Purification and properties of pea cotyledon and embryo diamine oxidase

Diamine oxidase was purified separately from cotyledon and embryo of pea seedlings germinated for 6 days. The K_m of the cotyledon enzyme for putrescine was 1.6 × 10^?4M while that for the embryo enzyme was 9 × 10^?5M. On heating for 15 min at 70° the embryo enzyme retained about 90% activity whereas the cotyledon enzyme retained only 20% activity. The electrophoretic mobility of the cotyledon enzyme was ca twice that of the enzyme from embryo.     

Purification and properties of agmatine iminohydrolase from groundnut cotyledons

Agmatine iminohydrolase was purified ca 375-fold from groundnut cotyledons. The enzyme exhibited an optimum pH between 5.5 and 8.5 and the energy of activation was 22 kcal/mol. The K_m for agmatine was (7.57 ± 0.77) × 10^?4 M. The enzyme was inhibited by tryptamine, putrescine, cadaverine, spermidine and spermine. Inhibition by cadaverine and spermidine was competitive. The K_i values for cadaverine and spermidine were 4.1 × 10^?3 and 7.5 × 10^?4 M, respectively.     

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.     

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.     

The Effect of Exogenous IAA and Kinetin on Nitrate Reductase,Nitrite Reductase and Glutamate Dehydrogenase Activities in Excised Pea Roots

Nitrate reductase (NO₃R) activity, nitrite reductase (NO₂R) activity and NADH₂ dependent glutamate dehydrogenase (GDH) activity were followed in extracts from excised pea roots incubated under aseptic conditions for 9 and 24 h in nitrate containing nutrient medium to which IAA was added in concentrations promoting lateral root formation (1 × 10^?5; 3 × 10^?5; 5 × 10^?5 M) and kinetin in concentrations which reduce lateral root formation (0.1; 1; 5 mg 1^?1, that is 4.65 × 10^?7;4.65 × 10^?6 and 2.3 × 10^?5 M). NO₃R activity was not influenced by IAA, NO₂R activity was slightly depressed by IAA after 24 h incubation and GDH activity was slightly increased after 24 h incubation in the presence of IAA. Kinetin decreased NO₃R activity significantly both after 9 h and 24 h incubation, slightly increased NO₂R activity after 9 h incubation but slightly decreased it after 24 h incubation, and did not affect GDH activity after 24 h incubation. However, when applied together with IAA, kinetin abolished the promoting effect of IAA on GDH activity. IAA neither reversed nor accentuated the effect of kinetin on NO₂R activity. Nevertheless the depressing effect of kinetin on NO₃R activity was emphasized by the presence of IAA after 9 h incubation. The results obtained indicate that reduced nitrate assimilation due to the depression of nitrate reductase activity caused by kinetin probably contributes to the negative growth effect of kinetin in pea root segments grown in nitrate medium.     

Polyamine oxidase of millet shoots

Polyamine oxidase was purified ca 168-fold from the acetone powder extract of millet shoots. The light yellow enzyme had maximum absorption at 278,380 and 460 nm. The absorption at 380 and 460 nm was decreased by the addition of spermidine. The enzyme (M, ca 80 000) showed a high specificity for spermine and spermidine (K_ms 6 × 10⁻⁵ M and 5 × 10⁻⁷ M respectively). The enzyme was inhibited by quinacrine and acriflavine.     

Effects of metal ions on benzylglucosinolate degradation in Lepidium sativum seed autolysates

The effects of varying concentrations of Fe²⁺ (5 × 10^?5 ?5 × 10^?1 M) on benzylglucosinolate degradation in Lepidium sativum seed autolysates were investigated. Increased glucosinolate decomposition was observed over the whole range with a maximum effect at ca 6 × 10^?3 M Fe²⁺, at which point glucosinolate degradation was more than three times that obtained in the absence of added Fe²⁺ . Nitrile formation was especially enhanced in the presence of all concentrations of Fe²⁺ studied, and maximum amounts were obtained at ca 6 × 10^?3 M Fe²⁺ when a more than four-fold increase over quantities produced in the absence of Fe²⁺ was observed. Thiocyanate formation was also promoted with a maximum at ca 4 × 10^?3 M Fe²⁺, but isothiocyanate production was considerably reduced in allcases. It is suggested that Fe²⁺ inhibits isothiocyanate formation by interfering with the availability of ascorbic acid which is a proven co-factor for most thioglucosidase isoenzymes, but that an Fe²⁺-ascorbate complex might then be responsible for promoting enzymic production of nitrile. The effects of a limited range of concentrations of Fe³⁺ and Cu⁺ were also studied, and results related to those for Fe²⁺. The relevance of the findings to natural systems and to glucosinolate-containing foods is briefly discussed.     

Activity of Various Growth Substances in the Avena First Internode Test

The elongation growth of the Avena first internode segments was studied in the presence of one or several of the following growth substances: indoleacetic acid (IAA), 6-fur-furylamino purine (FAP, kinetin), 6-benzylamino purine (BAP), gibberellin A₃ (GA₃) and A₄₊₇ (GA₄₊₇), and abscisic acid (ABA). The cytokinins at concentrations of 10^?7 to 10^?6M stimulated growth with 4 to 6 per cent but this effect was not statistically significant. Concentrations higher than 5 × 10^?6M inhibited growth. FAP and BAP (from 10^?8M to 10^?6M) had no significant interaction with any other growth substance used. The two-factor interactions of IAA × ABA, IAA × GA₃, and GA₃× ABA, as well as the three-factor interaction IAA × ABA × GA₃ were significant. However, the IAA × ABA interaction was significant only when high concentration (10^?6M) of ABA was used. The growth inhibition produced by 10^?7 and 10^?6M ABA was overcome by about equimolar concentrations of IAA. The stimulation of growth by GA₃ and GA₄₊₇ (10^?9 to 10^?7M) was prevented by simultaneous application of ABA, and it was reduced significantly by application of IAA (10^?7 to 10^?8M). GA3 at 10^?8M combined with different concentrations of IAA gave slightly higher elongation than IAA alone but the observed values were significantly lower than expected assuming independent additive action.     

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.     

Purification and characterisation of monoamine oxidase from Avena sativa

FAD-containing monoamine oxidase (MAO; EC 1.4.3.4) oxidises monoamines to their corresponding aldehydes, H₂O₂, and NH₃. It has been purified to homogeneity in mammals, but to our knowledge, there have been no reports of the enzyme in plants. MAO activity was detected in Avena sativa seedlings during germination using benzylamine as substrate. The enzyme was purified to homogeneity (as assessed by native PAGE) by Sephadex G-25, DEAE Sephacel, hydroxyapatite, Mono Q, and TSK-GEL column chromatographies. The molecular mass estimated by gel filtration using the TSK-GEL column was 220?kDa. SDS-PAGE yielded four distinct protein bands of 78, 58, 55, and 32?kDa molecular masses. The pI value of the enzyme was 6.3. The enzyme showed high substrate specificity for an endogenous amine, phenethylamine, which was oxidised to phenylacetaldehde, but not for ethylamine, propylamine, butylamine, pentylamine, dopamine, serotonin, tryptamine, or tyramine. The K _m values for benzylamine and phenethylamine were 2.7?×?10^?4 and 7.1?×?10^?4?M, respectively. Enzyme activity was not inhibited by pargyline, clorgyline, semicarbazide, or Na-diethyldithiocarbamate. Benzaldehyde, the product of benzylamine oxidation, exhibited strong competitive inhibition of enzyme activity with a Ki of 3???M. FAD was identified by ODS-column chromatography as an enzyme cofactor. The enzyme contained 2?mol of FAD per 220,000?g of enzyme.     

Coconut α-d-galactosidase isoenzymes: isolation,purification and characterization

α-d-Galactosidases (α-d-galactoside galactohydrolase, EC 3.2.1.22) from normal coconut endosperm were isolated and partially purified by a combination of ammonium sulfate fractionation, SP-Sephadex C50–120 ion-exchange chromatography and Sephadex G-200 and G-100 gel filtration. Two molecular forms of the enzyme, designated as A and B, were eluted after SP-Sephadex C50–120 ion-exchange chromatography. α-d-Galactosidase A, which is the major isoenzyme, was partially purified 43-fold on Sephadex G-200 and has a MW of about 23 000 whereas α-d-galactosidase B was partially purified 23-fold on Sephadex G-100 and has a similar MW of about 26 600. Both isoenzymes exhibited optimum activity at pH 7.5. The apparent K_m and V_max of α-d-galactosidase A were obtained at 3.46 × 10^?4M and 1.38 × 10^?3 M p-nitrophenyl α-<d-galactoside, respectively. A distinct substrate inhibition was noted. The enzyme was inhibited strongly by d-galactose and to a lesser extent by myo-inositol, d-glucose-6-phosphate, l-arabinose, melibiose and iodoacetic acid. Similarly, makapuno α-d-galactosidase was localized in the 40–70 % (NH₄)₂SO₄ cut but its optimum activity at pH 7.5 was considerably lower as compared to the normal. Its K_m was obtained at 6.75 × 10^?4 M p-nitrophenyl α-d-galactoside while the V_max was noted at 5.28 × 10^?3 M p-nitrophenyl α-d-galactoside. Based on the above kinetic data, the possible cause(s) of the deficiency of α-d-galactosidase activity in makapuno is discussed.     

Inhibition by indomethacin and aspirin of 15-hydroxy-prostaglandin dehydrogenase in vitro

15-Hydroxyprostaglandin dehydrogenase from bovine lung was purified 7.4 times to a specific activity of 1.4 mU/mg of protein. The isoelectric point was estimated to 5.4 and the molecular weight by gelfiltration to 40,000. K_m for prostaglandin E₁ and for NAD⁺ were found to be 3.4 μM and 1.1 × 10^?4M respectively. The enzyme was inhibited by indomethacin and aspirin. The indomethacin inhibition was found to be non-competitive to prostaglandin E₁ having a K_i=1.4 × 10^?4M and a K_i^′=1.6 × 10^?5M.     

Properties of a repressible alkaline phosphatase secreted by the wild-type strain 74a of neurospora crassa

A repressible extracellular alkaline phosphatase (with activity increasing steadily even up to pH 10.5) was purified from cultures of the wild-type strain 74A of Neurospora crassa, after growth on acetate and under limiting amounts of inorganic phosphate for 72 hr at 30°. The enzyme was homogeneous on polyacrylamide gel electrophoresis (PAGE) with or without sodium dodecyl sulphate (SDS). The MW was ca 172 000 and 82 000 as determined by Sephadex G-200 gel filtration and SDS-PAGE, respectively. The enzyme contained 23.6% neutral sugars, cations were not required for activity, and it was not inactivated by 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) at pH 8. Kinetic data showed Michaelian behaviour for the enzymatic hydrolysis of 4-nitrophenyl disodium orthophosphate (PNP-P) at pH 9 (the K_m value and Hill coefficient were 2.2 × 10^?4 M and 0.95, respectively). It was also shown that, at pH 9, the apparent number of Pi bound per dimer molecule equalled one, with a K_i value of 7.0 × 10^?4 M. The secreted enzyme showed half-lives of 23.5, 49.0 and 23.5 min at, pH 5.4, 7.4 and 9.0, respectively, after thermal inactivation at 60°. At pH 5.4, the half-life value was quite similar, while the others were respectively 2 and 4 times greater than those previously described for the repressible alkaline phosphatase retained by the mycelium at pH 5.6 or secreted by ‘slime’ cells.     

Purification of cassava linamarase

Linamarase was purified from parenchymal tissue of cassava by extraction with acetate buffer, fractional precipitation with ammonium sulphate, followed by column chromatography on DEAE-cellulose and Sepharose-6-B gel filtration. The specific activity is increased 350 fold with 35% recovery. The K_ms for linamarin and p-nitro-phenyl β-D-glucoside are 1.45 × 10^?3 M and 0.46 × 10^?3 M, respectively. The pH optimum in 50 mM NaPi is pH 6 and the specific activity is 26.5 nkat/mg. The enzyme can be prepared from cassava peel using the same procedure and has similar properties.     

Cerebral microvessels contain a beta 2-adrenergic receptor

Cerebral microvessels isolated from cat forebrain contain a specific β-adrenergic-sensitive adenylate cyclase. Among various compounds tested, the most potent activator of enzyme activity is isoproterenol (k_a = 1.4 × 10^?7M), followed in order by epinephrine (k_a= 1.5 × 10^?6M), norepinephrine (k_a= 1.4 × 10^?5M) and phenylephrine (k_a> 3 × 10^?4M). Isoproterenol-stimulated enzyme activity is blocked by propranolol (k_i= 2.4 × 10^?9M, IPS 339 (k_i= 4 × 10^?9M), H35/25 (k_i = 1.2 × 10^?7M), atenolol (k_i= 5.9 × 10^?6M) and practolol (k_i= 1.8 × 10^?5M). These agonist and antagonist properties are quite similar to those demonstrated by β₂-adrenergic receptors and β₂-stimulated adenylate cyclase present in other tissues and indicate that the majority of adenylate cyclase-associated adrenergic receptors in cerebral microvessels are β₂. The findings are relevant to physiological studies of cerebral blood flow and vascular permeability.     

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.     

Purification and characterization of diamine oxidase from rice embryos

Diamine oxidase of rice seedlings has been purified 1800-fold to homogeneity. The MW of the enzyme as determined by Sephadex G-100 gel filtration was 12.3 × 10⁴ and the enzyme contained two identical subunits each with a MW of 6.12 × 10⁴. The optimal temperature and pH for the enzyme were 30° and 7.5 respectively and the enzyme followed typical Michaelis kinetics with a K_m of 10^?5 M. Each enzyme molecule contained four molecules of FAD.     

Differential in vitro inhibition of polyphenoloxidase from a wild edible mushroom Lactarius salmonicolor

The polyphenol oxidase (LsPPO) from a wild edible mushroom Lactarius salmonicolor was purified using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. At the optimum pH and temperature, the K_M and V_Max values of LsPPO towards catechol, 4-methylcatechol and pyrogallol were determined as 0.025 M & 0.748 EU/mL, 1.809 × 10^{? 3} M & 0.723 EU/mL and 9.465 × 10^{? 3} M & 0.722 EU/mL, respectively.

Optimum pH and temperature values of LsPPO for the three substrates above ranged between the pH 4.5–11.0 and 5–50°C. Enzyme activity decreased due to heat denaturation with increasing temperature. Effects of a variety of classical PPO inhibitors were investigated opon the activity of LsPPO using catechol as the substrate. IC₅₀ values for glutathione, p-aminobenzenesulfonamide, L-cysteine, L-tyrosine, oxalic acid, β-mercaptoethanol and syringic acid were determined as 9.1 × 10^{? 4}, 2.3 × 10^{? 4} M, 1.5 × 10^{? 4} M, 3.8 × 10^{? 7} M, 1.2 × 10^{? 4} M, 4.9 × 10^{? 4} M, and 4 × 10^{? 4} M respectively. Thus L-tyrosine was by far the most effective inhibitor. Interestingly, sulfosalicylic acid behaved as an activator of LsPPO in this study.     

Zinc as a co-factor for an enzyme involved in exsheathment of Haemonchus contortus

The activity of concentrated exsheathing fluid of Haemonchus contortus against isolated sheaths was not inhibited by ethylenediamine tetra-acetic acid (EDTA), 10^?2 M, even when the concentrations of Mg and Mn were < 4 × 10^?4 M and < 0·9 × 10^?6 M respectively. Purified or diluted solutions of exsheathing fluid, even in the presence of Mg²⁺, 10^?3 M, were inhibited. Leucine aminopeptidase (LAP) in exsheathing fluid was active even at concentrations of Mg < 1·3 × 10^?5M. Concentrated solutions were partially inhibited by EDTA, 10^?2 M, at low concentrations of Mg; inhibition was increased in diluted and purified preparations.1,10-phenanthroline (Ophen) strongly inhibited exsheathing activity (Zn < 1 × 10^?6 M). When Zn²⁺, 10^?3 M was added, the inhibition was abolished. The hydrolysis of l-leucinamide was greatly increased in the presence of Ophen, 10^?4 M; this effect was abolished by adding Zn²⁺, 10^?3 M.It is suggested that exsheathing fluid from at least some ‘strains’ of H. contortus contains a Zn metallo-enzyme, probably LAP, which is involved in the process of exsheathment.     

Phosphofructokinase of Solanum tuberosum tuber

Potato tuber phosphofructokinase was purified 19·.6-fold by a combination of ethanol fractionation and DEAE-cellulose column chromatography. The enzyme was very unstable; its pH optimum was 8·0. K_m for fructose-6-phosphate, ATP and Mg²⁺ was 2·1 × 10^?4 M, 4·5 × 10^?5 M and 4·0 × 10^?4 M respectively. ITP, GTP, UTP and CTP can act as phosphate donors, but are less active than ATP. Inhibition of enzyme activity by high levels of ATP was reversed by increasing the concentration of fructose-6-phosphate; the affinity of enzyme for fructose-6-phosphate decreased with increasing concentration of ATP. 5′-AMP, 3′,5′-AMP, 3′-AMP, deoxy AMP, UMP, IMP, CMP, GMP, ADP, CDP, GDP and UDP did not reverse the inhibition of enzyme by ATP. ADP, phosphoenolpyruvate and citrate inhibited phosphofructokinase activity but Pi did not affect it. Phosphofructokinase was not reactivated reversibly by mild change of pH and addition of effectors.