Purification and metal requirements of 3-dehydroquinate synthase from Phaseolus mungo seedlings

Dehydroquinate synthase of Phaseolus mungo seedlings was purified 4400-fold from the (NH₄)₂SO₄ fraction of a crude extract, the specific activity being 810 nkat per mg protein. When the purified enzyme was subjected to electrophoresis with or without sodium dodecyl sulfate, a single band was observed. The MW of the enzyme was estimated to be 67 000 by Sephadex G-100 gel chromatography and the minimum MW of the enzyme 43 000 by gel electrophoresis with sodium dodecyl sulfate. Atomic absorption analysis revealed that the purified enzyme contained small amounts of copper. Cobalt was not detected, although it has been implicated as a cofactor requirement.     

UDP-glucose-4-epimerase from Poterioochromonas malhamensis

UDP-glucose-4-epimerase of Poterioochromonas malhamensis, Peterfi has been purified to apparent electrophoretic homogeneity. The enzyme has an apparent MW of 120 000 as determined by gel filtration of the active enzyme. Sodium dodecylsulfate polyacrylamide gel electrophoresis gave a MW of 59 000, thus indicating a dimeric structure. The epimerase does not require external NAD for activity. The apparent K_m values for UDP-glucose and UDP-galactose were calculated to be 1.67 mM and 0.26 mM, respectively. The pH optimum is at pH 8.7 and the isoelectric point is at pH 5.1 ± 0.15.     

Purification and properties of carbonic anhydrase from Chlamydomonas reinhardii

Carbonic anhydrase (CA) was purified from the unicellular green alga Chlamydomonas reinhardii, and the purity of the preparation was established by gradient gel electrophoresis. The purified enzyme exhibited a MW of 165 000 and contained 6 atoms of Zn. The subunit MW, as determined by dodecyl sulfate electrophoresis, was 27 000. These results are consistent with a quarternary structure which is hexameric, each monomer containing 1 g atom of Zn. Like spinach CA, and in contrast to other oligomeric plant CAs, a sulfhydryl reducing agent is not needed to stabilize the enzyme. CO₂-hydrase activity was inhibited by both acetazolamide (I₅₀ = 7.8 × 10^?9M) and sulfanilamide (I₅₀ = 1.3 × 10^?5M), as well as by certain inorganic anions. The purified enzyme showed relatively weak esterase activity with p-nitrophenyl acetate but was an extremely effective esterase with 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone as the substrate. Both esterase activities could be completely inhibited by adding acetazolamide. In its gross structural characteristics, the C. reinhardii enzyme resembles the CAs from higher plants. However, in its esterase activity and the inhibition by sulfonamides it is markedly different from plant CAs and bears more resemblance to erythrocyte CAs.     

Purification and characterization of L-mimosine synthase from Leucaena leucocephala

L-Mimosine synthase has been isolated from Leucaena leucocephala seedlings and purified 280-fold by heat treatment, ammonium sulphate fractionation, gel filtration and ion-exchange chromatography. The enzyme was shown to be homogeneous by gel electrophoresis (MW 64 000±2000) and to consist of two identical subunits with MWs of 32 000±2000. The purified enzyme has a K_m value of 6.25 x 10^?3 M for O-acetyl-L-serine and 5.0 x 10^?3 M for 3,4-dihydroxypyridine. In these and other properties, the enzyme differs from β-(pyrazol-1-yl)-L-alanine synthase from Citrullus vulgaris seedlings.     

UDP-galactose 4′-epimerase from vicia faba seeds

UDP-Galactose 4′-epimerase was purified ca 800-fold through a multi-step procedure which included affinity chromatography using NAD⁺ -Agarose. Three forms of the enzyme were separated by gel-filtration but only the major form was purified. The pH optimum of the enzyme was 9.5. Exogenous NAD⁺ was not required for enzymic activity but its removal caused inactivation. The enzyme was unstable below pH 7.0 but stable at pH 8.0 in the presence of glycerol and at ?20° for two months. The equilibrium constant for the enzyme-catalysed reaction was 3.2 ± 0.15. The K_m for UDP-galactose and UDP-glucose were 0.12 mM and 0.25 mM, respectively. The inhibition by NADH was competitive, with a K_i of 5 μM. The MW of the enzyme was 78 000; the two minor forms showed the values of 158 000 and 39 000, respectively.     

Purification and characterization of β-(pyrazol-1-yl)-l-alanine synthase from Citrullus vulgaris

From seedlings of Citrullus vulgaris the enzyme β-(pyrazol-1-yl)-l-alanine synthase was purified 200-fold, when it showed electrophoretic homogeneity (MW 58 000) and could be dissociated into identical subunits (MW 32 000) each containing one molecule of pyridoxal 5′-phosphate. The K_m value was 2.5 × 10^?3 M for O-acetyl-l-serine and 7.4 × 10^?2 M for pyrazole. The enzyme did not catalyse the formation of related β-substituted alanines, such as l-mimosine and l-quisqualic acid, and significant differences were found between the β-(pyrazol-1-yl)-l-alanine synthase and β-substituted alanine syntheses and cysteine synthase from other sources.     

Further characterization of phosphoinositol kinase isolated from germinating mung bean seeds

Phosphoinositol kinase isolated and purified from germinating mung bean seeds has been further characterized. The rate of phosphorylation varies with different inositol phosphates and this is consistent with the K_m and V_max for each of the substrates. The phosphate transfer from ATP has been found to be mediated by a phosphoprotein intermediate. In a particular step of the reaction the immediate product of the reaction has been found to be most inhibitory, other products being less or non-inhibitory. The inhibition has been found to be competitive in nature. The K_is have been found to range between 0.6 and 1 × 10^?4 M. ADP also inhibited non-competitively with respect to IP₅. K_i for this has been found to be 2.3 × 10^?4 M. The purified enzyme migrated as a single protein band on polyacrylamide gel electrophoresis. In the presence of sodium dodecyl sulphate it is dissociated into 3 subunits in the ratio 1 : 1 : 1. The MW of the three subunits are approx. 86 000, 56 000 and 35 000. The MW of the enzyme has been found to be approx. 177 000.     

Glutamine-dependent asparagine synthetase from Lupinus luteus

Asparagine synthetase (glutamine-hydrolyzing [l-aspartate: l-glutamine amido-ligase (AMP-forming), E.C. 6.3.5.4] was purified over 500-fold from cotyledon extracts of 1-week-old yellow lupin seedlings. The enzyme was labile and required protection by high levels of thiols; glycerol and the substrates also stabilized it. The reaction products were shown to be asparagine, AMP, PPi and glutamate. The limiting K_m values were for aspartate 1·3 mM, for MgATP 0·14 mM and for glutamine 0·16 mM. Positive homotropic cooperativity was observed for MgATP only, and gel filtration studies indicated that the substrate-free enzyme (MW 160 000) associated to a dimer (MW 320 000 in the presence of MgCl₂ and ATP. The purified enzyme, which had some glutaminase activity, catalyzed an aspartate- and glutamine-independent ATP-PPi exchange reaction at a rate 5–7-fold higher than the rate of asparagine synthesis. Initial velocity studies and exchange data indicated an overall ping-pong mechanism. Compared to similar enzymes isolated from mammalian tumor cells, the lupin enzyme appears to be unique with respect to MW, reaction mechanism and regulatory properties. The allosteric properties observed suggest an important role for this enzyme in the regulation of asparagine biosynthesis.     

Purification and properties of dehydroascorbate reductase from spinach leaves

Dehydroascorbate reductase was detected in the leaves of several plants and has been partially purified from spinach leaves. The enzyme has a MW of ca 25 000, a pH optimum of 7.5, a K_m for glutathione (GSH) of 4.43 ± 0.4 mM and a K_m for dehydroascorbate of 0.34 ± 0.05 mM. High concentrations of dehydroascorbate inhibit the enzyme. Cysteine cannot replace GSH as a donor. The purified dehydroascorbate reductase is extremely unstable and also inhibited by compounds which react with thiol groups. Dehydroascorbate does not protect the enzyme against such inhibition. GSH reduces dehydroascorbate non-enzymically at alkaline pH values.     

Purification and properties of sucrose-6-phosphatase from Pisum sativum shoots

Sucrose-6-phosphatase from pea shoots, which was purified to homogeneity, consists of two similar sub-units each with an MW of about 55 000. The pH optimum was at 6.8; the K_m for sucrose-6-phosphate was 250 μM and the K_m for magnesium was 175 μM. The enzyme was specific for sucrose-6-phosphate and was not inhibited by sucrose except at very high concentrations.     

Purification and properties of the arginase from Jerusalem artichoke tubers

Arginase [l-arginine amidinohydrolase] in Jerusalem artichoke tubers occurs in a particulate fraction from which it was released in active form by detergent treatment. The particulate enzyme was purified 450-fold with ca 3% yield. The enzyme has a MW of ca 140 000 and pI of 5.3. The enzyme required Mn²⁺ for activity and was unstable when Mn²⁺ was removed. In tissue extracts the K_m for arginine was ca 1OmM, but when purified the K_m (arginine) was 145 mM. The artichoke arginase was shown to be more substrate specific than other plant and animal arginases which have been described, and to be very sensitive to competitive inhibition by indospicine, ornithine and citrulline.     

An ethylene-forming enzyme in Citrus unshiu fruits

An ethylene-forming enzyme from Citrus unshiu fruits was purified some 630-fold. The enzyme catalysed ethylene formation from 1-aminocyclopropane-1-carboxylic acid in the presence of pyridoxal phosphate, β-indoleacetic acid, Mn²⁺ and 2,4-dichlorophenol. It behaved as a protein of MW 40 000 on Sephacryl S-200 gel filtration, and gave one band corresponding to a MW of 25 000 on SDS-PAGE. It had a specific activity of 0.025 μmol/min·mg protein. It exhibited IAA oxidase activity and had no guaiacol peroxidase or NADH oxidase activity. Its K_m for ACC was 2.8 mM, and its pH optimum was 5.7. It was inhibited by potassium cyanide n-propyl gallate and Tiron. d-Mannose, histidine, iodoacetate, PCMB, dimethylfuran and superoxide dismutase showed no inhibition. β-Indoleacrylic acid against IAA competitively inhibited ethylene formation. Other IAA analogues, such as β-indolepropionic acid, β-indolecarboxylic acid and β-indolebutylic acid, slightly stimulated ethylene formation. β-Indoleacrylic acid against 1-aminocyclopropane-1-carboxylic acid non-competitively inhibited ethylene formation. Ascorbate was a potent inhibitor. The inhibitory effects, however, were not always reproduced in vivo. It is difficult to identify this enzyme system as a natural in vivo system from the above observations. Nevertheless, the possible in vivo participation of this in vitro enzyme system is discussed.     

Isolation and characterization of the polyphenoloxidase from senescent leaves of black poplar

The polyphenoloxidase (PPO) from black poplar senescent leaves has been purified to almost complete homogeneity by a combination of ammonium sulphate precipitation, Sephadex G75 filtration and DEAE-cellulose chromatography. The purified enzyme has a MW of 60 000 and is probably a Cu⁺ enzyme. Peroxidase (PO) activity co-purifies with PPO and has the same MW as it. The two enzymes differ in pH optimum and in response to the effect of ionic strength. Natural phenols are either substrates, inhibitors or activators of black poplar PPO. This enzyme is an o-diphenoloxidase which binds substrates with K_m in the millimolar range. With caffeic and chlorogenic acids inhibition by excess substrate is observed. Benzoic acid phenols and cinnamic acid phenols are either competitive or non-competitive inhibitors of PPO. Hydroquinone is a highly potent non-competitive inhibitor of the enzyme (K_i  90 μM). Ferulic acid is a potent activator of the PPO-catalysed oxidation of catechol (K_a  0.34 mM, ν_sat/ν_o  7.7).     

α-Galactosidase from sweet chestnut seeds

The major sugars of fresh seeds of Castanea sativa were shown to be raffinose, stachyose and sucrose. Drying seeds at 25° for 14 weeks increased the ratio raffinose: stachyose from 1.1 to 3.5, reduced sucrose content by ca 50 % and decreased total extractable α-galactosidase. The enzyme activity was resolved into two peaks, a high MW form I (apparent MW215 000) and a low MW form II (apparent MW 53 000). The latter form was predominant in the extract of fresh seeds whereas the former was the main form in the 14-week dried seeds. An increase in the amount of enzyme I was also observed when a buffered extract (pH 5.5) of fresh seeds was stored at 4°. Enzymes I and II had pH optima of 4.5 and 6, respectively. Both enzymes hydrolysed p-nitrophenyl α-d-galactoside at a much greater rate than the natural substrates raffinose, stachyose, locust bean gum and carob gum. However, enzyme I showed preference for stachyose as compared to raffinose; the opposite order was observed for enzyme II.     

Purification and properties of caffeic acid O-methyltransferase from alfalfa root nodules

An O-methyltransferase which catalyses the methylation of caffeic acid to ferulic acid using S-adenosyl-l-methionine as methyl donor has been isolated and purified ca 70-fold from root nodules of alfalfa. The enzyme also catalysed the methylation of 5-hydroxyferulic acid. Chromatography on 1,6-diaminohexane agarose (AH-Sepharose-4B) linked with S-adenosyl-l-homocysteine (SAH) gave 35% recovery of enzyme activity. The K_m values for caffeic acid and S-adenosyl-l-methionine were 58 and 4.1 μM, respectively. S-Adenosyl-l-homocysteine was a potent competitive inhibitor of S-adenosyl-l-methionine with a K_i of 0.44 μM. The MW of the enzyme was ca 103 000 determined by gel filtration chromatography.     

Carbohydrate composition and physical properties of tobacco phosphodiesterase

Some physical and chemical properties of phosphodiesterase from cultured tobacco cells were studied. The enzyme contained ca 50% carbohydrate consisting of residues of arabinose, glucose, glucosamine, galactose, mannose and xylose. Analyses showed that the enzyme had a sedimentation coefficient, s_20,w of 16 S, a Stokes'radius of 5.7 nm and a calculated specific volume of 0.66 ml/g. The MW of the enzyme was calculated to be 300 000 from these values.     

GA3-Modulated multiple forms of monophenolase in wheat seed

Multiple forms of monophenolase in wheat half-seeds were separated by molecular sieving on Sephadex G-200. A single molecular form of monophenolase was observed in control, while two multiple forms were present in GA₃-treated wheat half-seeds. A high MW (200 000 or above) multiple form (activity peak I) which eluted soon after the void volume was exclusively present in GA₃-treated half-seeds. The second activity peak (peak II) was a low MW (45 000) multiple form and its elution profile coincided in control and GA₃-treated wheat half-seeds. Both the multiple forms of monophenolase in GA₃-treated wheat half-seeds showed a pH optimum at 9.0, while the optimum enzyme activity of the control molecular form (peak II) was at pH 7.0. This indicated that the treatment of wheat half-seeds with GA₃ brought about a structural modification in monophenolase. The in vitro addition of trypsin enhanced the control of the molecular form of monophenolase but this treatment failed to alter the activity of multiple forms in GA₃-treated half-seeds. This differential response of monophenolase towards trypsin could be ascribed to a conformational change of the enzyme in hormone-treated half-seeds. Brief exposure of the enzyme preparation to urea (6 M) brought about an irreversible activation of monophenolase both in control and GA₃-treated wheat half-seeds.     

Hydrophobic chromatographic purification of ethylene-enhanced chlorophyllase from Citrus unshiu fruits

Ethylene-enhanced chlorophyllase from Citrus unshiu fruits was purified to a homogeneous state after solubilization with sodium cholate, using acetone precipitation and hydrophobic chromatography. The enzyme adhered to phenyl Sepharose CL-4B in 3M KCl and was eluted with a linear gradient of Triton X-100 (0–0.5%). Its MW (SDS-PAGE) was 27 000. The enzyme behaved as a protein of MW 110 000 on Sephacryl S-200 gel filtration. The enzyme showed a specific activity of 0.069 μamol chlorophyllide a produced/min/ mg protein. This purification procedure is a rapid method for obtaining pure chlorophyllase.     

An inducible lactone hydrolase yielding 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid from pulvinic acid

The metabolism of vulpinic acid by an unclassified soil micro-organism was studied. A new compound, 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid (DHOHA) was isolated from the reaction mixture of a cell-free preparation and pulvinic acid. The existence of a hydrolase which catalyses the conversion of vulpinic acid to pulvinic acid was detected in cell-free preparation, and an inducible lactone hydrolase capable of converting pulvinic acid to DHOHA was purified 130-fold and characterized. This enzyme had a MW of ca 34 000, a K_m for pulvinic acid at pH optimum (pH 7.0) less than 10 ^{? 6} M, pI = 5.0, and was inhibited by p-chloromercuriphenylsulfonate and diethylpyrocarbonate. The enzyme was highly specific for pulvinic acid. The initial degradative steps proposed for this organism are vulpinic acid → pulvinic acid → DHOHA.     

Properties of polyphenol oxidase from tubers of the yam Dioscorea bulbifera

The subunit MW of Dioscorea bulbifera polyphenol oxidase (MW 115 000 ± 2000) determined by SDS-PAGE is ca. 31 000 indicating that the enzyme is an oligomeric protein with four subunits. K_i values of various inhibitors and their modes of inhibition have been determined with catechol and pyrogallol as substrates. p-Nitrophenol, p-cresol, quinoline and resorcinol are competitive inhibitors of catechol binding while only orcinol and p-nitrophenol behave in the same way towards pyrogallol as substrate. From the effect of pH on V_max, groups with pK values ca. 4.7 and 6.8 have been identified to be involved in catalytic activity. The Arrhenius activation energy (E_a) at pH 4.0 is 8.9 kcal/mol between 40–65°. At pH 7.0, the value is 22.1 kcal/mol between 40 and 60°. The enthalpies (ΔH) at pH 4.0 and pH 7.0 are 2.3 kcal/mol and 32.4 kcal/mol respectively. The results are discussed considering the conformational changes of the enzyme during substrate binding.