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.     

Sucrose metabolism by roots of Pisum sativum

The aim of this work was to relate [¹⁴C]-sucrose metabolism to the activities of sucrose synthetase and acid and alkaline invertases in roots of Pisum sativum. We fed [U-¹⁴C]-sucrose to 5-day-old plants and then excised the apical 6 mm of the roots and dissected the regions 6–24 mm from the root apices into stele and cortex. The detailed distribution of ¹⁴C in these parts of the roots was determined at the end of the feeding period and after a chase. The data indicate that sucrose arriving in the stele is divided between storage, conversion to polysaccharide, and consumption in respiration, whereas sucrose arriving in the rest of the root is used in respiration or converted to polysaccharide or hexose so rapidly that little is stored. Fractionation of carefully prepared extracts of pea roots, tubers of Solanum tuberosum, and spadices of Arum maculatum showed that sucrose synthetase was recovered in the soluble fraction. The results are discussed in relation to the roles of the aforementioned enzymes.     

Separation and properties of two ornithine carbamoyltransferases from Pisum sativum seedlings

Two ornithine carbamoyltransferases were separated from pea seedlings on DEAE-cellulose. The two enzymes have different pH-activity curves. Lineweaver-Burk plots for both enzymes are linear for carbamoyl phosphate and the Michaelis constants are of the same order of magnitude. The plot for ornithine was linear for one enzyme but a concave down for the other indicating negative cooperativity. The presence of two ornithine carbamoyltransferases is consistent with the presence of two pools for ornithine (one catabolic and the other anabolic previously suggested to exist in plant materials.     

Lipoxygenase in dark-grown Pisum sativum

Lipid oxidizing activity has been detected in acetone powders from both dark- and light-grown dwarf pea seedlings. This activity has been shown by several methods to be due to lipoxygenase. The enzyme from dark-grown seedlings has been purified 5·7-fold by ammonium sulphate precipitation and gel filtration. CM-cel-lulose chromatography of the purified enzyme yielded four active fractions. The properties of the four lipoxy-genase isoenzymes are described.     

Control of glutamate dehydrogenase from Pisum sativum roots

-Glutamate dehydrogenase (GDH) was found in soluble and particulate (mitochondrial) fractions of pea roots. The activity of NADH-dependent GDH in fresh mitochondrial extract was increased about 10-fold by addition of zinc, manganese or calcium, but high concentrations of zinc were inhibitory. During storage, GDH activity of the mitochondrial extract slowly increased. The NADH activity was inhibited by citrate and other chelating agents. NADH-dependent reductive amination was also inhibited by glutamate, the product of the reaction; by contrast NADPH dependent activity was relatively unaffected by zinc, chelating agents or glutamate. Sensitivity (of NADH-GDH) to glutamate was lost on purification, but was restored when the enzyme was immobilized by binding to an insoluble support (AE cellulose). Glutamate appears to change the affinity of the enzyme for 2-oxoglutarate.     

Threonine-sensitive aspartate kinase and homoserine dehydrogenase from Pisum sativum

Aspartate kinase and two homoserine dehydrogenases were partially purified from 4-day-old pea seedlings. A sensitive method for measuring aspartate kinase activity is described. Aspartate kinase activity was dependent upon ATP, Mg²⁺ or Mn²⁺, and aspartate. The aspartate kinase was inhibited in a sigmoidal manner by threonine and K_i for threonine was 0·57 mM. The enzyme could be desensitized to the inhibitor and threonine protected the enzyme against thermal inactivation. Aspartate kinase activity was enhanced by isoleucine, valine and alanine. Homoserine, methionine and lysine were without effect. The homoserine dehydrogenase activity which was associated with aspartate kinase during purification could be resolved into two peaks by gel filtration. The activity of both peaks was inhibited by aspartate and cysteine and one was inhibited by threonine.     

Homoserine dehydrogenase in Pisum sativum and Ricinus communis

Homoserine dehydrogenase was extracted from Ricinus communis and Pisum sativum. The kinetic parameters of the forward and reverse reactions were determined. In the forward reaction only the enzyme from Ricinus is inhibited by threonine. The response to K⁺ is different for the enzyme from the two sources.     

De novo synthesis of acetylcholinesterase in roots of Pisum sativum

Pisum sativum seeds contain a conserved acetylcholinesterase (AChE) which is active during the early stages of germination. The enzyme activity soon disappears and reappears after 72 hr of germination. A protein devoid of catalytic ability, but exhibiting similar chromatographic and electrophoretic properties as the active AChE, could be detected after 24 hr of germination. The pattern of incorporation of labelled amino acids into AChE and the influence of cycloheximide revealed that the AChE found in the roots from 72 hr onwards was entirely new. During this period of growth, the AChE protein accounts for 4–10% of the total proteins in the root tissue.     

d-Alanine in germinating Pisum sativum seedlings

Germinating pea seedlings (Pisum sativum var. Alaska) contain high concentrations of d-alanine, which occurs in the decotyledonized parts as the conjugates, N-malonyl-d-alanine and γ-l-glutamyl-d-alanine. By contrast, free alanine in pea seedlings is almost all l-isomer. During early stages of the germination, γ-l-glutamyl-d-alanine increased significantly and amounted to ca. 2.5 μmol/seedling at 8 days.     

Chloroplast cysteine synthases of Trifolium repens and Pisum sativum

About 68–86% of the cysteine synthase activity in leaf tissue of white clover (Trifolium repens) and peas (Pisum sativum cultivar Massey Gem) was associated with chloroplasts. The enzymes from white clover and peas were purified ca 66 and 12-fold respectively. For clover, the K_m values determined by calorimetric and S^2? ion electrode methods were: S^2? 0.51 and 0.13 mM; O-acetylserine (OAS), 3.5 and 2.O mM respectively. The analogous values for the pea enzyme were: S^2?, 0.24 and 0.06 mM; OAS, 3.1 and 0.24 mM. Both enzymes were inhibited by cystathionine and cysteine. Pretreatment with cysteine inactivated the enzyme, but addition of pyridoxal phosphate caused partial reactivation. Isolated pea chloroplasts (70–75 % intact) catalysed OAS-dependent assimilation of sulphide at a mean rate of 88 μmol/mg Chl/hr. About 85 % of the OAS-dependent sulphide assimilated was recovered as cysteine. The rates were unaffected by light and 2 μM DCMU. Sonicating the chloroplasts enhanced the rate by 1.3–2 fold. Cysteine synthase activity was associated with the chloroplast stroma. Similar results were obtained for clover chloroplasts except that both the intactness and the rates were lower.     

Properties of acetylcholinesterase from Pisum sativum

Acetylcholinesterase (AChE) from Pisum sativum purified 28 fold showed two closely moving protein bands on polyacrylamide gel electrophoresis, both     

Chromatographic analysis of isoflavonoid accumulation in stressed Pisum sativum

High-performance liquid chromatography has been used to study isoflavonoid accumulation in copper(II) chloride stressed Pisum sativum. Liquiritigenin, isoliquiritigenin, formononetin, pseudobaptigenin, afrormosin and anhydropisatin have been identified in addition to the pterocarpan phytoalexin pisatin. The relationships of these metabolites to isoflavonoid biosynthesis and stress response in pea are discussed.     

Polyamine oxidation by enzymes from Hordeum vulgare and Pisum sativum seedlings

The properties of the amine oxidases of barley leaves and pea seedling cotyledons have been compared using a colorimetric assay in which the hydrogen p     

Synthesis of wall glucan from sucrose by enzyme preparations from Pisum sativum

Radioactive sucrose, supplied through the cut base to Pisum sativum epicotyls, was transported to the growing apex (plumule and hook) and used there for the synthesis mainly of uridine diphosphoglucose (UDP- glucose), fructose and cell wall glucan. Enzyme extracts of the apical tissue contained sucrose synthetase activity which was freely reversible, i.e. formed UDP-glucose and fructose from sucrose (pH optimum = 6·6 for the cleavage reaction, K_m for sucrose = 63 mM). Particulate fractions of the same tissue contained a β-glucan synthetase which utilized UDP-glucose for formation of alkali-soluble and -insoluble products (pH optimum = 8·4, K_m for UDP-glucose = 1·9 mM). Values for V_max and yields of these two synthetase activities were sufficient to account for observed rates of cellulose deposition during epicotyl growth (15–25 μg/hr/epicotyl). When soluble pea enzyme was supplied with sucrose and UDP at pH 6·6 and then the preparation was supplemented with particles bearing β-glucan synthetase at pH 8·4, the glucose moiety of sucrose was converted to glucan in vitro. The results indicate that it is feasible for these synthetases to co-operate in vivo to generate β-glucan for expanding cell walls.     

Peroxidase changes during the cessation of elongation in Pisum sativum stems

The cessation of cell elongation in intact P. sativum epicotyls is accompanied by an increase in both soluble and cell wall peroxidases. These pero     

Starch synthases and starch branching enzymes from Pisum sativum

Concentrations of ADPglucose:α-1,4-glucan-4-glucosyltransferase (starch synthase) and α-1,4 glucan: α-1,4-glucan-6-glycosyltransferase (branching enzyme) from developing seeds of Pisum sativum were measured. Primed starch synthase activity increased from 8 to 14 days after anthesis and decreased by 50 % at 26 days. Citrate-stimulated starch synthase activity was highest at 10 days after anthesis decreasing to low levels by 22 days. Branching enzyme activity increased from 8 to 18 days after anthesis and decreased little by 26 days. Two fractions of starch synthase were recovered by gradient elution from DEAE-cellulose of extracts from 12- and 18-day-old seeds. The two fractions differed in primer specificity, K_m for ADPG and relative amounts of citrate-stimulated activity. A major and minor fraction of branching enzyme were observed in extracts from both 12- and 18-day-old seeds. Marked differences in the relative abilities ofthe two branching enzyme fractions to stimulate phosphorylase and to branch amylose as well as pH optima were found. Although the content of the starch synthase and branching enzyme fractions varied with seed age, little difference was seen in the properties of chromatographically similar fractions. Therefore, the changes in starch synthase and branching enzyme activity during pea seed development resulted from changes in the concentrations of a few enzyme forms, but not the appearance of different enzyme forms.     

Proteolytic and trypsin inhibitory activities in extracts of germinating Pisum sativum seeds

A rise in azoglobulytic activity and a fall in trypsin-inhibitory activity has been detected in extracts of germinating pea seeds free from microbial infection. By investigating the subcellular distribution of protease activity, trypsin-inhibitory activity and the effect of the inhibitor on endogenous protease activity, no convincing evidence could be obtained to suggest that the trypsin-inhibitory activity was involved in the regulation of proteases during germination.     

Identification of cadaverine in Pisum sativum

Cadaverine has been identified in normal young peas and in saline grown (necrotic) peas by GLC and MS. Its concentration was at least of 5 gm/g fresh plant in both cases. Analysis of the alkaloidal extracts failed to reveal any lupinine, anabasine or other pyridine alkaloids in normal or necrotic, salt grown, peas.     

De novo synthesis of d-alanine in germinating Pisum sativum seedlings

The amounts of d-alanine derivatives, γ-l-glutamyl-d-alanine and N-malonyl-d-alanine, increase rapidly during the early growth of pea seeds. Pyruvate-[1?¹⁴C], l-alanine-[U?¹⁴C], d-alanine-[U?¹⁴C], l-alanine-[¹⁵N] and ¹⁵NH₄Cl were therefore fed to the seedlings and the incorporation investigated. Labelling results revealed that pea seedlings can utilize these erogenous compounds to form d-alanine and that labelled l-alanine is effectively converted to the d-enantiomer with retention of ¹⁴C and, largely, ¹⁵N label. Enzyme analyses in vitro provided additional evidence that the extract of pea seedlings catalyzes the direct conversion of l-alanine to d-alanine. The data suggest that the de novo synthesis of d-alanine in pea seedlings occurs by a racemase reaction.