Isolation and characterization of two isoperoxidases from tobacco tissue cultures

Two isoperoxidases (A_f and C_n) from the medium of tobacco tissue suspension culture WR-132 grown in darkness have been purified to apparent homogeneity and partially characterized. C_n and A_f have MWs of ca 30 000 and 54 000, respectively. A_f has ca 5.1% carbohydrate, but none could be detected in C_n. Both isoperoxidases appear to follow simple Michaelis-Menten kinetics with respect to guaiacol as the substrate. The K_ms for guaiacol are 4 and 13.3 mM for Af and C_n, respectively, while both isoperoxidases have a pH optimum at 6.5. C_n, is dissimilar to other isoperoxidases from tobacco tissue cultures, but A_f is very similar to isoperoxidase A₃ from W-38 tobacco tissue culture.     

Isoperoxidases from tobacco tissue cultures

Two anodic isoperoxidases (A₁ and A₂) from tobacco tissue culture W-38 and two cathodic isoperoxidases (C₃ and C₄) from tobacco suspension culture WR-132 have been separated and characterized. Molecular weights for each of the isoperoxidases have been determined by two different methods. Only C₄ contained a carbohydrate component. The substrate specificity and the pH optima for the four enzymes with each of five substrates were determined.     

Comparison of tryptic peptide maps of eight isoperoxidases from tobacco tissue cultures

Eight isoperoxidases from tobacco suspension culture WR-132 (termed C_n, C₃, C₄, A_c, and A_f) and tobacco callus culture W-38 (termed A₁, A₂, and A₃) have been subjected to trypsin digestion followed by peptide mapping. The peptide maps of isoperoxidases A_f and A₃ are identical. All other isoperoxidases do not appear to be dramatically dissimilar in certain portions of their sequence, since many matching peptides have been found when various isoperoxidases are cross-compared. However, only two, and possibly three, highly homologous peptides are present in all of the isoperoxidases.     

Effect of darkness on isoperoxidases in tobacco tissue cultures

In order to study the effect of light on the tobacco tissue culture WR-132, 5 passages (10 days' growth per passage) of these cells were grown in darkness, and 3 passages were separately grown in intense light (16000 lx). All other growth conditions were the same. The resulting isoperoxidase patterns present in these cells and in their growth media were analyzed at 2-day intervals during this period and then compared with the isoperoxidase patterns of cells grown under dim light conditions (10 lx). A new cathodic isoperoxidase (C_n) appeared in the medium within 2 days after the cells were placed in the dark. C_n was present in all media of WR-132 cell cultures analyzed throughout the 5 passages grown in darkness. The fifth passage in darkness produced total cessation of growth (apparent death). C_n increased and new anodic isoperoxidases A_a, A_b, A_d and A_e appeared in the media as the cells approached death in darkness.     

Scopoletin: A substrate for an isoperoxidase from Nicotiana tabacum tissue culture W-38

Scopoletin was found to be a substrate for a single anodic isoperoxidase isolated from tobacco callus tissue W-38. Isolation of this peroxidase was accomplished using DEAE-cellulose chromatography. This isoperoxidase catalysed the destruction of scopoletin in the presence of H₂O₂ only. An enzyme assay for the scopoletin reaction was developed. The pH optimum of the enzyme was 5·5 and the apparent K_ms for scopoletin and H₂O₂ were 0·6 and 0·9 rnM respectively.     

Peroxidases of Solanum melongena leaves

Three major peroxidases, designated as A, B₂ and B₂ from Solanum melongena leaves have been reported. Peroxidases-A, -B₂ and -B₂ were considered to be true peroxidases on the basis of k₁:k₄ ratio. The pH optima for the three enzymes were found to be 7·0, 6·0 and 6.0 respectively. These peroxidases differ in their k₁:k₄ ratio, in the effect of pH on this ratio and in the uric acid/guaiacol and o-dianisidine/guaiacol activity ratio.     

Occurrence of some enzymes in starchy endosperm and hormonal regulation of isoperoxidase in aleurone of wheat

Peroxidase, indoleacetic acid-oxidase, alkaline, and acid phosphatases were detected in dry starchy endosperm (minus aleurone) of wheat grain. The isoperoxidase pattern differed in different parts of the dry grain. Several new isoperoxidases were found in embryos after soaking. The intensity of isoperoxidases in aleurones was enhanced in the presence of embryo or 2 μM GA₃ after 24 hours of soaking, but decreased after 72 hours. Indoleacetic acid and kinetin had no effect on isoperoxidase of aleurone. Actinomycin D and cycloheximide had no effect on isoperoxidases of aleurones from embryonectomized or naturally occurring embryoless grains. However, these two inhibitors increased the intensity of isoperoxidases in aleurones of intact embryonated grains after soaking.     

Peroxidase activity and isoenzyme patterns in wheat during ontogenesis

Protein content, total and specific peroxidase activity and isoperoxidase patterns were determined in crude protein preparations from individual parts of field-grown wheat (Triticum aestivum L., cv. Jubilar). Protein content in roots, leaves, and stalks increased at the beginning of ontogenesis and then decreased from 6th, 9th, and 10th development phase (according to Feekes), respectively. Steady increase of the protein content in the ears was observed. Highest peroxidase activity was found in the roots; it diminished from the onset of ontogenesis till maturity of the plants. In the leaves and stalks a slight decrease of peroxidase activity till the 10th development phase and then an increase till maturity was found. The ears exhibited a gradual increase of peroxidase activity. The course of specific peroxidase activity was found to be very similar to that of total activity. Isoperoxidase patterns did not change significantly. In the leaves, a decrease of activity of C₄ and C₅ isoperoxidases was recorded. In the stalks, C l isoenzyme emerged at the end of ontogenesis. A gradual increase of A₁ and A₅ isoperoxidase intensity took place both in the leaves and stalks.     

Properties of peroxidases from tobacco cell suspension culture

An enzyme preparation from suspension cultured tobacco cells oxidized IAA only in the presence of added cofactors, Mn²⁺ and 2,4-dichlorophenol, and showed two pH optima for the oxidation at pH 4·5 and 5·5. Effects of various phenolic compounds and metal ions on IAA oxidase activity were examined. The properties of seven peroxidase fractions separated by column chromatography on DEAE-cellulose and CM-Sephadex, were compared. The peroxidases were different in relative activity toward o-dianisidine and guaiacol. All the peroxidases catalysed IAA oxidation in the presence of added cofactors. The pH optima for guaiacol peroxidation were very similar among the seven isozymes, but the optima for IAA oxidation were different. The anionic and neutral fractions showed pH optima near pH 5·5, but the cationic isozymes showed optima near pH 4·5. With guaiacol as hydrogen donor, an anionic peroxidase (A-1) and a cationic peroxidase (C-4) were very different in H₂O₂ concentration requirements for their activity. Peroxidase A-1 was active at a wide range of H₂O₂ concentrations, while peroxidase C-4 showed a more restricted H₂O₂ requirement. Gel filtration and polyacrylamide gel studies indicated that the three cationic peroxidases have the same molecular weight.     

Preliminary crystallographic data for Azotobacter cytochrome c5.

Two closely related crystal forms of dimeric cytochrome c₅ from Azotobacter rinelandii have been grown. The crystals belong to space groups (C2 with $\text{a = 45·0, b = 38·4, c = 41·3}\text{A}\text{?}\text{and}\text{β = 101 ° 0′}$; and C1 (a centered triclinic cell) with $\text{a = 46·0, b = 37·6, c = 49·4}\text{A}\text{?}$, α = 87 ° 20′, β = 96 ° 40′ and γ = 90 ° 0′. In C2 the 24,000 molecular weight dimer lies on a Crystallographic 2-fold axis; in C1 the entire dimer occupies the asymmetric unit.     

Evidence for long-range interactions in DNA. Analysis of melting curves of block polymers d(C15A15)·d(T15G15), d(C20A15)·d(T15G20), and d(C20A10)·d(T10G20)

The influence of one DNA region on the stability of an adjoining region (telestability) was examined. Melting curves of three block DNA's, d(C₁₅A₁₅)·d(T₁₅G₁₅), d(C₂₀A₁₅)·d(T₁₅G₂₀), and d(C₂₀A₁₀)·d(T₁₀G₂₀) were analyzed in terms of the nearest neighbor Ising model. Comparisons of predicted and experimental curves were made in 0.01 M and 0.1 M sodium ion solutions. The nearest neighbor formalism was also employed to analyze block DNA transition in the presence of actinomycin, a G·C specific molecule. The results show that nearest neighbor base-pair interaction cannot predict the melting curves of the block DNA's. Adjustments in theoretical parameters to account for phosphate repulsion assuming a B conformation throughout the DNA's do not alter this conclusion. Changes in the theoretical parameters, which provide good overall agreement, are consistent with a substantial stabilization of the A·T region nearest the G·C block. The melting temperature T A·T for the average A·T pari in d(C₂₀A₁₀)·d(T₁₀G₂₀), with 10 A·T pairs, appears to be 4°C greater than T_A·T for d(C₁₅A₁₅)·d(T₁₅G₁₅) and d(C₂₀A₁₅)·d(T₁₅G₂₀), both with 15 A·T pairs. Actinomycin bound to the G·C end effectively stabilizes the A·T end by 9°C. These results indicate a long-range contribution to the interactions governing DNA stability. A possible mechanism for these interactions will be discussed.     

Simultaneous separation of acidic and basic isoperoxidases in wounded potato tissue by acrylamide gel electrophoresis

Preparation and use of a newly developed pH 4.3 horizontal thin layer acrylamide gel which permits the simultaneous separation of acidic and basic isoperoxidases in up to 30 samples is described. Use of cytochrome c, horseradish peroxidase, and a purified potato isoperoxidase as internal standards for a range in isoelectric points of peroxidases from pH 3 to 11 is introduced to facilitate comparison of results obtained with different materials and different methods. Distribution of tissue-specific isoperoxidases in different cell layers of wounded potato (Solanum tuberosum L.) tissue is shown and their purification described. Evidence for the in vitro degradation of basic potato isoperoxidases resulting in more acidic forms similar to isoperoxidases occurring in wounded potato tissue is presented. The significance of this observation for the postulated differential function of different isoperoxidases is discussed.     

Purification and Characterization of Isoperoxidases Elicited by Aspergillus flavus in Cotton Ovule Cultures

Two anionic isoperoxidases were isolated from media of Aspergillus flavus-inoculated cotton (Gossypium hirsutum L.) ovule cultures and purified about 150-fold to apparent homogeneity by treatment with Cell Debris Remover and ion exchange chromatography on Accell QMA medium. These isoperoxidases were present in noninoculated cotton ovule cultures at low levels. The major activity peak (B) represented 90% of the recovered peroxidase activity and was electrophoretically homogeneous. The minor activity peak (A) was about 95% pure. Isoelectric focusing analysis showed that B was greater than 95% pure with respect to other peroxidase isozymes, while the enzyme in A was about 90% isozymically pure. Each isoperoxidase displayed a molecular mass of 56 kilodaltons by interpolation from denaturing gel electrophoresis. The B isozyme displayed a molecular mass of 55 kilodaltons by gel filtration chromatography. The pH optima for the cotton ovule isoperoxidases were similar, 5.0 for isozyme A and 6.0 for isozyme B. The isoelectric points for isozymes A and B were 4.2 and 4.4, respectively. Eugenol, guaiacol, and 3,3′,5,5′-tetramethylbenzidine were good electron donor substrates, whereas 4-aminoantipyrine was a poor substrate. The absorption spectrum of the material in B revealed a major peak at 400 nanometers and a minor peak at 280 nanometers. The molar extinction coefficient at 400 nanometers (pH 7.0) was calculated to be 1.07 × 10⁵ per square centimeter per mole. Amino acid analysis of isozyme B confirmed the acidic nature of this protein and identified a number of similarities to the anionic peroxidases from tobacco and potato. This glycoprotein was found to contain 12 to 14% sugar (by weight), mainly in the form of galactose and mannose.     

Purification and characterization of an anionic isoperoxidase from scented-geranium callus

Secretory anionic isoperoxidase (EC 1.11.1.7), named PA1, was 68-fold purified from scented-geranium (Pelargonium graveolense) callus by using ion exchange chromatography and gel filtration. Isoperoxidase PA1 was a glycoprotein with an isoelectric point (pI) of 4.0. The molecular weight of PA1 was approximately 42.5 and 44 kDa, estimated by SDS-PAGE and Sephadex G-150 gel filtration, respectively. The optimum pH of the enzyme was 5.0 for guaiacol and H2O2, and the Km values for guaiacol and H2O2 were 1.96 and 8.5mM, respectively. Substrate studies in terms of optimum pHs and Km values with various synthetic and naturally occurring phenolic compounds were performed. In comparison with cationic isoperoxidase, PC3, which has been already characterized, anionic isoperoxidase PA1 had much lower Km values for synthetic phenolic compounds and much higher Km values for naturally occurring phenolic compounds than PC3. Moreover, anionic isoperoxidase PA1 could utilize ferulic acid as a substrate very well, while cationic isoperoxidase PC3 could not utilize ferulic acid as a substrate.     

Specific peroxidase isoenzymes are correlated with organogenesis

We have examined isoperoxidase patterns obtained from buffer-, salt-, and enzyme-extractable fractions and correlated them with histological changes in tobacco (Nicotiana tabacum L., cv Wisc. 38) `epidermal' explants induced to produce either callus, vegetative buds, or floral buds. By utilizing a combination of extraction and electrophoretic procedures different from any hitherto used for this kind of investigation, we were able to resolve 47 isoperoxidases distributed between the three types of fractions. The majority of these isoperoxidases were common to all explants regardless of their developmental fate. Correspondingly, a number of histological changes were observed in all explants (e.g. the initiation of cell division by day 2, lignin deposition by day 4, and the formation of clustered tracheary elements by day 8). We have made correlations between 25 isoperoxidases and specific developmental events based on the time when certain isoperoxidases were detected relative to observed histological changes: 3 were correlated with desuppressed/sustained cell division, 3 to 6 with lignification/tracheary element maturation, 7 with callus formation, 1 with localized suppression of growth, 3 with determinate axial organization, 4 with leaf development, and 1 with stamen development. These results suggest that a continued investigation using this system could lead to a better understanding of the role of specific isoperoxidases in different developmental processes.     

Conformational Preferences of Modified Nucleoside N(4)-Acetylcytidine, ac4C Occur at “Wobble” 34th Position in the Anticodon Loop of tRNA

Conformational preferences of modified nucleoside, N(4)-acetylcytidine, ac⁴C have been investigated using quantum chemical semi-empirical RM1 method. Automated geometry optimization using PM3 method along with ab initio methods HF SCF (6-31G**), and density functional theory (DFT; B3LYP/6-31G**) have also been made to compare the salient features. The most stable conformation of N(4)-acetyl group of ac⁴C prefers “proximal” orientation. This conformation is stabilized by intramolecular hydrogen bonding between O(7)···HC(5), O(2)···HC2′, and O4′···HC(6). The “proximal” conformation of N(4)-acetyl group has also been observed in another conformational study of anticodon loop of E. coli elongator tRNA^Met. The solvent accessible surface area (SASA) calculations revealed the role of ac⁴C in anticodon loop. The explicit molecular dynamics simulation study also shows the “proximal” orientation of N(4)-acetyl group. The predicted “proximal” conformation would allow ac⁴C to interact with third base of codon AUG/AUA whereas the ‘distal’ orientation of N(4)-acetyl cytidine side-chain prevents such interactions. Single point energy calculation studies of various models of anticodon–codon bases revealed that the models ac⁴C₍₃₄₎(Proximal):G₃, and ac⁴C₍₃₄₎(Proximal):A₃ are energetically more stable as compared to models ac⁴C₍₃₄₎(Distal):G₃, and ac⁴C₍₃₄₎(Distal):A₃, respectively. MEPs calculations showed the unique potential tunnels between the hydrogen bond donor–acceptor atoms of ac⁴C₍₃₄₎(Proximal):G₃/A₃ base pairs suggesting role of ac⁴C in recognition of third letter of codons AUG/AUA. The “distal” conformation of ac⁴C might prevent misreading of AUA codon. Hence, this study could be useful to understand the role of ac⁴C in the tertiary structure folding of tRNA as well as in the proper recognition of codons during protein biosynthesis process.     

Double-helical structures for polyxanthylic acid

Polyxanthylic acid has been found to exist in two different duplex forms, A_I and A_II. a_I, formed at pH5·7, occurs in a compact lattice with nearest neighbor molecules spaced at 2.11 nm. It has an axial translation per residue, h = 0·301 nm, and a rotation per residue, t = 36·0 °. The intensity distribution in its X-ray fiber diffraction pattern is analogous to that of A-RNA (h = 0·281 nm, t = 32·7 °). On the other hand A_II, formed at pH 8·0, has a less compact, statistically disordered crystal packing with nearest neighbors 2·35 nm apart. It has h = 0·252 nm and t = 32·7 ° and gives an X-ray intensity distribution essentially identical to A-DNA (h = 0·256 nm, t = 32·7 °). Similar right-handed helical duplex models, with flexible C-3′-endo sugar rings, have been developed for each molecular structure. Both have purine purine base-pairs, possibly triply hydrogen-bonded, and certainly with the same symmetry as Watson-Crick pairs but with a 0·2 nm greater C-1′ … C-1′ separation.     

Isoperoxidase pattern and internode length genotype in Pisum

Inbred Pisum sativum lines of known constitution for the intemode length genes Le, La and Cry, and representing four height phenotypes, were grown to the 7-intemode stage in the light. Six cationic isoperoxidases, making up ca. 90% of the activity of stem extracts, were resolved by concave gradient elution from Dowex 50 columns and shown to run as single peroxidase bands on starch gel electrophoresis. They were all able to oxidise IAA in the presence of 2,4-dichlorophenol, but fell into two groups with widely differing IAA oxidase/peroxidase ratios. The isoperoxidase patterns were independent of both genotype and phenotype for internode length, thus making it unlikely that these loci exert their effect on internode extension via control of synthesis of a particular isoperoxidase. Amongst the lines screened polymorphism was detected involving two of the isoperoxidases, and limited F₂ data suggest that these two variants fire determined by alleles of a single gene. Isoperoxidase patterns of stem extracts of 6 other Pisum species did not differ significantly from the two found in P. sativum.     

The electronic structure of the C2H4O···2HF tri-molecular heterocyclic hydrogen-bonded complex: a theoretical study

The geometries of three isomers of the C₂H₄O···2HF tri-molecular heterocyclic hydrogen-bonded complex were examined through B3LYP/aug-cc-pVDZ calculations. Analysis of structural parameters, determination of CHELPG (charge electrostatic potential grid) intermolecular charge transfer, interpretation of infrared stretching modes, and Bader’s atoms in molecules (AIM) theory calculations was carried out in order to characterize the hydrogen bonds in each isomer of the C₂H₄O···2HF complex. The most stable structure was determined through the identification of hydrogen bonds between C₂H₄O and HF, (O···H), as well as in the hydrofluoric acid dimer, (HF^D–R···HF^D). However, the existence of a tertiary interaction (F^λ···H^α) between the fluoride of the second hydrofluoric acid and the axial hydrogen atoms of C₂H₄O was decisive in the identification of the preferred configuration of the C₂H₄O···2HF system. Figure Geometries of three isomers of the C₂H₄O···2HF tri-molecular heterocyclic hydrogen-bonded complex