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 characterization of thylakoid-bound Mn-superoxide dismutase in spinach chloroplasts

Thylakoid-bound superoxide dismutase (SOD; EC 1.15.1.1) was solubilized by Triton X-100 from spinach and purified to a homogeneous state. The molecular weight of thylakoid-bound SOD was 52000; the enzyme was composed of two equal subunits. Its activity was not sensitive to cyanide and hydrogen peroxide, and the isolated SOD contained Mn, but neither Fe nor Cu. Thus, the thylakoid-bound SOD is a Mn-containing enzyme. The subunit molecular weight of thylakoid Mn-SOD is the highest among Mn-SODs isolated so far, a fact which might reflect its binding to the membranes.     

Purification and characterization of 33 kilodalton protein of spinach chloroplasts

A protein was prepared from spinach chloroplasts in a highly purified form. The isoelectric point of the protein was 5.2. The apparent molecular weight was estimated to be 33 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea, and 34 000 by gel filtration column chromatography with Sephadex G-100. The protein was provisionally named '33 kilodalton protein' according to the molecular weight. The absorption spectrum of the protein did not show any absorption band in the visible region. No histidine was found in the amino acid analysis of the protein. The 33 kilodalton protein was released from the thylakoid membrane by EDTA-treatment and also by sonic oscillation. The protein was bound to System II particles, but not to System I particles.     

Purification and characterization of a salinity induced alkaline protease from isolated spinach chloroplasts

In cynobacteria and higher plants, salinity is known to inhibit the activity of several enzymes involved in photosynthesis and hence decreases the overall photosynthetic rate. This gave us an impetus to search for a protease, which may be involved in the turnover of non-functional enzymes produced under salinity stress. Taking the possible changes in pH gradient of the chloroplast under consideration, we have tried to identify a protease, which is induced under salinity and characterized it as an alkaline protease using spinach (Spinacia oleracea) leaves as a model system. The HIC-HPLC purified homogeneous alkaline serine protease from the isolated spinach chloroplasts had two subunits of molecular weight 63 and 32 kDa. The enzyme was maximally active at pH 8.5 and 50°C. The enzyme showed the property to hydrolyze the synthetic substrate like azocaesin and had sufficient proteolytic activity in gelatin bound native PAGE. The enzyme activity was also dependent upon the presence of divalent cations and reduced environment. The active site residues were identified and the homogeneous alkaline serine protease had cysteine, lysine and tryptophan residues at its active site.     

Purification and properties of protoporphyrinogen oxidase from spinach chloroplasts

Protoporphyrinogen oxidase (Protox), an enzyme that catalyzes the common step of chlorophyll and heme biosynthetic pathways, was purified from spinach chloroplasts. The molecular weight of purified protein was estimated to be approximately 60,000 by SDS-PAGE. Protox activity was stimulated by addition of FAD, suggesting that chloroplast Protox requires FAD as a cofactor. Furthermore, the Protox-inhibiting herbicide, S23142, specifically inhibited the purified Protox activity at an IC50 value of 1 nM.     

Cytochrome f from spinach and cyanobacteria. Purification and characterization

Cytochrome f has been purified from spinach chloroplasts and from the photosynthetic membranes of the cyanobacterium Spirulina maxima. The spinach protein has an isoelectric point of 5.2 and gives a single band on isoelectric focusing gels. The S. maxima cytochrome shows a major band with a pI of 4.01 and a minor band with a pI of 3.97. S. maxima cytochrome f has a molecular weight approximately 38,000 and is monomeric, while the spinach protein is slightly smaller, approximately 36,000 daltons, and aggregates to form an octamer. S. maxima cytochrome f has an E'0 of +339 mV which is close to that of cytochromes f from higher plants. The NH2-terminal amino acid sequences of the cytochromes show striking similarities. Spinach cytochrome f shows a clear preference for oxidation by spinach plastocyanin and S. maxima cytochrome f is more readily oxidized by its in vivo reaction partner, cytochrome c553.     

Hexosediphosphatase from spinach chloroplasts: Purification,crystallization and some properties

Hexosediphosphatase (d-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) has been isolated, purified, and crystallized, from previously isolated spinach chloroplasts. The effects of various anions, cations, and sulfhydryl compounds were tested, and activation by Mg²⁺, glycine, HCO₃^?, and sulfhydryl compounds is described. The purified enzyme is very specific for fructose 1,6-diphosphate and does not attack sedoheptulose-1,7-bisphosphate. The s₂₀ value of the enzyme was 7.7, from which the molecular weight of the enzyme was estimated as 140 000.     

Fructose-1,6-diphosphatase from spinach leaf chloroplasts: Purification and heterogeneity

The enzyme fructose- 1,6-diphosphatase (FDPase), involved in the reductive cycle of the pentose phosphate pathway, has been purified from spinach leaves by heating (30 min at 60°), “salting out” with ammonium sulphate (between 30–70% of saturation), filtration through Sephadex G-100 and G-200, fractionation on DEAE-52 cellulose and preparative electrophoresis on polyacrylamide gel. Filtration through DEAE-cellulose led to the isolation of two active fractions (fractions I and II) with very close MWs and isoelectric points. By electrophoresis on acrylamide gel, both fractions gave two active fractions (fractions I_a-I_b and II_a-II_b). The fractions with low electrophoretic migration rate—I_b and II_b—are stable in acid and neutral pH, have a MW between 90 000 and 110 000 and constitute the native form of the photosynthetic enzyme. The fractions of faster migration rate—I_a and II_a-originate from the corresponding fractions I_b and II_b under alkaline conditions, show half the MW of the respective fractions, and behave as subunits of the original dimer form. Measured by electrofocusing, the four active fractions have isoclectric points in the range 4·10–4.30.     

Purification and reconstitution of the N,N'-dicyclohexylcarbodiimide-sensitive ATPase complex from spinach chloroplasts.

The dicyclohexylcarbodiimide-sensitive ATPase from spinach chloroplast has been isolated. On sodium dodecyl sulfate gels, seven different polypeptides were seen. Five of these polypeptides coincided with the CF1 subunits, a 7,500-dalton peptide was identified as the proteolipid which interacts with [14C]dicyclohexylcarbodiimide, and there was a 15,500-dalton hydrophobic polypeptide with unknown function. In two-dimentional gels, two additional peptides were resolved, one 17,500 daltons (co-migrating in sodium dodecyl sulfate gels with subunit delta) and one 13,500 daltons (co-migrating with subunit epsilon). Reconstitution was obtained by freezing and thawing the complex with a crude mixture of phospholipids. After reconstitution the complex catalyzed 32P1-ATP exchange (rates of 200 to 400 nmoles x mg-1 x min-1) and ATP formation during acid-to-base transition. These reactions were inhibited by dicyclohexylcarbodiimide and uncouplers. Uncouplers at low concentrations stimulated and at high concentrations inhibited the Mg2+-ATPase activity. ATP hydrolysis and 32P1-ATP exchange were catalyzed by the complex in the presence of either Mg2+ or Mn2+ but not with Ca2+ or Co2+. ATP and GTP were substrates for the exchange reaction but not ADP or CTP.     

Serine hydroxymethyltransferase from spinach leaf mitochondria. Purification and characterization

A mitochondrial serine hydroxymethyltransferase (EC 2.1.2.1) has for the first time been purified close to homogeneity from a photosynthetically active tissue, spinach ( Spinacea oleracea L. cv Viking II) leaves. The specific activity of the enzyme was 7.8 μmol (mg protein)⁻¹ min⁻¹ using L-serine as substrate. The enzyme was stable for at least 8 weeks at 4°C in the presence of folate. The pH optimum was at pH 8.5 where the enzyme had a K_m for L-serine of 0.9 m M . Carboxymethoxylamine was a strong competitive inhibitor with a K₁ of 1.4 μM. An absorption spectrum taken of the enzyme in the presence of glycine and tetrahydrofolate showed a peak at 492 nm, probably originating from a substrate-enzyme complex. The molecular weight obtained by gel filtration was 209 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified enzyme showed that the apparent molecular weight of the subunit was 53 kDa, indicating four subunits.     

Purification and characterization of nucleoside diphosphate kinase from spinach leaves.

Two types of nucleoside diphosphate kinase (NDP kinase I and NDP kinase II) have been purified from spinach leaves to electrophoretic homogeneity. The enzymes were copurified with apparent [35S]GTP-gamma S-binding activities. NDP kinase I, which was not adsorbed to a hydroxyapatite column, and NDP kinase II, which was adsorbed, had molecular weights of 16,000 and 18,000, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weights determined by gel filtration were 92,000 and 110,000, respectively, suggesting that both enzymes are composed of six identical subunits. Minor differences in some amino acids between NDP kinase I and NDP kinase II were observed when both enzymes were analyzed for amino acid composition. The apparent [35S]GTP gamma S-binding activity of purified NDP kinase I and NDP kinase II was found to be due to the formation of a [35S]thiophosphorylated enzyme, which is the intermediate of the NDP kinase reaction.     

Purification and comparative characterization of an enolase from spinach

An enolase has been purified to apparent homogeneity, as measured by gel electrophoresis, some 400-fold from spinach (Spinacia oleracea). This is the first plant enolase that has been purified to homogeneity. At moderate ionic strengths, the 5,5-dithio-bis-2-(nitrobenzoate) (DTNB)-or parachloromercuribenzoate-reacted enzyme elutes from a Bio-Gel P-200 column with somewhat greater volumes than the yeast enzyme (M_r = 93,000) indicating a greater size. Its elution volume from Ultrogel in 50% ammonium sulfate, however, suggests it exists as an active monomer (M_r = 47,000). Sodium dodecyl sulfate-gel electrophoresis indicates the subunit molecular weight is 50,000 ± 3,000, like that of yeast enolase.

The enzyme contains 23 ± 4 half-cystines per mole of subunit. Titrations with DTNB in guanidine hydrochloride or nondenaturing media indicate that most of these, if not all, are in the reduced state. Reaction of one or more of the sulfhydryls with DTNB or parachloromercuribenzoate stabilizes the enzyme.

The kinetic parameters of the reaction catalyzed by spinach enolase, as well as the inhibitions by transition metal ions and fluoride, are similar to those properties of the yeast and rabbit muscle enzymes.

     

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.     

Purification and characterization of ribulose-5-phosphate kinase from spinach

An efficient purification procedure utilizing affinity chromatography is described for spinach ribulose-5-phosphate kinase, a light-regulated chloroplastic enzyme. Gel filtration and polyacrylamide gel electrophoresis of the purified enzyme reveal a dimeric structure of 44,000 Mr subunits. Chemical crosslinking with dimethyl suberimidate confirms the presence of two subunits per molecule of native kinase, which are shown to be identical by partial NH2-terminal sequencing. Based on sulfhydryl titrations and on amino acid analyses, each subunit contains four to five cysteinyl residues. The observed slow loss of activity during spontaneous oxidation in air-saturated buffer correlates with the intramolecular oxidation of two sulfhydryl groups, presumably those involved in thioredoxin-mediated regulation.     

Purification and characterization of chloroplast dehydroascorbate reductase from spinach leaves

Green leaves of plants require the high-level activity that can regenerate ascorbate during photosynthesis. One of such enzyme is dehydroascorbate reductase (DHAR), but the molecular and enzymological properties of the enzyme remain to be fully characterized. In this study, we showed that two major DHAR existed in spinach leaves. The two DHARs occupied at least over 90% of total DHAR activity. The amount of the two DHARs was almost the same. We purified both DHARs from spinach leaves. One form of DHAR originated in chloroplasts; the other occurred in the subcellular compartment other than chloroplasts. The chloroplast DHAR had Km values of 70 microM and 1.1 mM for dehydroascorbate and reduced glutathione, respectively. The specific activity of the purified enzyme corresponded to 360 micromol of ascorbate formed per milligram of protein per minute. These properties were quite different from those of trypsin inhibitor, which has been reported to be the plastid DHAR. The other DHAR had the very similar properties to those of chloroplast DHAR. Chloroplast and the other DHARs functioned as a monomer with molecular masses of 26 kDa and 25 kDa, respectively. cDNA for the chloroplast DHAR was cloned with the determined amino-terminal amino acid sequence. The primary sequence predicted from the cDNA included the plastid-targeting sequence. Finally, the significance of chloroplast DHAR in the regeneration of ascorbate is discussed.     

Purification and characterization of glutathione reductase from corn mesophyll chloroplasts

Differences in the apparent molecular weights of the subunits of glutathione reductase (EC 1.6.4.2) from pea chloroplasts and corn mesophyll chloroplasts have been recently reported. In order to more fully describe the differences between the enzymes from these two sources, glutathione reductase from the mesophyll chloroplasts of corn seedlings ( Zea mays L. cv. G-4507) has been purified 200-fold by affinity chromatography using adenosine 2',5'-disphosphate agarose. The purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)^-1 min^-1. The native enzyme had a relative molecular weight of 190 ± 30 kDa and exhibited polypeptides of 65, 63, 34, and 32 kDa when separated on sodium dodecylsulfate-polyacrylamide gels. Comparisons of the results from electroblotting, native molecular weight and subunit molecular weight analyses suggest that the enzyme exists as a heterotetramer. Optimal enzyme activity was obtained at pH 8 in N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES-NaOH) buffer. The sulfhydryl reagent, n -ethylmaleimide, inhibited enzymatic activity when incubated in the presence of NADPH while no inhibition was detected with oxidized glutathione in the incubation mixture. Reduced glutathione (5 m M ) inactivated the enzyme by 50%. This inactivation followed first order kinetics with a rate constant of 0.0028 s^-1. The enzyme was also inactivated by NADPH. The inactivation reached ca 90% within 30 min and followed first order kinetics with a rate constant of 0.0015 s^-1.     

Purification and characterization of a membrane-bound protein kinase from spinach thylakoids

A protein kinase was isolated from spinach thylakoid membranes by solubilization with octyl glucoside and cholate. The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, and sucrose density centrifugation, followed by affinity chromatography on either Affi-Gel blue (yielding denatured enzyme) or on histone cross-linked to Sepharose (yielding active enzyme). Electrophoresis on denaturing polyacrylamide gels, followed by staining with silver, revealed the kinase as a single band corresponding to an apparent molecular mass of 64 kDa. The active enzyme underwent autophosphorylation and could be detected by autoradiography following incubation with [gamma-32P]ATP and Mg2+ ion. The specific phosphotransferase activity of purified kinase was approximately 30 nmol of phosphate min-1 (mg protein)-1 with lysine-rich histone (III-S or V-S) as substrate; casein was phosphorylated at approximately 30% of this rate. The physiological substrate for the kinase is presumed to be light-harvesting chlorophyll a/b protein complex. In solubilized form, this was phosphorylated at approximately 10% of the rate observed with histone III-S as substrate, or 10-100 times slower than the estimated rate of phosphorylation of the light-harvesting complex in situ. Possible reasons for this shortfall are considered. The kinase is proposed as the principal effector of thylakoid protein phosphorylation and associated State transition phenomena.     

Isolation and characterisation of 14-S RNA from spinach chloroplasts.

14-S RNA was purified from spinach chloroplasts. It has a molecular weight of 0.43 . 10(6) and the following nucleotide composition: 20% CMP, 23.9% AMP, 24.2% GMP and 31.9% UMP. The accumulation of 14-S RNA in chloroplasts of cotyledons of dark-grown plants is stimulated by light. Conditions are described for the isolation of 14-S RNA in the absence of appreciable fragmentation of chloroplast 23-S rRNA and the evidence that it represents a distinct type of chloroplast RNA is discussed. Translation of 14-S RNA in a protein synthesising system from Escherichia coli gives rise to two polypeptides with molecular weights of 13 200 and 12 600 and the possible role of 14-S RNA as a chloroplast messenger is discussed.