Accepted March 25, 1988)     

Subcellular Distribution of O-Acetylserine(thiol)lyase in Cauliflower (Brassica oleracea L.) Inflorescence

The subcellular localization of O-acetyiserine(thiol)lyase (EC 4.2.99.8) in nongreen tissue from higher plants has been studied using purified proplastids, mitochondria, and protoplasts from cauliflower (Brassica oleracea L.) buds as a source of subcellular fractions. O-Acetylserine(thiol)lyase has been detected in both organelles (proplastids and mitochondria) and a cytosolic extract obtained by protoplast fractionation. We confirmed these observations, demonstrating that a form of the enzyme different in global charge and separated from others by anion-exchange chromatography corresponded to each subcellular location. Our observations are consistent with the need for cysteine biosynthesis in each subcellular compartment where the synthesis of proteins occurs.     

Purification and characterization of O-acetylserine (thiol) lyase from spinach chloroplasts.

O-Acetylserine (thiol) lyase, the last enzyme in the cysteine biosynthetic pathway, was purified to homogeneity from spinach leaf chloroplasts. The enzyme has a molecular mass of 68,000 and consists of two identical subunits of Mr 35,000. The absorption spectrum obtained at pH 7.5 exhibited a peak at 407 nm due to pyridoxal phosphate, and addition of O-acetylserine induced a considerable modification of the spectrum. The pyridoxal phosphate content was found to be 1.1 per subunit of 35,000, and the chromophore was displaced from the enzyme by O-acetylserine, leading to a progressive inactivation of the holoenzyme. Upon gel filtration chromatography on Superdex 200, part of the chloroplastic O-acetylserine (thiol) lyase eluted in association with serine acetyltransferase at a position corresponding to a molecular mass of 310,000 (such a complex called cysteine synthase has been characterized in bacteria). The activity of O-acetylserine (thiol) lyase was optimum between pH 7.5 and 8.5. The apparent Km for O-acetylserine was 1.3 mM and for sulfide was 0.25 mM. The calculated activation energy was 12.6 kcal/mol at 10 mM O-acetylserine. The overall amino-acid composition of spinach chloroplast O-acetylserine (thiol) lyase was different than that determined for the same enzyme (cytosolic?) obtained from a crude extract of spinach leaves. A polyclonal antibody prepared against the chloroplastic O-acetylserine (thiol) lyase exhibited a very low cross-reactivity with a preparation of mitochondrial matrix and cytosolic proteins suggesting that the chloroplastic isoform was distinct from the mitochondrial and cytosolic counterparts.     

Purification and Molecular Properties of the Thylakoid-Bound Ascorbate Peroxidase in Spinach Chloroplasts

The hydrogen peroxide that is photoproduced in thylakoids isscavenged by the thylakoid-bound ascorbate peroxidase (tAPX)[Miyake and Asada (1992) Plant Cell Physiol. 33: 541]. tAPXwas purified from spinach thylakoids to homogeneity as judgedby SDS-polyacrylamide gel electrophoresis, and its molecularproperties were studied. Spinach tAPX was a monomer with a molecularweight of 40,000, which is about 10,000 higher than that ofthe stromal ascorbate peroxidase (sAPX) from spinach chloroplasts.tAPX cross-reacted with the antibody raised against sAPX fromtea leaves, as determined by Western blotting, which also providedevidence for the higher molecular weight of tAPX from spinachthylakoids than that of tea sAPX. The amino acid sequence ofthe amino-terminal region of tAPX showed a low degree of homologyto those of cytosolic APXs from spinach, pea and Arabidopsisthaliana, but a high degree of homology to that of stromal APXfrom tea. Thus, the amino-terminal region of tAPX seems notto be a domain required for binding of the enzyme to the thylakoidmembranes. tAPX contained protoheme IX, as identified by itspyridine hemochromogen, and gave a Soret peak at 403 nm and433 nm with an a band at 555 nm in its oxidized and reducedforms, respectively. Resembling sAPX but differing from cytosolicAPX, tAPX showed high specificity for ascorbate as the electrondonor. tAPX was inhibited by cyanide, thiol-modifying reagents,thiols and several suicide inhibitors, such as hydroxyurea andp-aminophenol. ¹Present address: Beijing Vegetable Research Centre, PO Box2443, Beijing, China.     

Purification and Properties of Formyltetrahydrofolate Synthetase from Spinach

Formyltetrahydrofolate synthetase (E. C. 6. 3. 4. 3) was found in fresh spinach leaves and purified about 60-fold by treatments of ammonium sulfate, protamine sulfate, dialysis, and DEAE-cellulose column chromatography. Some properties of the enzyme were investigated. Optimum pH was found to be 7.5, and optimum temperature was observed to be at 37°C. In the enzyme reaction, FAH₄ and formate were required specifically as the substrates, and Mg⁺⁺ and ATP were essential components. The Michaelis constants for dl-FAH₄, formate, ATP and magnesium chloride were 1.7×10^?3 m, 1.7×10^?2 m, 4.1×10^?4 m and 3.3×10^?3 m, respectively. The primary product formed in the reaction catalyzed by the enzyme was suggested as N¹⁰-formyl-FAH₄ spectrophotometrically. It was observed that the enzyme also catalyzed the reverse reaction. The possible role of the enzyme in plants was discussed.     

Properties of a Membrane-bound Phosphatase from the Thylakoids of Spinach Chloroplasts

A 3-phosphoglycerate phosphatase activity of about 2 micromoles per minute per milligram chlorophyll is associated with the thylakoid membranes of spinach chloroplasts. The K_m for 3-phosphoglycerate is 3 millimolar. The enzyme can be solubilized from thylakoid membranes by treatment with 0.33 molar MgCl₂ or sodium deoxycholate. The activity is not stimulated by sulfhydryl reagents or the addition of 10 millimolar MgCl₂. The enzymic activity is insensitive to ethylenediaminetetraacetate. The pH optimum is broad, between 5.5 to 7.5. Although the substrate specificity is broad, 3-phosphoglycerate is the best substrate of those tested at neutral pH. However, p-nitrophenyl phosphate was a more effective substrate at pH 5.5. The enzyme exhibits the general characteristics of an acid phosphatase.     

Successful Fertilization Requires the Presence of at Least One Major O-Acetylserine(thiol)lyase for Cysteine Synthesis in Pollen of Arabidopsis

The synthesis of cysteine (Cys) is a master control switch of plant primary metabolism that coordinates the flux of sulfur with carbon and nitrogen metabolism. In Arabidopsis (Arabidopsis thaliana), nine genes encode for O-acetylserine(thiol)lyase (OAS-TL)-like proteins, of which the major isoforms, OAS-TL A, OAS-TL B, and OAS-TL C, catalyze the formation of Cys by combining O-acetylserine and sulfide in the cytosol, the plastids, and the mitochondria, respectively. So far, the significance of individual OAS-TL-like enzymes is unresolved. Generation of all major OAS-TL double loss-of-function mutants in combination with radiolabeled tracer studies revealed that subcellular localization of OAS-TL proteins is more important for efficient Cys synthesis than total cellular OAS-TL activity in leaves. The absence of oastl triple embryos after targeted crosses indicated the exclusiveness of Cys synthesis by the three major OAS-TLs and ruled out alternative sulfur fixation by other OAS-TL-like proteins. Analyses of oastlABC pollen demonstrated that the presence of at least one functional OAS-TL isoform is essential for the proper function of the male gametophyte, although the synthesis of histidine, lysine, and tryptophan is dispensable in pollen. Comparisons of oastlABC pollen derived from genetically different parent plant combinations allowed us to separate distinct functions of Cys and glutathione in pollen and revealed an additional role of glutathione for pollen germination. In contrast, female gametogenesis was not affected by the absence of major OAS-TLs, indicating significant transport of Cys into the developing ovule from the mother plant.Sulfur assimilation in plants is hallmarked by two reaction sequences, namely sulfate reduction and Cys synthesis. The sulfate reduction pathway consists of three steps and produces sulfide from sulfate, which is available in the soil and transported into the roots by specific transporters (Takahashi et al., 2011). Sulfide is subsequently incorporated into the amino acid O-acetylserine (OAS) by O-acetylserine(thiol)lyase (OAS-TL; EC 2.5.1.47) to produce Cys (Hell and Wirtz, 2011). Cys then serves as the sulfur source for all organic metabolites containing reduced sulfur in plants, including proteins, cofactors, and secondary metabolites. The tripeptide glutathione (GSH) is one of the most important Cys-derived metabolites, since it has an important function in redox homeostasis and the control of development (Meyer and Rausch, 2008). Impaired GSH synthesis negatively affects growth of the shoot and root system of Arabidopsis (Arabidopsis thaliana; Vernoux et al., 2000; Xiang et al., 2001), and loss-of-function mutants for the first enzyme (GSH1, Glu-Cys ligase; EC 6.3.2.2) or the second enzyme (GSH2, glutathione synthase; EC 6.3.2.3) of the two-step pathway leading to GSH formation show an embryo- and seedling-lethal phenotype, respectively (Cairns et al., 2006; Pasternak et al., 2008).Cys synthesis by OAS-TL constitutes the direct link between carbon and nitrogen (OAS) as well as sulfur (sulfide) metabolism and, therefore, can be designated as one of the central reactions in plant primary metabolism. The genome of the model plant Arabidopsis encodes nine OAS-TL-like enzymes: OAS-TL A1 (At4g14880), OAS-TL B (At2g43750), and OAS-TL C (At3g59760) are the major isoforms and are localized in the cytosol, plastids, and mitochondria, respectively (Jost et al., 2000). OAS-TL A2 (At3g22460) encodes a truncated and nonfunctional protein (Jost et al., 2000). In the following, therefore, OAS-TL A1 is referred to as OAS-TL A. CYS D1 (At3g04940) and CYS D2 (At5g28020) show OAS-TL activity in vitro (Yamaguchi et al., 2000). Whether they contribute to net Cys synthesis in vivo is unknown (Heeg et al., 2008). CS26 (At3g03630) encodes a plastidic S-sulfocysteine synthase, which prefers thiosulfate instead of sulfide as substrate and produces S-sulfocysteine (Bermúdez et al., 2010). Whether thiosulfate is taken up from the soil or formed within the plant is unclear, but its presence in Arabidopsis was demonstrated (Tsakraklides et al., 2002). However, the synthesis of S-sulfocysteine from thiosulfate potentially constitutes an alternative sulfur fixation pathway. So far, CS26 was shown to be important for the regulation of redox homeostasis in plastids under certain stress conditions (Bermúdez et al., 2010). DES1 (At5g28030; formerly known as CS-LIKE) is a Cys desulfhydrase (EC 4.4.1.15) that releases sulfide in the cytosol (Alvarez et al., 2010). As a Cys-consuming enzyme, it contributes to Cys homeostasis, especially in late vegetative development and under certain stress conditions (Alvarez et al., 2010, 2012). CYS C1 (At3g61440), finally, encodes a mitochondrial β-cyanoalanine synthase (EC 4.4.1.9), which detoxifies cyanide by incorporation into Cys (Yamaguchi et al., 2000; Watanabe et al., 2008a; García et al., 2010). The major isoforms OAS-TL A, OAS-TL B, and OAS-TL C as well as CYS D1 and CYS D2 can interact with serine acetyltransferase (SAT; EC 2.3.1.30) in the cysteine synthase complex (CSC; Heeg et al., 2008). Although SAT acetylates Ser at the hydroxyl group to form OAS, the direct substrate of OAS-TL, formation of the CSC has no substrate-channeling function but contributes to the demand-driven regulation of Cys synthesis (Hell and Wirtz, 2011).The subcellular compartmentation of Cys precursor formation is a remarkable feature of Cys synthesis in higher plants that implies a high degree of regulation between the participating compartments: while sulfate is exclusively reduced to sulfide in plastids (Takahashi et al., 2011), the synthesis of OAS and the incorporation of sulfide take place in all three compartments where SAT and OAS-TL are present, namely in the cytosol, plastids, and mitochondria. Reverse genetics approaches proved a certain redundancy between the different SAT and OAS-TL isoforms, which demonstrates that sulfide, OAS, and Cys can be exchanged between these compartments (Haas et al., 2008; Heeg et al., 2008; Watanabe et al., 2008a, 2008b). Indeed, sulfide can easily diffuse through membranes (Mathai et al., 2009), but OAS and Cys need to be actively transported. However, the identity of these transporters is unknown. Although sulfide, OAS, and Cys can pass the mitochondrial membrane (Wirtz et al., 2012), the loss-of-function mutant for mitochondrial OAS-TL C is the only single oastl knockout mutant that displays a significant growth phenotype (Heeg et al., 2008). This result was astonishing, since OAS-TL C contributes only 5% to extractable foliar OAS-TL activity (Heeg et al., 2008). The retarded growth of the oastlC mutant, however, cannot be explained by the lack of sulfide detoxification in mitochondria by OAS-TL C, due to an alternative detoxification mechanism for sulfide in mitochondria (Birke et al., 2012). These data question the total redundancy between the different OAS-TL isoforms and suggest specific functions in the different subcellular compartments.Despite its central position in the primary metabolism of higher plants, fundamental questions about Cys synthesis are still unanswered. First, the contribution of OAS-TL-like proteins, especially CYS D1, CYS D2, and CS26, to the fixation of sulfur in planta is unknown. Second, the significance of Cys synthesis by the major OAS-TL proteins in the different subcellular compartments during sporophyte and gametophyte development is unclear. In this study, we addressed these questions using a reverse genetics approach. We were able to prove that fixation of sulfur is carried out exclusively by the major OAS-TL isoforms OAS-TL A, OAS-TL B, and OAS-TL C and elucidated specific functions for OAS-TL A in the cytosol and OAS-TL C in mitochondria of leaf cells. Furthermore, we demonstrate that Cys can be supplied by the mother plant for the development of female gametophytes lacking OAS-TL activity. In contrast, the presence of at least one functional OAS-TL isoform is essential in the male gametophyte.     

Purification and Some Properties of Alcohol Acetyltransferase from Banana Fruit

Isoamyl acetate, one of the major characteristic aroma compoundsof banana fruit (Musa sapientum L.), was synthesized by thecondensation of acetyl-CoA and isoamyl alcohol under the actionof alcohol acetyltransferase, which was found to be localizedin the soluble fraction of pulp cells. The activity of thisenzyme increased with the ripening of banana fruit. The enzyme was purified about 62-fold. The purified enzyme wasvery labile at pHs lower than pH 7.0 but relatively stable atpH 7.5{small tilde}9. The enzyme was most active at 30C andpH 8.5. The molecular weight was estimated to be about 40,000by gel filtration. Its K_m values for acetyl-CoA and isoamylalcohol were 50 µM and 0.4 mM, respectively. (Received January 28, 1985; Accepted May 30, 1985)     

Purification and Properties of Nitrite Reductase from Spinach Leaves

Nitrite reductase (EC 1.6.6.4) has been purified 730-fold from spinach leaves. The enzyme catalyzes the reduction of nitrite to ammonia, with the use of reduced form of methyl viologen and ferredoxin. A stoichiometry of one molecule of nitrite reduced per molecule of ammonia formed has been found. KCN at 2.5×10^-4 m inhibited nitrite reductase activity almost completely. Purified enzyme was almost homogeneous by disk electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 61,000 from gel filtration. Nitrite reductase, in the oxidized form, has absorption maxima at 276, 388 and 573 mμ. Both methyl viologen and ferredoxin linked nitrite reductase activities of the enzyme were inactivated on exposure to low ionic strength.     

Characteristics of Prompt and Delayed Fluorescence from Spinach Chloroplasts

Effects of ferricyanide, dichlorophenyldimethylurea (DCMU), and uncouplers of phosphorylation on the prompt and delayed fluorescences from spinach chloroplasts are described. Any factor that affects the yield of prompt fluorescence will similarly influence the intensity of delayed fluorescence. This idea, recently investigated by Lavorel, should be expressed in terms of a “live” component of fluorescence; that is, the component from chlorophyll associated with the photochemical traps of System II. Some of the effects of ferricyanide and DCMU on delayed fluorescence can then be explained in terms of effects on the yield of prompt fluorescence. From the internal consistency of the explanation, applied to various observations, a judgment can be made that most of the prompt fluorescence observed initially when dark-adapted chloroplasts are first illuminated is “dead,” coming from chlorophyll not associated with trap II. The live fluorescence is represented almost entirely by the time-varying component that develops during illumination. The observed intensity of delayed fluorescence can be divided by the yield of live prompt fluorescence to give an intrinsic delayed fluorescence. This intrinsic delayed fluorescence is proportional to the square root of exciting light intensity (as long as the excitation is not saturating) and decays with second order kinetics. This behavior may reflect the photochemical formation and second order dissipation of an oxidized product of Photosystem II.     

Purification and Characterization of Leu-Proteinase, the Leucine Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves

The leucine specific serine proteinase present in the soluble fraction of leaves from Spinacia oleracea L. (called Leu-proteinase) has been purified by acetone precipitation and a combination of gel-filtration, ion exchange, and adsorption chromatography. This enzyme shows a molecular weight of 60,000 ± 3,000 daltons, an isoelectric point of 4.8 ± 0.1, and a relative electrophoretic mobility of 0.58 ± 0.03. The Leu-proteinase catalyzed hydrolysis of p-nitroanilides of N-α-substituted(-l-)amino acids as well as of chromogenic macromolecular substrates has been investigated between pH 5 and 10 at 23 ± 0.5°C and I = 0.1 molar. The enzyme activity is characterized by a bell-shaped profile with an optimum pH value around 7.5, reflecting the acid-base equilibrium of groups with pK_a values of 6.8 ± 0.1 and 8.2 ± 0.1 (possibly the histidyl residue present at the active site of the enzyme and the N-terminus group). Among the substrates considered, N-α-benzoyl-l-leucine p-nitroanilide shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 1 × 10⁻⁹ molar. In agreement with the enzyme specificity, only N-α-tosyl-l-leucine chloromethyl ketone, di-isopropyl fluorophosphate and phenylmethylsulfonyl fluoride, among compounds considered specific for serine enzymes, strongly inhibit the Leu-proteinase. Accordingly, the enzyme activity is insensitive to cations, chelating agents, sulfydryl group reagents, and activators.     

Properties and Partial Purification of Choline Acetyltransferase from the Nematode Caenorhabditis elegans

We have stabilized and studied choline acetyltransferase from the nematode Caenorhabditis elegans. The enzyme is soluble, and two discrete forms were resolved by gel filtration. The larger of these two forms (MW approximately 154,000) was somewhat unstable and in the presence of 0.5 M NaI was converted to a form indistinguishable from the "native" small form (MW approximately 71,000). We have purified the small form of the enzyme greater than 3,300-fold by a combination of gel filtration, ion-exchange chromatography, and nucleotide affinity chromatography. The purified preparation has a measured specific activity of 3.74 mumol/min/mg protein, and is free of acetylcholinesterase and acetyl-CoA hydrolase activities. The Vmax of the purified enzyme is stimulated by NaCl, with half-maximal stimulation at 80 mM NaCl. The Km for each substrate is also affected by salt, but in different manners from each other and the Vmax; the kinetic parameter Vmax/Km thus changes significantly as a function of the salt concentration.     

Release of Free Fatty Acids and Loss of Hill Activity by Aging Spinach Chloroplasts

The free fatty acid content of spinach chloroplasts, isolated at pH 5.8 to 8.0, has been found to vary between 3.1 and 5.5% of the total chloroplast fatty acids. When chloroplasts were incubated at room temperature for 2 hours, the free fatty acids increased by 42% and the Hill activity decreased by 70%. After 2 hours of incubation at 37 degrees , the free fatty acids increased about 3-fold and the Hill activity decreased to almost 0. The addition of crystalline bovine serum albumin largely prevented the loss of Hill activity at room temperature and at 5 degrees , but had little effect during incubation at 37 degrees . Both the release of free fatty acids and the loss of Hill activity were pH dependent. The losses were the least during incubation at pH 5.8 and the greatest during incubation at pH 8.0. The major free fatty acids released at pH 5.8 were saturated, while those released at pH 7.0 or 8.0 were mainly the unsaturated acids, alpha-linolenic acid and hexadecatrienoic acid.