Effect of 2,4-dichlorophenoxyacetic acid on invertases in chicory root

The invertase present in roots of chicory (Cichoriun intybus) has a pH optimum of 7.5 and a MW of ca 260 000. It requires relatively high ionic strength to remove it from DEAE cellulose. Treatment of chicory root tissue with 2,4-dichlorophenoxyacetic acid gives rise to a highly active invertase with pH optimum of 5.6 and MW of ca 61 000. It is more easily removed from DEAE cellulose.     

Epicuticular wax of Pinus radiata needles

Epicuticular wax isolated from the cotyledons and primary needles of 10-week-old Pinus radiata seedlings is similar in composition and contains 86% neutral compounds, viz. alkyl esters (25%, C₂₄–C₆₄), nonacosan-10-ol (52%), heptacosane-5,10-diol (2%), nonacosane-4,10-diol, nonacosane-5,10-diol, and nonacosane-10,13-diol (total 12%) and estolides, MW ca 800 (2%), MW ca 1100 (6%), and MW ca 1500 (1%). The acidic fraction (14%) contains n-acids (78%, C₁₂–C₃₂) and diterpene acids (22%, mainly abieta-8,11,13-trien-18-oic, with lesser amounts of pimara-8(14),15-dien-18-oic, isopimara-7,15-dien-18-oic and hydroxylated aromatic, diene and mono-ene acids). Wax isolated from primary needles of 1-yr-old seedlings had a similar neutral fraction composition, but the acidic fraction contained predominantly the diterpene acid mixture, with only trace amounts of n-acids. The wax from 1-yr-old secondary, needles from P. radiata forest trees aged 5 yr and 40 yr contained an acid fraction (12% 5 yr, 17% 40 yr trees) comprising the diterpene acid mixture, with trace amounts of n-acids together with ω-hydroxy acids (C₁₂, C₁₄ and C₁₆). The neutral fraction from both young and old trees had a similar composition containing alkyl esters (7%, C₂₄–C₆₆), estolides (90%, MW 566-ca 1500), nonacosan-10-ol (2%) and the heptacosane and nonacosane diols (1%). During growth and maturation of P. radiata, the nonacosan-10-ol content of the needle wax decreases while the proportion of estolides and diterpene acids increases, the latter probably being located around the stomatal pore.     

Arginine Decarboxylase of oat seedlings

Arginine decarboxylase activity in the shoots of seedlings was high in oats, intermediate in barley and low in rice, maize, wheat and rye. After partial purification, the arginine decarboxylase from the shoots of potassium deficient oat seedlings was separated into two fractions, A (MW 195 000) and B (MW 118 000), by gel chromatography. On gel electrophoresis, the mobilities of these fractions were respectively 0.12 and 0.55 relative to bromophenol blue at pH 9.5. Fraction A was twice as active as fraction B in extracts of seedlings grown with both normal and potassium deficient nutrition, despite the greater activity ( × 5) of the potassium deficient plants. The properties of the two fractions were similar with respect to pH optimum (7–7.5), K_m (3 × 10 ^?5M) and the effect of inhibitors. Fraction A was purified to apparent homogeneity by DEAE-cellulose chromatography. The enzyme was specific for l-arginine and it was strongly inhibited by NSD 1055, d-arginine and canavanine. Mercaptoethanol and dithiothreitol stimulated the enzyme by ca 50% and p-chloromercuribenzoate was an inhibitor. Pyridoxal phosphate stimulated activity by ca 30% and EDTA stimulated activity by 30%. Ca²⁺ and Mg²⁺ inhibited the enzyme by 50% at ca 20 mM. Putrescine and the polyamines showed only moderate inhibition at 10 mM, but agmatine reduced activity to 30% at this concentration.     

Urease of Spirulina maxima

Urease (EC 3.5.1.5) was purified from Spirulina maxima by ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-200. The enzyme had maximum activity at pH 8.7, a K_m for urea of 0.12 mM and a MW of ca 232 000. A MW of 38 000 was determined for the subunits. The enzyme was inactivated by p-hydroxymercuribenzoate.     

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.     

Characterization of wheat o-diphenolase isoenzyme

A highly purified isoenzyme of wheat o-diphenolase was characterized. The isoenzyme had a MW of ca 115 000, as determined by Sephadex G-100 gel filtration. The copper content was 0.20%, and the amino acid composition was determined. Two subunits (MWs ca 30 000 and 23 500) were detected by SDS gel electrophoresis. The K_m was found to be 5.1 mM for 4-methylcatechol and kinetic analysis showed that the isoenzyme exhibited substrate inhibition. The isoenzyme was characterized by its response to some inhibitors.     

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.     

Amino acid sequence of the small subunit of ribulose-1,5-bisphosphate carboxylase of Pisum sativum

The amino acid sequence of the small subunit of ribulose-1, 5-bisphosphate carboxylase from pea consists of a single polypeptide chain of 123 residues with a calculated MW of ca 14 480. The N-terminus was ‘ragged’ and both methionine and glutamine were determined in residue position 1. No heterogeneity was found even though two isofocussing variants were observed. The amino acid sequence confirms the nucleic acid sequence of cDNA of mRNA determined independently.     

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.     

Sulphides and furanones from steam volatile oils of Allium fistulosum and Allium chinense

The constituents of the steam volatile oils from two kinds of Allium fistulosum, A. fistulosum var. caespitosum and A. chinense, have been investigated by GC and spectral techniques (IR, UV, GC/MS, ¹H NMR and ¹³C NMR). The compounds identified from the neutral fraction of each volatile oil included sulphides, thiolanes, alcohols, aldehydes, ketones, furanones and others. Among the sulphur compounds, dipropyl disulphide comprised ca 28% of A. fistulosum oil, ca 23% of A. fistulosum var. caespitosum oil and ca 30% of A. chinense oil. A. fistulosum oil was characterized by a large quantity of tridecan-2-one (ca 52%) and 2,3-dihydro-2-octyl-5-methylfuran-3-one (ca 16%). Also, a large amount of 2,3-dihydro-2-hexyl-5-methylfuran-3-one (ca 20%) was isolated from A. chinense oil.     

Proteinase inhibitors from lonchocarpus capassa (appleleaf) seed

Four proteinase inhibitors (DE-1 to DE-4) were purified from L. capassa seed by chromatographic procedures involving Sephadex G-50 and DEAE-cellulose. They comprise each 80 amino acids (MW ca 10 000) including fourteen half-cystine residues. The partial amino acid sequence of inhibitor DE-4 was determined; 60 of the 80 residues have been sequenced. The MW, cystine content and partial sequence of DE-4 resemble those of the Bowman-Birk-type proteinase inhibitors. The properties of inhibitors DE-1 and DE-4 are very similar. Each contains a potent inhibitor for porcine trypsin but they inhibit bovine α-chymotrypsin only weakly.     

Isolation and properties of a ferredoxin from Anabaena Flos-Aquae

Ferredoxin was isolated from the blue-green alga Anabaena flos-aquae. Its homogeneity was shown by conventional and SDS-polyacrylamide gel electrophoresis, and isoelectric focusing on polyacrylamide gel columns, the latter indicating a pI at ca pH 3·7. The absorption spectrum had, in the oxidized state, maxima at 462, 421, 327 and 276 nm, with a shoulder at 284 nm, a spectrum characteristic of plant-type ferredoxins. The 421 : 276 nm absorbance ratio was typically 0.49. The ferredoxin effectively mediated the photoreduction of NADP⁺ by barley chloroplasts depleted of native ferredoxin. The MW obtained by sedimentation-equilibrium and sedimentation velocity-diffusion coefficient studies was ca 12 000 daltons, a value somewhat higher than suggested by amino acid composition data. The ferredoxin contained 2Fe and 2S per molecule.     

Purification and properties of glycollate oxidase from Pisum sativum leaves

A rapid and efficient method for the isolation of glycollate oxidase from pea leaves is described. The method utilizes the unusually high isoelectric point (pH 9·6) which has been determined for the enzyme using isoelectric focusing. The enzyme is apparently homogeneous by polyacrylamide gel electrophoresis and has a MW of ca 100000. Some properties of the enzyme are described.     

Purification et propriétés d'une protéase neutre de Tricholoma columbetta

An endopeptidase was partially purified from the fruiting bodies of Tricholoma columbetta. The enzyme, MW ca 14 500, pH optimum 7.0, hydrolyses the natural substrates, casein and fibrin, but is inactive with the usual synthetic substrates. It is relatively insensitive to most inhibitors of proteases except for high concentrations of KCN, whereas high concentrations of EDTA greatly increase its caseinolytic activity.     

Further properties of the polyamine oxidase from oat seedlings

After purification, the polyamine oxidase from the leaves of oat seedlings grown in the dark appeared to be homogeneous on electrophoresis. The MW determined by density gradient centrifugation was 119 000. The enzyme would not oxidise diaminodipropylamine and neither diaminodipropylamine nor diaminopropane were inhibitors at concentrations up to 1 mM. With spermidine as substrate, the energy of activation was 19.7 kJ/mol and activity was reduced to 50% on heating for 10 min at 50°. With spermine as substrate, activity was increased up to 3-fold in the presence of M sodium chloride. This stimulation was not observed with spermidine as substrate The enzyme was also stimulated by sodium phosphate and sodium citrate at high concentrations. The pH for optimal stability was 6.5, the same as the pH for maximum activity with both spermidine and spermine as substrates. For spermidine and spermine the K_ms were 8 × 10 ^?6 M and 2 × 10 ^?6 M respectively. Loss of activity on storage of leaves at ? 15° was ca 5 % per week and in extracts the loss was ca 10 % per week.     

Affinity chromatography of malic enzyme from grape berries

NADP-dependent malic enzyme from grape berries is associated with NAD-dependent malate dehydrogenase. A two step procedure, involving affinity chromatography on 2′,5′-ADP-Sepharose 4B, followed by gel- permeation on Bio-Gel A- 1.5 m, was used to separate malic enzyme from malate dehydrogenase and other proteins. The yield was ca 60% Malic enzyme and malate dehydrogenase migrated respectively as three bands and one band during disc electrophoresis in polyacrylamide gel. The MW resulting from gel-permeation was 220 000 for malic enzyme and 53 000 for malate dehydrogenase.     

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.     

Xylans from the tropical grass Panicum maximum

Two xylans have been isolated from the mature tissues of the tropical grass Panicum maximum—an arabino(4-O-methylglucurono)xylan and an acidic galactoarabinoxylan. Both consist of a main chain of β(1 → 4) linked d-xylopyranosyl residues. The former has average of ca 46 such residues to which are attached ca 7 l-arabinofuranosyl and (ca 2 4-O-methyl-d-glucopyranuronosyl residues at C3 and C2 positions respectively. The acidic galactoarabinoxylan has a $\text{DP}$_n of ca 90 and contains arabinose, galactose, xylose and uronic acid residues in the molar ratio 10:5:22:4. Methylation analysis and periodate oxidation indicated the highly branched nature of this polysaccharide.     

Hydrocarbons from the essential oil of Cymbopogon martinii

The composition of the hydrocarbon fraction of the essential oil from Cymbopogon martinii, which represents less than 5% of the oil, has been studied. Using well-established techniques, 11 monoterpenes (ca 46 %), 28 sesquiterpenes (ca 52%) and 16 n-alkanes (ca 1.6%) have been identified. The major constituents are limonene, α-terpinene, myrcene, β-caryophyllene, α-humulene, β- and δ-selinenes. The study of the n-alkanes of C. martinii revealed the presence of all members of the homologous series C₁₅C₃₀.     

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.