Partial purification and some biochemical properties of nonspecific alkaline phosphatase in germinating chick-pea (Cicer arietinum) seeds

A procedure for the partial purification of a non-specific alkaline phosphatase (EC 3.1.3.1.) from the embryonic axes of chick-pea seeds is described. Ammonium sulphate precipitation, DEAE-cellulase chromatography, Sephacryl S-200 chroma-tography and polyacrylamide gel electrophoresis are the most important steps. The molecular weight of this non-specific enzyme, as determined by Sephacryl S–200 gel filtration and SDS–polyacrylamide gel electrophoresis, was estimated as being 68 and 78 kDa respectively; the optimum pH for p-nitrophenylphosphate hydrolysis was 7.5, and the K_m for this artificial substrate was 0.5 mM. The enzyme catalyzes the hydrolysis of a variety of organic phosphate esters. The best substrates are: phos-phoenolpymvate (K_m= 2.4 m M ), NADP⁺ (K_m= 4.0 m M ), 5'-AMP (K_m= 4.5 m M ), 5'-ADP (K_m= 6.1 m M ) and ribose-5P (K_m= 5.8 m M ); but it is unable to hydrolyze 5'-ATP, phosphocreatine and tripolyphosptiate. Phospate was a competitive inhibitor. Zn²⁺, K⁺, Hg²⁺ and Mo⁶⁺ were strong inhibitors, whereas F⁻ and Ca²⁺ inhibited weakly; Co²⁺ and Ni²⁺ were activators.     

GTP-specific pyrophosphorylation of thiamin in dark-grown soybean (Glycine max) seedling axes

ATP:thiamin pyrophosphotransferase (TPT: EC 2.7.6.2) was purified 5 900-fold from 48 h dark-grown soybean [ Glycine max (L.), Merr. cv. Ransom II] seedling axes. TPT activity was monitored during purification by measuring the formation of thiamin pyrophosphate (TPP) from [2-¹⁴C]-thiamin at optimal pH (7.3). Although other nucleoside triophosphates were active as pyrophosphate donors (apparent K_ms from 21 to 138 m M ), GTP was the preferred nucleotide with an apparent K_m of 0.021 m M . TPT activity was extremely sensitive to TPP formation, suggesting product feedback inhibition of TPT activity in vivo. Sulfhydryl, H⁺ and Mg²⁺ concentrations, either independently or in concert, were found to affect TPT activity.     

Purification and kinetic properties of a castor bean seed acid phosphatase containing sulfhydryl groups

An acid phosphatase (EC 3.1.3.2) has been identified and purified from castor bean ( Ricinus communis L., IAC-80 ) seed through sulphopropyl (SP)-Sephadex, diethylaminoethyl (DEAE)-Sephadex, Sephacryl S-200, and Concanavalin A-Sepharose chromatography. The enzyme was purified 2 000-fold to homogeneity, with a final specific activity of 3.8 μkat mg⁻¹ protein. The purified enzyme revealed a single diffuse band with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis, at pH 8.3. The relative molecular mass, determined by high-performance liquid chromatography (HPLC), was found to be 60 kDa. The acid phosphatase had a pH optimum of 5.5 and an akpparent K_m value for p -nitrophenylphosphate of 0.52 m M . The enzyme-catalyzed reaction was inhibited by inorganic phosphate, fluoride, vanadate, molybdate, p -chloromercuribenzoate ( p CMB), Cu²⁺ and Zn²⁺. The strong inhibition by p CMB, Cu²⁺ and vanadate suggests the presence of sulfhydryl groups essential for catalysis. The castor bean enzyme also recognized tyrosine-phosphate and inorganic pyrophosphate (KPP_i) as substrate. The highest specificity constant (V_max/K_m) was observed with KPP_i, making it a potential physiological substrate.     

Resolution and characterization of multiple cytosolic phosphatases capable of hydrolyzing fructose-1,6-bisphosphate in spinach and soybean leaves

The apparent activity of cytoplasmic fructose bisphosphatase (EC 3.1.3.11) in crude extracts of spinach ( Spinacia oleracea L.) and soybean ( Glycine max [L.] Merr.) leaves was only partially dependent on Mg²⁺. At least two major non-chloroplastic fructose bisphosphatases that differed in dependence on Mg²⁺ were chromatographically resolved from spinach leaves. The Mg²⁺-dependent enzyme had an apparent Michaelis constant of 4 μM for fructose-1,6-P₂, was highly specific, and was strongly inhibited by fructose-2,6-P₂. Enzyme activity was inhibited by physiological levels of fructose-6-P.
Both species also contained at least one major enzyme, the activity of which was independent of Mg²⁺. These enzymes had pH optima near neutrality, Michaelis constants of 25 to 30 μM for fructose-1,6-P₂, and were inhibited by AMP. Although hexose monophosphates were not metabolized, the enzymes were not specific for fructose-1,6-P₂: phosphate was released from phosphoenolpyruvate and ribulose-1, 5-P₂, and with fructose-1,6-P₂, as substrate, Pi release was about 1.5-fold greater than fructose-6-P production. It is concluded that only the Mg²⁺-dependent fructose bisphosphatase, previously characterized, functions in the photosynthetic sucrose formation pathway. Inhibition of the Mg²⁺-dependent enzyme by fructose-6-P may be involved in regulation of sucrose formation.     

Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana

Adenine phosphoribosyltransferase (APRT; EC 2. 4,2. 7) from Arabidopsis thaliana was purified approximately 3800-fold, to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, (NH₄)₂SO₄ precipitation, Sephadex G-25 salt separation, ultracentrifugation and liquid chromatography on Diethylaminoethyl Sephacel, Phenyl Sepharose CL-4B, Blue Sepharose CL-6B and adenosine 5'-monophosphate-Agarose. The purified APRT was a homodimer of approximately 54 kDa and it had a specific activity of approximately 300 μmol (mg total protein)^-1 min^-1. Under standard assay conditions, the temperature optimum for APRT activity was 65°C and the pH optimum was temperature dependent. High enzyme activity was dependent upon the presence of divalent cations (Mn²⁺ or Mg²⁺). In the presence of MnCl₂₊ other divalent cations (Mg²⁺, Ca²⁺, Ba²⁺, Hg²⁺ and Cd²⁺) inhibited the APRT reaction. Kinetic studies indicated that 5-phosphoribose-1-pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The K_m for adenine was 4.5±1.5 μ M , the K_m for PRPP was 0.29±0.06 m M and the K_i for PRPP was 1.96±0.45 m M . Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The K_m for benzyladenine was approximately 0.73±0.06 m M     

Phosphoenolpyruvate carboxylase in root nodules of Vicia faba: Partial purification and properties

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) was purified 56-fold from Vicia faba root nodules to a specific activity of 24.8 units mg^-1 protein. Native molecular mass was determined to be 443 kDa by gel permeation chromatography, whereas a molecular mass of 113 kDa was obtained for the subunit by means of SDS-PAGE, indicating that the enzyme is a homotetramer. One peak of activity was obtained by ion-exchange chromatography or gel filtration, and thus there was no evidence of isoenzymes. The effect of pH on PEPC activity was studied, the pH optimum found at 8.25. The effect of substrate (phosphoenolpyruvate, PEP) on the enzyme activity was studied at five different pH values from 6.5 to 9.5. The K_m(PEP) at pH 8.25 proved to be 0.064 m M. Inhibition by malate or activation by glucose-6-phosphate was dependent on the pH of the reaction mixture. Malate behaved as a non-competitive mixed-type inhibitor with a K_i of 0.76 m M , a K_i(s) of 1.15 m M and a K_i(i) of 0.72 m M , at pH 7.0 while at pH 8.25 K_i was about 140 m M. Activation by glucose-6-P was 70% with 4 m M PEP at pH 7, whereas no effect was found at pH 8.25. Experiments with mixed effectors at pH 7 and 1 m M PEP, showed that glucose-6-P can reverse the inhibition caused by L-malate on the PEPC activity.     

Purification and properties of a neutral invertase from the roots of Cichorium intybus

Multiple activity peaks of neutral invertase (EC 3.2.1.26) were found in chicory roots ( Cichorium intybus L. var. foliosum cv. Flash). The main activity peak was purified by a combination of anion-exchange chromatography, hydrophobic interaction chromatography, chromatofocusing and gel filtration. This protocol produced a 77-fold purification and a specific activity of 1.6 μmol (mg protein)⁻¹ min⁻¹. The mass of the enzyme was 260 kDa as estimated by gel filtration and 65 kDa on SDS-PAGE. Optimal activity was found between pH 7 and 7.5. The purified enzyme exhibited hyperbolic saturation kinetics with a K_m between 10 and 20 mM for sucrose. No other products than glucose and fructose could be detected. Raffinose was hydrolyzed at a rate of 2.4% relative to sucrose whereas the enzyme did not hydrolyze maltose, cellobiose, trehalose, 1-kestose, 1.1-nystose or inulin. Neutral invertase activity was completely inhibited by HgCl₂ and AgNO₃ and partially inhibited by CoCl₂, and ZnSO₄ (1 mM). Pyridoxal phosphate (K_i≅ 500 μ M ), Tris (K_i≅ 1.2 m M ), glucose and fructose (K_i≅ 16 m M ) were strong inhibitors of the enzyme. Fructose and Tris behaved as competitive inhibitors. A possible role for the enzyme's activity in vivo is discussed.     

γ-Glutamyl-transpeptidase in tobacc suspension cultures: Catalytic properties and subcellular localization

γ-Glutamyl-transpeptidase activity (EC 2.3.2.2) was found in ammonium sulfate precipitates of extracts from cultured cells of Nicotiana tabacum L. var. Samsun. Specific activity up to 3.2 nmol (mg protein)⁻¹ min⁻¹ was achieved, using the artificial substrate γ-glutamyl- p -nitroanilide (K_m 0.6 m M ) instead of glutathione. Optimal enzyme activity was obtained at pH 8.0–8.5 and 45°C. The enzyme reaction was inhibited competitively by γ-glutamyl analogs (6-diazo-5-oxo-L-norleucine: K_i 0.76 μ M ; L-azaserine: K_i 0.23 m M ) or the inorganic ion m -periodate (K_i 0.43 m M ). Cell fractionation and in vivo experiments revealed that 77% of the γ-glutamyl-transpeptidase activity is localized in the soluble cytoplasmic fraction, while 20–23% of the enzyme is found on the outer surface of the plasmalemma.     

Invertase and sucrose synthase activity, carbohydrate status and endogenous IAA levels during Citrus leaf development

Levels of activity of the sucrose catabolizing enzymes, acid invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13), were measured during development of new leaves of Citrus sinensis (L.) Osbeck cv. Shamouti. Soluble acid invertase showed a peak activity of 32 nkat (g fresh weight)⁻¹ at ca 60% of full leaf expansion and rapidly declined toward and after full expansion. There was no concomitant increase in an insoluble form of the enzyme. Sucrose synthase activity, measured in the synthesis direction, declined from 33% of full leaf expansion [10 nkat (g fresh weight)⁻¹] 10, and following, full expansion. Highest sucrose synthase activity, measured in the cleavage direction, was 6 nkat (g fresh weight)⁻¹ and showed little change during development. Acid invertase has a K_m of 5 m M for sucrose, while sucrose synthase had a K_m of 118 m M for sucrose. Changes in acid invertase activity correlated with changes in the reducing sugar:sucrose ratio. These results suggest that soluble acid invertase activity is the primary enzyme responsible for sucrose catabolism in the expanding Citrus leaf. Changes in leaf expansion rate and invertase activity did not correlate positively with changes in endogenous free IAA level, as determined by enzyme linked immunoassay.     

Ornithine decarboxylase activity in Helianthus tuberosus

Ornithine decarboxylase (ODC, EC 4.1.1.17) was studied in crude extracts of parenchyma slices of dormant tubers activated for 12 h, tuber shoots and shoot apices. It was highest in shoot apices. The enzyme activity was measured by the production of ¹⁴CO₂ from labelled ornithine; V_max was 450 nmol (mg protein)^-1h^-1, K_m for ornithine and pyridoxal phosphate were, respectively, 30 m M and 5μ M . Only when partially purified, the ¹⁴CO₂ production was inhibited by α-difluoromethylornithine, while in crude extracts dithiothreitol was inhibitory. Ornithine and arginine decarboxylase (ADC, EC 4.1.1.19) activities from parenchyma tubers were not greatly altered by exogenously supplemented ornithine, even though its endogenous pool increased. Exogenously supplemented arginine enhanced ornithine decarboxylase activity, whereas putrescine decreased it slightly. The possibility of artifactual activities in the crude extract is also discussed.     

Characteristics of β-galactosidase purified from cell suspension cultures of carrot

Five glycosidase activities from cell homogenate of carrot ( Daucus carota L. cv. Kintoki) cell cultures were assayed after extraction successively by phosphate buffer (pH 7.0) and the buffer plus 2 M NaCl. A β-galactosidase (EC 3.2.1.23) was isolated in a highly purified state from the buffer-soluble protein fraction by ammonium sulfate fractionation and chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50 and Sephadex G-200. The molecular weight of this enzyme was ca 104 000 and the isoelectric point was pH 7.8. The optimal activity occurred at pH 4.4 with McIlvaine buffer. The K_m and V_max values were 1.67 m M and 201 units (mg protein)⁻¹, respectively, for p -nitrophenyl β- d -galactopyranoside. The enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺, Hg²⁺ and d -galactono-1,4-lactone. The enzyme acted on the β-1,4-linked galactan prepared from citrus pectin in an exo-fashion. Furthermore, the enzyme was slightly involved in the hydrolysis of the pectic polymer and cell walls purified from carrot cell cultures.     

Isolation and characterization of cytoplasmic NADP+-malic enzyme from Lupinus luteus leaves

NADP⁺-dependent malic enzyme (L-malate : NADP⁺ oxidoreductase, decarboxylating, EC 1.1.1.40) was extracted from the leaves of yellow lupine. The purification procedure included fractionation with (NH₄)₂SO₄ and Sephadex G-25 chromatography, followed by purification on DEAE-cellulose and Sephadex G-200 columns. The enzyme was purified 122-fold. The enzyme affinity towards L-malate was found to be significantly higher with Mn²⁺ than with Mg²⁺. The Hill coefficient for Mg²⁺ depended on concentration and was 1.6 for the lower and 3.9 for the higher concentrations. The dependence of the enzyme activity on NADP⁺ followed a hyperbolic curve. K_m values and Hill coefficients for NADP⁺ were similar with both Mn²⁺ and Mg²⁺. The enzyme activity was strictly dependent on divalent cations and followed a sigmoidal curve at least for Mg²⁺. The enzyme had 4-fold higher affinity towards Mn²⁺ than towards Mg²⁺, the K_m values being 0.3 and 1.15 m M respectively. Of several tested organic acids, oxalate was the most effective inhibitor followed by oxaloacetate while succinate was the strongest activator.     

Genetic transformation using Agrobacterium rhizogenes

UDP-glucose pyrophosphorylase (EC 2.7.7.9) has been highly purified from the plant fraction of soybean ( Glycine max L. Merr. cv Williams) nodules. The purified enzyme gave a single polypeptide band following sodium docecyl sulphate polyacryla-mide gel electrophoresis, but was resolved into three bands of activity in non-denaturing gels. The enzyme appeared to be a monomer of molecular weight between 30 and 40 kDa. UDP-glucose pyrophosphorylase had optimum activity at pH 8.5 and displayed typical hyperbolic kinetics. The enzyme had a requirement for divalent metal ions, and was highly specific for the substrates pyrophosphate and UDP-glucose in the pyrophosphorolysis direction, and glucose-1-phosphate and UTP in the direction of UDP-glucose synthesis. The K_m values were 0.19 m M and 0.07 m M for pyrophosphate and UDP-glucose, respectively, and 0.23 m M and 0.11 m M for glucose-1-phosphate and UTP. The maximum velocity in the pyrophosphorolysis direction was almost double that for the reverse reaction. UDP-glucose pyrophosphorylase did not appear to be subject to a high degree of fine control, and activity in vivo may be regulated mainly by the availability of the substrates.     

Pyrophosphate-dependent phosphofructokinase from pollen: properties and possible roles in sugar metabolism

To evaluate the role of pyrophosphate-dependent phosphofructokinase (PFP. EC 2.7.1.90) in the sugar metabolism of pollen. its occurrence and properties were studied in pollen grains of several plants including camellia ( Camellia japonica L.). In all pollen samples, PFP was strongly activated by fructose-2,6-bisphosphate (F2,6BP), and the activity of F2,6BP-activated PFP was higher than that of phosphofructokinase (PFK. EC 2.7.1.11). PFP partially purified from camellia pollen required Mg²⁺ for activity with an optimum at 1 m M . and was almost unaflected by a variety of metabolites at 1 m M . Its molecular mass was around 220 kDa, and apparent K_m values for F6P, PP_i. F1, 6BP and P_i were 294, 4, 20 and 580 u M , respectively. The levels of F2.6BP. PP_i and F6P in camellia pollen were sufficent to support the forward reaction by PFP, and PFP, was 20- to 40-fold more active than PFK during pollen growth. These results suggest that pollen PFP plays a role in glycolysis but not gluconeogenesis. and the possible relevance of this to pollen tube growth is discussed.     

Purification and properties of NADP-dependent glutamate dehydrogenase from Sphaerostilbe repens

NADP-dependent glutamate dehydrogenase (EC 1.4.1.4) extracted from Sphaerostilbe repens was purified to homogeneity by using ammonium sullate fractionation hydroxyapatite and DEAE-cellulose column chromatography and, finally, preparative polyacrylamide gel electrophoresis. The turnover number of the enzyme for the amination reaction was about 66000 mol substrate transformed min^-1 (molecule of GDH)^-1. Molecular weight of the native enzyme was estimated to be 280000 dalton by polyacrylamide gradient gel electrophoresis. The same technique in the presence of sodium dodecyl sulfatc gave a single protein band that corresponded to the subunit molecular weight of 48000 dalton. Thus, it is concluded that NADP-GDH is composed of six identical polypeptidic chains.
The pH optimums were 6.9 and 8.4 for the forward and reverse reactions respectively. The NADP-GDH lost practically none of its activity for ten days at 4°C and for 15 h at room temperature, but was inactivated by higher temperatures. Thiol compounds such as 2-mercaptoethanol and dithiolhrcitol protected the enzyme from rapid inactivation. The Michaelis constants for GDH were 0.64, 0.049. 0.043 and 5.5 m M for α-ketoglutaratc. NADPH, NADP and glutamate, respectively. The enzyme had a negative cooperativity for ammonium (Hill number of 0.66), and its K_m value increased from 2.6 to 21.2 m M when the ammonium concentration exceeded 16 m M . The deamination reaction was highly sensitive to inhibition by ammonium, while the amination reaction was only slightly inhibited by glutamate. These results, considered together with the K_m values, indicate that the NADP-GDH in Sphaerostilbe repens is primarily concerned with glutamate biosynthesis.     

Shikimate dehydrogenase from pepper (Capsicum annuum) seedlings. Purification and properties

Shikimate dehydrogenase (SKDH, EC 1.1.1.25) was extracted from seedlings of pepper ( Capsicum annuum L.) and purified 347-fold. The purification procedure included precipitation with ammonium sulphate and chromatography in columns of Reactive Red-agarose, Q-Sepharose and Sephadex G-100. Pepper SKDH isozymes are separable only using PAGE. The purified enzyme has a relative molecular mass of 67 000 as estimated by gel filtration. The optimum pH of enzyme activity is 10.5 and the optimum temperature is 50°C, but the enzyme is quickly inactivated at temperatures higher than 40°C. The purified enzyme exhibited typical Michaelis-Menten kinetics and K_m values are 0.087 m M for shikimic acid and 0.017 m M for NADP. The mechanism of reaction is sequential considering NADP as a cosubstrate. Ions such as Ca²⁺, Mg²⁺ and Mn²⁺ activate the enzyme, but Zn²⁺ and Cu²⁺ are strong inhibitors. Some phenolic compounds such as guaiacol, protocatechuic acid and 2,4-D are competitive inhibitors of pepper SKDH, showing K_i values of 0.38 m M , 0.27 m M and 0.16 m M , respectively.     

Alanine aminotransferase in Leptosphaeria michotii. Isolation, properties and cyclic variations of its activity in relation to polypeptide level

Alanine aminotransferase (EC 2.6.1.2) was obtained from the fungus Leptosphaeria michotii (West) Sacc, and enriched 714-fold by a 5-step purification procedure as a dimer of M_r 110000, associated with a polypeptide of M_r 25000. Its isoelectric point was 5.25. The enzyme was active from pH 3.5 to 9.5 with a maximum at pH 7.5. Its specific activity was 6000 nkat (mg protein)⁻¹; the K_m was 6.85 m M for L-alanine and 0.2 m M for 2-oxoglutarate. The enzyme did not show any detectable activity in the presence of L-aspartate, cysteine sulfinate, α-aminobutyrate or cyclic amino acids as substrates. It did not express alanine:glyoxylate aminotransferase activity. Alanine aminotransferase in L. michotii has previously been shown to have an activity rhythm in constant temperature and darkness. The enzyme level was quantified along the activity rhythm by enzyme-linked immunosorbent assay (ELISA), using a monospecific polyclonal antibody against the purified enzyme. The cyclic variations of alanine aminotransferase activity were correlated with cyclic variations in the enzyme level.     

Properties and localization of the homoglutathione synthetase from Phaseolus coccineus leaves

The synthesis of homoglutathione (hGSH) by several plants of the tribe Phaseoleae is shown to be catalysed by a β-alanine-specific hGSH synthetase, Properties of the enzyme from Phaseolus coccineus L. cv. Preisgewinner were studied, using ammonium sulfate precipitates of primary leaf extracts. The hGSH synthetase showed a broad pH optimum at pH 8–9, an absolute requirement for Mg²⁺, a stimulation by K⁺, and a high affinity for γ-glutamylcysteine [K_m(app.) 73 μ M ]. The enzyme exhibited a high specificity for β-alanine [K_m(app.) 1.34 m M ] compared to glycine [K_m(app.) 98 m M ]. Chloroplasts, isolated from the leaves of Phaseolus coccineus , contained about 17% of the hGSH synthetase activity in the leaf cells.     

Soybean leaf urease: Comparison with seed urease

Soybeans, Glycine max (L.) Merr., from ureides for transport of nitrogen from the root nodule to the shoot. The most direct routes for ureide utilization include the degradation of ureide-derived urea to NH₃ and CO₂. Ureolytic activity was found in leaf disks of soybean and exhbited optimal activity at pH 7 in the presence of a high concentration of urea (250 m M ). In vitro studies showed neither urea amidolyase nor urea dehydrogenase activity in soybean leaves and the ureolytic activity was characterized as urease. Several biochemical properties of soybean leaf urease were determined and compared to seed urease properties. Soybean leaf urease differed from that of seed in five ways: pH optima (5.25 and 8.75), apparent K_m (0.8 m M ), no inhibition by hydroxyurea, faster electrophoretic mobility and no cross-reactivity with soybean seed urease antibodies. The data suggest that urease is the primary urea metabolizing enzyme present in soybean leaves. The properties of soybean leaf urease support the conclusion that a unique isozyme of urease is present in leaf tissue.