Pyrrolooxygenases from pepper and poinsettia leaves

Leaf extracts of pepper (Capsicum annuum) and poinsettia (Euphorbia pulcherrima) contained pyrrolooxygenases which varied in activity according to the age of the leaves and the origin and physiological condition of the plants. An inhibition of the pyrrolooxygenases was present in the crude extracts of senescent leaves. Fruiting enhanced pyrrolooxygenase activity and added a new ionic form of greater negative charge to the usual cationic form of the enzymes. Pyrrolooxygenases of C. annuum leaves from greenhouse-grown plants showed three forms of different ionic charge which exhibited multiple MW forms for porphobilinogen oxygenase and skatole pyrrolooxygenase. The cationic form of porphobilinogen oxygenase had sigmoidal kinetics, while the anionic forms had Michaelis kinetics. Skatole and tryptophan pyrrolooxygenase showed Michaelis kinetics. Pyrrolooxygenase activities in E. pulcherrima were lower than those in C. annuum and the former were also found to be more unstable.     

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.     

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.     

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.     

Separation of tryptophan pyrrolooxygenase into three molecular forms. A study of their substrate specificities using tryptophyl-containing peptides and proteins

Tryptophan pyrrolooxygenase from wheat germ was separated into three molecular forms by microgranular DEAE-cellulose using a stepwise or a linear gradient elution procedure. In the first case molecular forms A and B were eluted with 10 mM Tris/HCl buffer (pH 7.4) and molecular form C was eluted with 50 mM KCl in the same buffer. The same separation could also be achieved with a linear KCl gradient (0-100 mM) in 10 mM Tris/HCl buffer (pH 7.4). The three molecular forms of tryptophan pyrrolooxygenase oxidized L-, D-, DL-Trp as well as many Trp derivatives with formation of N-formylkynurenyl derivatives. They also efficiently oxidized Trp-Phe, Trp-Tyr, Trp-Ala, Ala-Trp, Trp-Gly, Gly-Trp, Trp-Leu, Leu-Trp, Pro-Trp and Val-Trp, although the dipeptides were oxidized at different rates by the three molecular forms. A number of tryptophyl-containing tetra-, penta-, octa-, nona- and decapeptides were also oxidized. The oligopeptides which were known to have a helical conformation were better substrates than the smaller oligopeptides which were devoid of the conformational factor. The three molecular forms of tryptophan pyrrolooxygenase oxidized the tryptophyl residues of lysozyme, pepsin, chymotrypsin, trypsin and bovine serum albumin. It was found that molecular form A oxidized the more exposed (or hydrophilic) Trp residues of the proteins, while molecular form C also oxidized the Trp residues of a more hydrophobic nature. The three molecular forms were inhibited by chelating agents (alpha, alpha'-dipyridyl, EDTA and omicron-phenanthroline), although they differed in their sensitivities to these agents. Their optimum temperatures and inactivation rates at 65 degrees C was also different.     

Onset of deoxyribonucleic Acid synthesis in germinating wheat embryos

Germinating wheat embryos (Triticum vulgare var. Florence) synthesize proteins before the onset of DNA synthesis. The onset of DNA replication occurs at about 15 hours of germination and was shown to depend on proteins synthesized before 9 hours of germination with the use of blasticidin S, a specific inhibitor of protein synthesis. A 10-fold increase in the activity of DNA-dependent DNA polymerase was found in extracts derived from germinated embryos, as compared to the activity found in extracts from ungerminated embryos.     

Indolic compounds in the leaves of Tecoma stans

Indole, tryptophan, tryptamine and skatole were isolated from the leaves of Tecoma stans. Anthranilic acid was also identified in its free form, in contrast to its glucoside, in Jasminum grandiflorum. The presence of both indole and anthranilic acid in the leaves of Tecoma stans indicates that they are the true substrate and product of indole oxygenase from the leaves of Tecoma stans.     

Catabolic Pathway for the Production of Skatole and Indoleacetic Acid by the Acetogen Clostridium drakei, Clostridium scatologenes, and Swine Manure

Skatole (3-methylindole) is a malodorous chemical in stored swine manure and is implicated as a component of foul-tasting pork. Definitive evidence for the skatole pathway is lacking. Deuterium-labeled substrates were employed to resolve this pathway in the acetogenic bacterium Clostridium drakei and Clostridium scatologenes and to determine if a similar pathway is used by microorganisms present in stored swine manure. Indoleacetic acid (IAA) was synthesized from tryptophan by both bacteria, and skatole was synthesized from both IAA and tryptophan. Microorganisms in swine manure produced skatole and other oxidation products from tryptophan, but IAA yielded only skatole. A catabolic mechanism for the synthesis of skatole is proposed.     

The dual oxygenase and peroxidase activities of porphobilinogen oxygenase and horseradish peroxidase: a study using the reaction with phenylhydrazine.

Porphobilinogen oxygenase and horseradish peroxidase show dual oxygenase and peroxidase activities. By treating porphobilinogen oxygenase with phenylhydrazine in the presence of H2O2 both activities were inhibited. When horseradish peroxidase was treated in the same manner only the peroxidase activity was lost while its oxygenase activity toward porphobilinogen remained unchanged. The phenylhydrazine treatment alkylated the prosthetic heme group of porphobilinogen oxygenase and N-phenylheme as well as N-phenylprotoporphyrin IX were isolated from the treated hemoprotein. In horseradish peroxidase the modified heme was mainly 8-hydroxymethylheme. The apoproteins of the alkylated enzymes were isolated and recombined with hemin IX. The oxygenase and peroxidase activities of porphobilinogen oxygenase were entirely recovered in the reconstituted enzyme, while the reconstituted horseradish peroxidase regained 75% of its peroxidase activity.     

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.     

Effects of Allopurinol [4-Hydroxypyrazolo(3,4-d)Pyrimidine] on the Metabolism of Allantoin in Soybean Plants

Some studies on the effects of xanthine oxidase inhibitor allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] on allantoin metabolism of soybean plants (Glycine max cv. Tamanishiki) are reported. Soybean seedlings, aseptically germinated for 96 hours on agar containing 1 millimolar allopurinol, contained only slight amounts of allantoin, allantoic acid, and urea as compared with controls. Analysis of purines and pyrimidines of the allopurinol-treated seedlings showed marked accumulation of xanthine both in the cotyledons and seedling axes. No hypoxanthine accumulation was found. Xanthine accumulation due to allopurinol treatment was relatively low after the cotyledons had fallen. For nodulated plants, allopurinol caused a significant drop in allantoin (+allantoic acid) in the stems and nodules, accompanied by a striking accumulation of xanthine in the nodules. The xanthine concentration in the nodules far exceeded that in the germinated seedlings. Allopurinol at a concentration of 50 micromolar strongly inhibited xanthine oxidase prepared from soybean nodules.

The results suggested that the main pathway of allantoin formation in soybean plants was through purine decomposition, via xanthine-uric acid. It was specially noted that a very active purine-decomposing system existed in soybean nodules.

     

Porphobilinogen oxygenase. Purification and evidence of its hemoprotein structure

Porphobilinogen oxygenase oxidizes porphobilinogen to 2-hydroxy-5-oxo-porphobilinogen. This enzyme isolated from wheat germ has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis under both nondenaturing and denaturing conditions. The molecular weight of the enzyme formed from two identical (or very similar) polypeptide chains is 70,000. It has a pI of 9.0 indicating its cationic nature. The pure enzyme contains 1 mol of high-spin heme and 2 mol of non-heme iron. It requires both of these as well as molecular O2 and a reducing agent for catalytic activity. Although the enzyme has many characteristics of a peroxidase, hydrogen peroxide cannot substitute for oxygen and dithionite for catalysis. The catalytic reaction is not affected by catalase, superoxide dismutase, or by hydroxyl radical scavengers. A comparison between porphobilinogen oxygenase and a commercial preparation of horseradish peroxidase was made. The latter also catalyzes aerobic porphobilinogen oxidation, with dithionite as electron donor. Here the oxidation of porphobilinogen is inhibited by superoxide dismutase and was not affected by catalase.     

Subunit Structure Differences in RNA Polymerase II Purified from Ungerminated versus Germinated Wheat Embryos

DNA-dependent RNA polymerase II (RNAP II) was purified from wheat embryos germinated for 0, 12, 24, and 36 hours and examined with several polyacrylamide gel electrophoretic systems. A changing electrophoretic pattern of RNAP II was observed on nondenaturing polyacrylamide gels. Subunit structure analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that from ungerminated embryos, RNAP IIA was almost exclusively obtained which has a subunit structure identical to that established for wheat germ RNAP II previously (Jendrisak, Burgess 1977 Biochemistry 16: 1959-1964). Twelve polypeptides with molecular weights × 10⁻³ of 220, 140, 42, 40, 27, 25, 21, 20, 17.8, 17.0, 16.3, and 16.0 were routinely found to be associated with the purified enzyme. From embryos germinated for 36 hours, RNAP IIB was almost exclusively obtained which has a largest subunit of 180,000 mol wt instead of 220,000. From embryos germinated for 24 hours, an approximately equimolar mixture of RNAP IIA and IIB was obtained. Peptide maps of the 220,000 and 180,000 mol wt polypeptides of RNAP IIA and IIB were virtually identical, indicative of a precursor-product relationship for the two polypeptides. In addition to these results, SDS-PAGE indicated that the stoichiometry of the 27,000 mol wt polypeptide increased at the expense of the 25,000 mol wt polypeptide during germination and concomitantly with the appearance of the 180,000 molecular weight polypeptide. No modifications (e.g. gain, loss, or altered mobilities on analytical gels) in any of the other RNAP II subunits were observed in enzyme purified from embryos after various times of germination as determined by a variety of electrophoretic analyses under denaturing conditions.     

Relationship between seed quality and changes in conjugated polyamine in plasma membrane purified from wheat embryos during grain ripening

In order to explore the relationship between seed quality and changes in conjugated polyamine in plasma membrane purified from wheat embryos during grain ripening. Plasma membrane (PM) vesicles were isolated from the embryos of ripening wheat (Triticum aestivum L.) grains by the gradient centrifugation method. The contents of polyamines conjugated (covalently and non-covalently) to the PM vesicles were investigated. Results showed that after pollination, from the 22nd to the 32nd day, the embryos of wheat grains underwent dehydration, as judged by the decrease of embryo relative water content (ERWC). During embryo ripening, non-covalently conjugated (NCC)-Spd and NCC-Spm, covalently conjugated (CC)-Put and CC-Spd contents increased markedly, while relative embryo cell vigor (RECV) decreased slightly. The treatment with methylglyoxyl-bis (guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), inhibited the increases of the NCC-Spd and NCC-Spm contents, enhanced the decrease of RECV simultaneously, and decreased mature seed relative germination potential (RGP). The effects of MGBG were reversed by exogenous Spd and Spm. Phenanthrolin (o–Phen), an inhibitor of transglutaminase (TGase), inhibited the increases of CC-Put and CC-Spd contents, enhanced the decrease of RECV simultaneously, and decreased mature seed RGP. These results suggest that during embryo ripening, the levels of NCC-Spd, NCC-Spm, CC-Put, and CC-Spd increase, and these increases might affect embryo cell vigor and seed germination potential.     

Study of the role of tryptophan residues in streptokinase molecules using a chemical modification method

Incubation of streptokinase in an H2O2-dioxane-bicarbonate buffer (pH 8.5) system leads to the oxidation of tryptophan residues as can be evidenced from the changes in absorption and tryptophan fluorescence spectra. A complete oxidation of tryptophan residues of the protein takes place within 3 hours, the number of the residues is 4. The first tryptophanyl of the protein is oxidized the most easily; the activity of streptokinase decreases thereby by 50%. Modification of the second residue leads to complete inactivation of streptokinase. The rate constants for the oxidation of the first, of the two first and of the third plus fourth tryptophanyls are equal to 1.5.10(-2) min-1, 1,1.10(-2) min-1 and 0.5.10(-2) min -1, respectively. The complete oxidation of tryptophan residues is concomitant with the inability of streptokinase to form stable equimolar complexes with human plasminogen, but in does not result (as can be judged from the CD spectroscopy data) in the breakdown of the protein secondary structure. The specificity of oxidation of the protein tryptophan residues is discussed. The importance of readily oxidized tryptophan residues for the streptokinase function is postulated.     

De novo synthesis of 3'-nucleotidase in germinating wheat embryo

The enzyme 3′-AMP nucleotidase was purified 2,500- to 5,000-fold from extracts of an acetone powder of wheat (Triticum aestivum) embryonic axes germinated for 40 hours. Sodium dodecyl sulfate acrylamide gel electrophoresis and chromatography on Biogel-P100 indicate that the enzyme is monomeric with a molecular weight of 39,000. Extracts of embryos germinated up to 6 hours have only 1% of the 40-hour level of enzyme activity. To see if the increase to 40 hours represents de novo synthesis, extracts were compared for their ability to react with a rabbit antibody prepared against the enzyme. In immunodiffusion tests, 40-hour extracts showed a strong precipitin line coincident with that of the purified enzyme, whereas no precipitation was observed with 1-hour extracts. When the enzyme present in 40-hour extracts was partially inactivated by EDTA, it still blocked the ability of the antibody to inhibit enzyme activity. Extracts of 1-hour embryos, in contrast, were not able to block the inhibitory activity of the antibody. Embryos allowed to take up ³⁵SO₄ between 40 and 46 hours of germination synthesized ³⁵S-labeled 3′-nucleotidase. In contrast, no radioactive protein synthesized by embryos during the first 6 hours of germination coincided on gel electrophoresis with the enzyme. These results indicate that the increase in 3′-nucleotidase activity is a consequence of de novo synthesis of the enzyme.     

Tryptophanyl adenylate formation by tryptophanyl-tRNA synthetase from Escherichia coli

By gel filtration and titration on DEAE-cellulose filters we show that Escherichia coli tryptophanyl-tRNA synthetase forms tryptophanyl adenylate as an initial reaction product when the enzyme is mixed with ATP-Mg and tryptophan. This reaction precedes the synthesis of the tryptophanyl-ATP ester known to be formed by this enzyme. The stoichiometry of tryptophanyl adenylate synthesis is 2 mol per mole of dimeric enzyme. When this reaction is studied either by the stopped-flow method, by the fluorescence changes of the enzyme, or by radioactive ATP depletion, three successive chemical processes are identified. The first two processes correspond to the synthesis of the two adenylates, at very different rates. The rate constants of tryptophanyl adenylate synthesis are respectively 146 +/- 17 s-1 and 3.3 +/- 0.9 s-1. The third process is the synthesis of tryptophanyl-ATP, the rate constant of which is 0.025 s-1. The Michaelis constants for ATP and for tryptophan in the activation reaction are respectively 179 +/- 35 microM and 23.9 +/- 7.9 microM, for the fast site, and 116 +/- 45 microM and 3.7 +/- 2.2 microM, for the slow site. No synergy between ATP and tryptophan can be evidenced. The data are interpreted as showing positive cooperativity between the subunits associated with conformational changes evidenced by fluorometric methods. The pyrophosphorolysis of tryptophanyl adenylate presents a Michaelian behavior for both sites, and the rate constant of the reverse reaction is 360 +/- 10 s-1 with a binding constant of 196 +/- 12 microM for inorganic pyrophosphate (PPi).(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphology and anatomy of mature embryos and seedlings in parasitic angiospermCuscuta japonica

Morphological and anatomical features of mature embryos and seedlings were observed at different growth stages in the parasitic angiospermCuscuta japonica Choisy. The spirally coiled embryos from scarified seeds had no cotyledons but possessed blunt radicles. Seeds germinated at 30°C in the dark. Although most embryo cells incubated for 16 h did not have starch grains, the shoot cells of three-day-old seedlings possessed numerous starch grains. After these seedlings were transferred to a lightened growth chamber, all the shoot apical regions of seedlings grown for 6,8, and 10 days became greenish and hooked. Most of the shoot cells, including the green apical parts, contained abundant starch grains. The hooks opened only when one seedling made contact with another seedling. This suggested that the green and hooked shoot apical regions played an important role in searching for and twining about their host plants. In some two-day-old seedlings, the massive roots were circular or semi-circular. This enabled the shoot axes to stand erect on some substratum. It would assist the shoots in making contact with the host plant. In eight-day-old seedlings, the green apical regions also were hooked and the roots were considerably degraded.