Changes in the Activities of Pyrrolooxygenases during the Germination of Wheat Grains

Porphobilinogen oxygenase, skatole pyrrolooxygenase, and tryptophan pyrrolooxygenase were found in the different parts of germinating wheat (Triticum aestivum) grain seedlings. In the embryos of grains germinated for 24 hours, the activities of PBG oxygenase and skatole pyrrolooxygenase were inhibited by a labile inhibitor. Tryptophan pyrrolooxygenase activity was not inhibited. Embryos of grains germinated for 48 hours showed higher activities for the three enzymes. The latter were also present in the radicles and coleoptiles of 96-hour germinated wheat grains. A DEAE-cellulose analysis of a crude enzymic preparation from embryos allowed the separation of two molecular forms of the three pyrrolooxygenases. The more cationic forms of porphobilinogen oxygenase and skatole pyrrolooxygenase were associated with the inhibitor. This form of porphobilinogen oxygenase had allosteric kinetics while the more anionic form had Michaelis kinetics. Both forms of skatole pyrrolooxygenase had Michaelis kinetics. The activity of tryptophan pyrrolooxygenase was highest in seedling roots and was found to be inhibited in seedling young leaves. This enzyme oxidized tryptophanyl dipeptides, as well as a nonapeptide, to N-formylkynurenine-containing peptides. The pyrrolooxygenase also oxidized the tryptophanyl residues of lysozyme, chymotrypsin, and trypsin.     

Pyrrolooxygenase isoenzymes from wheat germ

Both porphobilinogen oxygenase and skatole pyrrolooxygenase of wheat germ have isoenzyme forms of different charge. The more cationic isoenzymes were eluted from DEAE-cellulose with 10 mM Tris-HCl buffer (pH 7.6) and the less cationic were eluted with 50 mM NACl in the same buffer. The former had almost twice as many free amino groups (per mg of protein) as the latter. The more cationic isoenzyme was more sensitive to chelating agents and to acid treatment. They were differently inhibited by sodium dodecyl treatment and by temperature inactivation. Porphobilinogen oxygenase isoenzymes showed different activities with different buffers and also differed in their kinetics.     

Disappearance of porphobilinogen deaminase activity in leaves before the onset of senescence

The activity of porphobilinogen deaminase was measured in young and senescent or mature leaves of pepper (Capsicum annuum), and poinsettia (Euphorbia pulcherrima). Whereas high activity was found in the crude extracts of the young leaves, almost no activity was found in the extracts of senescent or mature leaves. The decrease in deaminase activity was not due to the presence of an isolatable inhibitor. By purifying the crude enzyme extracts from leaves of different ages on DEAE-cellulose columns it was shown that the decrease in deaminase activity was due to a real decrease in the amount of enzyme. Fruiting also decreased porphobilinogen deaminase activity. Several kinetic constants of the C. annuum deaminase were determined.     

Pyrrolooxygenases: A new type of oxidases

Summary A new type of oxygenases was isolated from plant and animal sources which oxidized pyrrole and indole derivatives. They had a broad substrate specificity and were called pyrrolooxygenases. Three different enzymes within the group were identified; skatole pyrrolooxygenase, tryptophan pyrrolooxygenase and porphobilinogen oxygenase. The first two oxidized the pyrrole ring of the various indole derivatives affording substituted o-formanidophenacyl derivatives as the main oxidation products. Tryptophan pyrrolooxygenase also oxidized the tryptophanyl residues of peptides and enzymes. When those residues were essential for the activity of the tryptophan containing enzymes, then inactive enzymes were obtained.Porphobilinogen oxygenase oxidized porphobilinogen and related alkylpyrrole compounds affording 3-pyrrolin-2-one derivatives. The pyrrolooxygenases acted as mixed-function oxidases, since they required the presence of oxygen and of a reducing agent. The substrate, the oxygen and the reductant were consumed in equimolar amounts. The best artificial reducing agent was sodium dithionite. Illuminated active chloroplasts were the natural reducing agent of the plant enzymes and NADPH was the reducing agent of the animal enzymes. Pyrrolooxygenases were located in the chloroplasts of green leaves and in the microsomes in the case of the mammalian enzymes. The activity of the enzymes in the crude extracts was usually low, due to the presence in the same of a protein inhibitor. When the inhibitor was separated by protein fractionation methods, full enzymatic activity was recovered. Destruction of the inhibitor by aging or by temperature had the same effect. The very low oxygenase activity present in the microsomal rat liver preparations could be strongly enhanced by previous administration to the rats of phenobarbital or steroids. This induction of the oxygenase activity was coincident with a drop in the amount of inhibitor present in the extracts.The properties and metabolic role of the pyrrolooxygenases are discussed.     

Pyrrolooxygenases from Argentine wheat varieties

Pyrrolooxygenase activities were examined in different varieties of Argentine wheat (Triticum aestivum) which included the traditional Klein varieties and the new mixed Mexican and traditional varieties (DeKalb and Cargill). The enzymatic activities were variety-dependent and were more inhibited in some varieties than in others, while some (Cargill) were devoid of the proteic inhibitor. The enzymes were isolated from the flours as two isoenzymes of different charge whose relative proportions were dependent on the variety of wheat used. The more cationic isoenzymes were eluted with 10 mM Tris-HCl buffer (pH 7.6 from DEAE-cellulose and the less cationic were eluted with 50 mM NACl in the same buffer. The protein inhibitor, when present, was associated with the more cationic isoenzymes. Porphobilinogen oxygenase and skatole pyrrolooxygenase activities were higher in the endosperm, while tryptophan pyrrolooxygenase activity was higher in the embryo. The proteic inhibitors were mainly concentrated in the embryo.     

Characterization of <Emphasis Type="Italic">Capsicum annuum</Emphasis> L. leaf and root antimicrobial peptides: antimicrobial activity against phytopathogenic microorganisms

This study aimed to detect and characterize antimicrobial proteins, especially antimicrobial peptides (AMPs) from leaves and roots of Capsicum annuum and to evaluate their inhibitory activities against different phytopathogenic fungi and the bacterium Xanthomonas euvesicatoria. Two methodologies were used for the extraction of peptides from leaves and roots of C. annuum: acid and ethanolic extraction. Extracts were subjected to reversed-phase chromatography on HPLC. The extraction and purification procedures were analysed by uni- and bi-dimensional electrophoresis in tricine gels. Our results show that alcoholic and acid extracts from both tissues can inhibit the growth of the phytopathogenics fungi C. lindemuthianum and C. gloeosporioides. The acid extracts from both tissues are active against X. euvesicatoria and only leaf extracts displayed specific inhibitory activity towards trypsin and α-amylase activity. The data compiled here aim to contribute to establish the multiplicity of potential uses of plant AMPs for the control of pests and pathogens of agricultural relevance.     

Purification and chemical modifications of porphobilinogen oxygenase

Porphobilinogen oxygenase from wheat germ was purified and was found to be a cationic protein containing 8 mol of nonheme iron and 8–10 mol of labile sulfide per mole of enzyme (M_r, 100,000). The enzyme isolated from either wheat germ or rat liver microsomes was found to exist in multiple molecular weight forms. When succinylated, only one molecular weight form of 25,000 was obtained and it retained full activity. It had lost all of the sigmoidal kinetics characteristic of the native enzyme. While the native enzyme had an n = 3.5, the succinylated enzyme showed Michaelian kinetics. A K_m of approximately 1.70 mm was determined for the succinylated wheat germ enzyme, and a K_m of approximately 2.5 mm was found for the succinylated microsomal enzyme. Acetylation of the enzyme afforded an active acetylated enzyme which showed allosteric kinetics and multiple molecular weight forms. The products formed by the succinylated enzyme were the same as those formed by the native enzyme.     

Antiviral activity of extracts of transgenic chicory and lettuce plants with the human interferon α2b gene

This paper studies the biological activity of protein extracts of the Cichorium intybus L. and Lactuca sativa L. transgenic plants with the human interferon ??2b gene againsf vesicular stomatitis virus. Extracts from transgenic lettuce and chicory roots, which were obtained after A. rhizogenes-mediated transformation, had antiviral activity in the range 1620?C5400 IU/g of weight; extracts from leaves of chicory plants transformed by A. tumefaciens, up to 9375 IU/g. The dependence of the antiviral activity of plant extracts from roots or leaves on the vector used for plant transformation is shown. The extracts of plant roots obtained by A. rhizogenes-mediated transformation had antiviral activity; at the same time, such activity was absent in the extracts from leaves.     

Characterization of cholinesterases from the parasitic nematode Trichinella spiralis

1. Trichinella cholinesterases occur in multiple molecular forms which differ in size, kinetics, activity with butyrylthiocholine, and effects of inhibitors.2. The 5.3 and 13S forms identified in Trichinella extracts are also found in C. elegans and other nematodes but the 7S form which occurs in other nematodes was absent from Trichinella detergent extracts. Differences in kinetic and inhibition properties among nematode species were also evident.3. The level of cholinesterases in excretory/secretory products is low.4. Trichinella cholinesterases did not elicit a detectable antibody response in mice.     

Adenine phosphoribosyltransferase in plant tissues: some effects of kinetin on enzymic activity

Adenine phosphoribosyltransferase activity was measured in extracts of soybean (Glycine max var. Acme) callus and of senescing barley leaves (Hordeum distichon c.v. Prior). The enzyme from soybean callus had Michaelis constants for adenine and 5-phosphoribosyl pyrophosphate of 1.5 and 7.5 μm respectively and was inhibited by AMP and stimulated by ATP. The presence of kinetin was found to considerably increase the activity of adenine phosphoribosyltransferase in extracts of soybean callus and senescing barley leaves.     

Normal growth of transgenic tobacco plants in the absence of cytosolic pyruvate kinase

The coding sequence of the cytosolic isozyme of potato tuber pyruvate kinase (PK) was attached to the transit peptide of the small subunit of pea ribulose-1,5-bisphosphate carboxylase oxygenase and placed under the control of the cauliflower mosaic virus 35S promoter. This construct was transformed into Nicotiana tabacum. Unexpectedly, two primary transformants were recovered in which PK activity in leaves was greatly reduced. The reduction in PK activity appeared to result from the complete absence of the cytosolic form of the enzyme (PK_c). In addition, no PK_c could be detected on western blots of leaf extracts. Metabolite analyses indicated that the levels of phosphoenolpyruvate are substantially higher in PK_c-deficient leaves than in wild-type leaves, consistent with a block in glycolysis at the step catalyzed by PK. PK_c deficiency in the leaves does not appear to adversely affect plant growth. Analysis of progeny indicates that PK_c deficiency is a heritable trait. The leaves of PK_c-deficient transformants have normal rates of photosynthetic O₂ evolution and respiratory O₂ consumption, indicating that these plants are using alternative pathways to bypass PK.     

Expression of Tobacco Carbonic Anhydrase in the C4 Dicot Flaveria bidentis Leads to Increased Leakiness of the Bundle Sheath and a Defective CO2-Concentrating Mechanism

Flaveria bidentis (L.) Kuntze, a C₄ dicot, was genetically transformed with a construct encoding the mature form of tobacco (Nicotiana tabacum L.) carbonic anhydrase (CA) under the control of a strong constitutive promoter. Expression of the tobacco CA was detected in transformant whole-leaf and bundle-sheath cell (bsc) extracts by immunoblot analysis. Whole-leaf extracts from two CA-transformed lines demonstrated 10% to 50% more CA activity on a ribulose-1,5-bisphosphate carboxylase/oxygenase-site basis than the extracts from transformed, nonexpressing control plants, whereas 3 to 5 times more activity was measured in CA transformant bsc extracts. This increased CA activity resulted in plants with moderately reduced rates of CO₂ assimilation (A) and an appreciable increase in C isotope discrimination compared with the controls. With increasing O₂ concentrations up to 40% (v/v), a greater inhibition of A was found for transformants than for wild-type plants; however, the quantum yield of photosystem II did not differ appreciably between these two groups over the O₂ levels tested. The quantum yield of photosystem II-to-A ratio suggested that at higher O₂ concentrations, the transformants had increased rates of photorespiration. Thus, the expression of active tobacco CA in the cytosol of F. bidentis bsc and mesophyll cells perturbed the C₄ CO₂-concentrating mechanism by increasing the permeability of the bsc to inorganic C and, thereby, decreasing the availability of CO₂ for photosynthetic assimilation by ribulose-1,5-bisphosphate carboxylase/oxygenase.     

Dark/Light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories

Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from lightexposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO₃⁻ and Mg²⁺ concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C₃); P. maximum (C₄ phosphoenolpyruvate carboxykinase); P. milioides (C₃/C₄); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C₃); P. miliaceum (C₄ NAD malic enzyme); Zea mays and Sorghum bicolor (C₄ NADP malic enzyme); Moricandia arvensis (C₃/C₄); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C₃ species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO₂ and Mg²⁺ activation, but which can be converted to an activatable state upon exposure of the leaf to light.     

Factors affecting interconversion between kinetic forms of ribulose diphosphate carboxylase-oxygenase from spinach

Ribulose diphosphate carboxylase was found to exist in two distinct kinetic forms in spinach leaf extracts. One form displayed an apparent K_m for CO₂ in excess of 200 μm and is likely to be the form purified and studied by many previous workers. However, if leaf extracts were prepared in the presence of Mg²⁺ and atmospheric levels of CO₂, the recently described high-affinity form was obtained. It had a K_m for CO₂ of about 20 μm, was quite stable even at 25 °C, and its properties were consistent with it being the form which operates in photosynthesis in vivo. Mg²⁺ was also able to convert the high-K_m (CO₂) form to the low-K_m (CO₂) form when it was added to an extract which had been prepared in its absence. Mg²⁺ was more effective in causing this conversion if bicarbonate was added as well. This activating effect of bicarbonate is a probable cause of previously reported apparent homotropic effects of bicarbonate on ribulose diphosphate carboxylase activity. It is possible that the apparently high-K_m (CO₂) form is not intrinsically active and appears to have activity only by virtue of the low-K_m (CO₂) form produced by contact with Mg²⁺ and bicarbonate (or CO₂) during the course of the assay. Extracts prepared with ribose 5-phosphate in the absence of Mg₂₊ also showed low-K_m (CO₂) carboxylase activity initially, but the presence of this sugar phosphate was deleterious during storage at 25 °C, where it promoted conversion to the apparently high-K_m (CO₂) form.Effects on the affinity of ribulose diphosphate carboxylase for CO₂ were paralleled by effects on the activity of the associated ribulose diphosphate oxygenase. Treatments which produced the low-K_m (CO₂) form of the carboxylase also resulted in high oxygenase activity, and it is possible that the apparently high-K_m (CO₂) form of the carboxylase has little, if any, oxygenase activity associated with it.The carboxylase and oxygenase activities of the low-K_m (CO₂) form showed broad and quite similar responses to pH variation, and the oxygenase had a K_m for O₂ of 0.22 mm.The stability of the low-K_m (CO₂) form in the presence of Mg²⁺ and bicarbonate was quite sufficient for it to be partially purified by Sepharose chromatography. The significance of the low-K_m (CO₂) form is discussed with respect to activation of photosynthesis by Mg²⁺.     

The Association of d-Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase with Phosphoribulokinase

When Ribulose- 1,5-bisphosphate carboxylase/oxygenase was purified from spinach leaves (Spinacia oleracea) using precipitation with polyethylene glycol and MgCl₂ followed by DEAE cellulose chromatography, 75% of phosphoribulokinase and 7% of phosphoriboisomerase activities copurified with ribulose- 1,5-bisphosphate carboxylase/oxygenase. This enzyme preparation showed ribose-5-phosphate and ribulose-5-phosphate dependent carboxylase and oxygenase activities which were nearly equivalent to its corresponding ribulose- 1,5-bisphosphate dependent activity. The ribose-5-phosphate and ribulose-5-phosphate dependent reaction rates were stable and linear for much longer time periods than the ribulose- 1,5-bisphosphate dependent rates. When sucrose gradients were used to purify ribulose- 1,5-bisphosphate carboxylase/oxygenase from crude stromal extracts, phosphoribulokinase was found to cosediment with ribulose- 1,5-bisphosphate carboxylase. Under these conditions most of the phosphoriboisomerase activity remained with the slower sedimenting proteins. Ammonium sulfate precipitation resulted in separation of the ribulose- 1,5-bisphosphate carboxylase peak from phosphoribulokinase peak. Crude extracts of peas Pisum sativum and spinach contained 0.725 to 0.730 milligram of phosphoribulokinase per milligram of chlorophyll, respectively, based on an enzyme-linked immunosorbent assay.     

Visualization of Cavitated Vessels in Winter and Refilled Vessels in Spring in Diffuse-Porous Trees by Cryo-Scanning Electron Microscopy

The regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity by 2-carboxyarabinitol 1-phosphate (CA1P) was investigated using gas-exchange analysis of antisense tobacco (Nicotiana tabacum) plants containing reduced levels of Rubisco activase. When an increase in light flux from darkness to 1200 μmol quanta m⁻² s⁻¹ was followed, the slow increase in CO₂ assimilation by antisense leaves contained two phases: one represented the activation of the noncarbamylated form of Rubisco, which was described previously, and the other represented the activation of the CA1P-inhibited form of Rubisco. We present evidence supporting this conclusion, including the observation that this second phase, like CA1P, is only present following darkness or very low light flux. In addition, the second phase of CO₂ assimilation was correlated with leaf CA1P content. When this novel phase was resolved from the CO₂ assimilation trace, most of it was found to have kinetics similar to the activation of the noncarbamylated form of Rubisco. Additionally, kinetics of the novel phase indicated that the activation of the CA1P-inhibited form of Rubisco proceeds faster than the degradation of CA1P by CA1P phosphatase. These results may be significant with respect to current models of the regulation of Rubisco activity by Rubisco activase.     

Inhibition of Pepper mild mottle virus with Commercial Cellulases

Pepper mild mottle virus (PMMoV) is one of the most important pathogens of pepper crops (Capsicum annuum L.) worldwide. We have found that commercial cellulases from Trichoderma reesei and Trichoderma viride strongly inhibit PMMoV infection of plants. When purified PMMoV was mixed with the cellulases, it greatly lost the capacity to induce local lesions on leaves of Nicotiana glutinosa. Pretreatment of sweet pepper leaves (C. annuum L.) with cellulase solutions before PMMoV inoculation greatly reduced the number of infected plants. These effects were superior to the effects of known viral inhibitors such as skim milk or Lentinula edodes culture filtrates. Although the mechanism of inhibition remains unknown, this is the first report of antiviral activity ascribed to commercially available cellulases.     

Ribulose bisphosphate carboxylase/oxygenase content determined with [C]carboxypentitol bisphosphate in plants and algae

As is the case with spinach ribulose bisphosphate carboxylase/oxygenase (Rubisco), [¹⁴C]carboxyarabinitol bisphosphate (CABP) bound to purified Chlorella Rubisco with a molar ratio of unity to large subunit of the enzyme. The concentration of binding sites in extracts of photosynthetic organisms was determined by reacting the extracts with [¹⁴C]-carboxypentitol bisphosphate (CPBP) and precipitating the resultant Rubisco-[¹⁴C]CABP complex with a combination of polyethylene glycol-4000 and MgCl₂. Plots of the relationship between concentrations of [¹⁴C] CPBP in the reaction mixture and the precipitated [¹⁴C]CPBP gave a straight line and the concentration of binding sites were estimated by extrapolation to zero [¹⁴C]CPBP since the dissociation constant of CABP with Rubisco is 10⁻¹¹ molar. Spinach, pea, and soybean leaves contained 6.4 to 6.8 milligrams Rubisco per milligram chlorophyll, corresponding to 92 to 97 ribulose bisphosphate-binding sites per milligram chlorophyll. The Rubisco content of sunflower and wheat leaves was 5.3 to 5.5 milligrams per milligram chlorophyll. The concentrations in C₄ plants were not uniform and corn and Panicum miliaceum leaves contained 3 and 7 milligrams Rubisco per milligram chlorophyll. The Rubisco content of green algae was one-fifth to one-sixth that of C₃ plant leaves and was affected by the CO₂ concentration during growth. The content of Euglena and blue-green algae is also reported.     

Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : I. Purification, Kinetics, and Physical Properties

NADH:nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max [L.] Merr., cv Williams) and nr₁-mutant soybean plants. Purification included Blue Sepharose- and hydroxylapatite-column chromatography using acetone powders from fully expanded unifoliolate leaves as the enzyme source.

Two forms of constitutive nitrate reductase were sequentially eluted with NADPH and NADH from Blue Sepharose loaded with extract from wild-type plants grown on urea as sole nitrogen source. The form eluted with NADPH was designated c₁NR, and the form eluted with NADH was designated c₂NR. Nitrate-grown nr₁ mutant soybean plants yielded a NADH:nitrate reductase (designated iNR) when Blue Sepharose columns were eluted with NADH; NADPH failed to elute any NR form from Blue Sepharose loaded with this extract. Both c₁NR and c₂NR had similar pH optima of 6.5, sedimentation behavior (s_20,w of 5.5-6.0), and electrophoretic mobility. However, c₁NR was more active with NADPH than with NADH, while c₂NR preferred NADH as electron donor. Apparent Michaelis constants for nitrate were 5 millimolar (c₁NR) and 0.19 millimolar (c₂NR). The iNR from the mutant had a pH optimum of 7.5, s_20,w of 7.6, and was less mobile on polyacrylamide gels than c₁NR and c₂NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.

Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants.