The screening of the enzyme and isoenzyme patterns in seeds ofAllium cepa L.

The screening of enzyme patterns in seeds ofAllium cepa cv. Všetatská revealed the presence of the following enzymes: alcohol dehydrogenase, lactate dehyd ogenase, NAD+- and NADP+-glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase NAD+- and NADP+-malate dehydrogenase, NADH₂- and NADPH₂-tetrazolium reductase catalase, Superoxide dismutase, acid and alkaline phosphatase, L-leucine aminopeptidase, glutamate dehydrogenase, non-specific esterase, and cholinesterase. Altogether 17 enzymes were detected in onion seeds, nine of which had more than three isoenzymes, NAD+-malate dehydrogenase had 8, and non-specific esterase 9 isoenzymes. The demonstration of cholinesterase and Superoxide dismutase activities is remarkable.     

Immunochemical quantitation of isoenzymes. Alpha-amylase isoenzymes in barley malt

Two α-amylase isoenzymes were studied in germinating seeds from three varieties of barley by means of crossed immunoelectrophoresis, and amounts of corresponding isoenzymes could be compared. The method permits distinction between activator control and change in enzymatic protein quantity, de novo synthesis, and degradation. During germination of seeds variety Carlsberg II, two α-amylase isoenzymes were found to increase in amount to reach a maximum on Day 7 and then decrease.     

Biomass Enhancement in Maize and Soybean in Response to Glutamate Dehydrogenase Isomerization

The relationship between nutrient composition, crop biomass, and glutamate dehydrogenase (GDH) isoenzyme pattern was investigated in soybean (Glycine max) and maize (Zea mays) by monitoring the nutrient induced isomerization of the enzyme from the seedling stage to the mature crop. GDH was extracted from the leaves of the plants, and the isoenzymes were fractionated by isoelectric focusing followed by native polyacrylamide gel electrophoresis. The isomerization V_max values for soybean GDH, similar to maize GDH increased curvilinearly from 200 – 400 μmol mg⁻¹ min⁻¹ as the inorganic phosphate nutrient applied to the soil decreased from 50 − 0 mM. In soybean, combinations of N and K, P, or S nutrients induced the acidic and neutral isoenzymes, and gave biomass increases 25 – 50 % higher than the control plant. GDH isoenzymes were suppressed in soybean that received nutrients without N, K, or P and accordingly the biomass was about 30 % lower than the control. Treatment of maize with NPK nutrients increased the GDH V_max values from 138.9 at the vegetative to 256.4 μmol mg⁻¹ min⁻¹ at the reproductive phase, and suppressed the basic isoenzymes, but induced both the acidic and neutral isoenzymes thereby inducing seed production (27.0 ± 1.4 g per plant); whereas both the acidic and basic isoenzymes were suppressed in the control maize, and seeds did not develop. Simultaneous induction of the acidic, neutral, and basic isoenzymes of GDH indicated the occurrence of senescence. Therefore in maize and soybean, the induction of the acidic and basic isoenzymes of GDH led to the enhancement of biomass. This revised version was published online in July 2006 with corrections to the Cover Date.     

马肝醇脱氢酶基因的克隆及其在大肠杆菌中的表达

利用RT-PCR技术从马肝扩增HLADH-E和HLADH-S基因,通过基因工程方法构建表达质粒pLY115E和pLY115S,在大肠杆菌中表达,并利用Ni柱分离纯化。利用紫外检测辅酶NADH在340nm的吸光值,来考察表达产物转化环己醇的活性。试验结果证明马肝醇脱氢酶HLADH-E和HLADH-S基因均能在大肠杆菌中表达,并且可溶性表达产物都具有氧化环己醇的活性,为马肝醇脱氢酶的进一步研究开发奠定了基础。     

The lipoxygenases in developing soybean seeds, their characterization and synthesis in vitro

A number of lipoxygenase isoenzymes were identified in developing soybean (Glycine max L. Merrill cv Provar) seeds and two have been partially characterized. In a study of lipoxygenase level in developing soybean seeds, the enzyme content increased markedly during development. Comparisons of the lipoxygenases from mature soybean seeds and immature seeds by isoelectric focusing, chromatofocusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping identified two categories of isoenzyme. The isoenzymes from immature seeds were found by electron paramagnetic resonance spectroscopy to be isolated at least in part as the high spin iron(III) or active form of the enzyme in contrast to lipoxygenases from mature seeds which were isolated as electron paramagnetic resonance silent, high spin iron(II) species. The discovery of increased levels of lipoxygenases during seed development and their isolation in an active form suggests that the enzyme may play a physiological role during the maturation process. The incorporation of iron-59 from the nutrient medium into lipoxygenase during culture of immature seeds was indicative of de novo synthesis of the enzyme. The efficiency of the iron uptake was high, as indicated by the level of radioactivity found in the enzyme (one gram atom of iron per mole of lipoxygenase).     

Separation and characterization of the isoenzymes of wall-bound peroxidase from cultured Zinnia cells during tracheary element differentiation

Cell wall-bound peroxidase (EC 1.11.1.7) isoenzymes (P1-P5) from cells of Zinnia elegans L. that were differentiating into tracheary elements were separated and characterized to obtain information about the relationships between these isoenzymes and the biosynthesis of lignin. Fractionation of Zinnia cells by centrifugation in solutions of Percoll revealed that P1, P2, and P5 were present in differentiated tracheary elements. These peroxidase isoenzymes were separated by several column-chromatographic steps. During hydrophobic chromatography on Phenyl Superose, P5 activity was separated into activities P5A and P5B. Enzymatically pure preparations of P1, P3, P5A, and P5B were finally obtained and used for the characterization of each isoenzyme. The optimum pH was 5.5–6.0 for P1, 5.0–7.5 for P3, 5.0 for P5A, and 4.0 for P5B. Each of the isoenzymes oxidized coniferyl alcohol efficiently, whereas p-coumaryl alcohol and sinapyl alcohol were poor substrates for all the isoenzymes. An absolute requirement for Ca²⁺ ions was demonstrated for P3. Based on these results, possible roles of peroxidase isoenzymes in the formation of lignin during the differentiation of tracheary elements are discussed.Abbreviations DAB diaminobenzidine - GTA equal proportions of 3,3-dimethylglutaric acid, tris(hydroxymethyl)aminomethane, and 2-amino-2-methyl-1,3-propanediol - TE tracheary element The authors are very grateful to Professor M. Tanahashi of Gifu University for providing hydroxycinnamyl alcohols. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan to H.F.     

Application of seed esterase isoenzymes in testing hybridity of tomato

Expression of esterase isoenzymes in 1272 seeds was used to estimate hybridity of Lycopersicon esculentum Mill. Individual seeds (440) of the parental cultivars taken from different experimental stations in Bulgaria were also analysed. The banding patterns were obtained by means of vertical block electrophoresis in polyacrylamide gels. It was established that quantitative variation of locus Est-1 can be applied to prove hybridity of F₁ tomato seeds. This marker is related to the genetic nature of tomatoes and is not the result of the environmental influence. The reason for this conclusion is the fact that the isoenzymes of the Est-1 locus are indicative for hybridity determination of all examined seeds taken from different regions in Bulgaria. Use of this locus is to be recommended for both its universality and the fact that the reagents for esterase isoenzymes staining are not expensive. This revised version was published online in July 2006 with corrections to the Cover Date.     

Glucan-phosphorylase forms in cotyledons of Pisum sativum L.: Localization,developmental change,in-vitro translation,and processing

The occurrence, location, and biosynthesis of glucan-phosphorylase (EC 2.4.1.1) isoenzymes were studied in cotyledons of developing or germinating seeds of Pisum sativum L. Type-I and type-II isoenzymes were detected, and were also localized by indirect immunofluorescence using polyclonal anti-type-I or anti-type-II phosphorylase antibodies. Type-I isoenzyme was found in the cytosol of parenchyma cells whereas the type-II enzyme form is a plastid protein which resides either in amyloplasts (in developing seeds) or in proplastids (in germinating seeds). During seed development, type-II phosphorylase was the predominant isoenzyme and the type-I isoenzyme represented a very minor compound. During germination, the latter increased whilst type-II phosphorylase remained at a constant level. In in-vitro translation experiments, type-I isoenzyme was observed as a final-size product with an apparent molecular weight of approx. 90 kDa. In contrast, type-II phosphorylase was translated as a high-molecular-weight precursor (116 kDa) which, when incubated with a stromal fraction of isolated intact pea chloroplasts, was processed to the size of the mature protein (105 kDa).Abbreviations IgG immunoglobulin G - kDa kilodalton - poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work has been made possible by grants from the Deutsche Forschungsgemeinschaft. The authors are endebted to Mrs. Karin Niehüser for help in the immunocytochemical studies.     

Differential induction of cinnamyl alcohol dehydrogenase during defensive lignification in wheat (Triticum aestivum L.): characterisation of the major inducible form

The induction and substrate specificity of cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) was investigated in relation to the deposition of a defensive, syringyl-rich lignin at wound margins in wheat (Triticum aestivum L. cv. Brigadier). Column chromatography of untreated, wounded and elicitor-treated tissues revealed three major CAD forms (CAD-A, -B and -C) of which only CAD-C was responsive to elicitors. Examination of the substrate preference of these fractions indicated p-coumaryl alcohol to be the preferred substrate of CAD-A and CAD-B, whereas sinapyl alcohol was favoured by CAD-C. Activity-stained isoelectric focussing gels revealed in untreated and wounded leaves four CAD isoenzymes with isoelectric points of 4.59 (i), 4.67 (ii), 4.81 (iii), 4.93 (iv). Elicitor treatment generally enhanced the staining of all isoenzymes and resulted in the appearance of two new isoenzymes of 5.22 (v) and pI 5.31 (vi). In activity stained non-denaturing PAGE gels, CAD-C further resolved into two distinct zones of CAD activity. Cinnamyl alcohol dehydrogenase-C was purified to apparent homogeneity and characterisation revealed a 45-kDa subunit peptide which in its native form demonstrated a marked substrate preference for sinapyl alcohol. Overall, the differential induction and substrate preference of CAD-C are consistent with a defensive role during defensive lignification at wound margins in wheat. Received: 20 September 1998 / Accepted: 22 October 1998     

The role of two isoenzymes of alpha-amylase of Araucaria araucana (Araucariaceae) on the digestion of starch granules during germination

Starch is the principal reserve of Araucaria araucana seeds, and it is hydrolysed during germination mainly by alpha-amylase. There are several alpha-amylase isoenzymes whose patterns change in the embryo and in the megagametophyte from the one observed in quiescent seeds (T(0)) to a different one observed 90 h after imbibition (T(90)). The objective of this research was to study the roles of two purified alpha-amylase isoenzymes by in vitro digestion of starch granules extracted from the tissues at two times of imbibition: one is abundant in quiescent seeds and the other is abundant after 90 h of imbibition. The isoenzymes digested the starch granules of their own stage of germination better, since the isoenzyme T(0) digested starch granules mainly from quiescent seeds, while the isoenzyme T(90) digested starch mainly at 90 h of imbibition. The sizes of the starch granule and the tissue from which these granules originated make a difference to digestion by the isoenzymes. Embryonic isoenzyme T(0) digested large embryonic starch granules better than small and medium-sized granules, and better than those isolated from megagametophytes. Similarly isoenzyme T(90) digested small embryonic starch granules better than medium-sized and large granules, and better than those isolated from megagametophytes. However, a mixture of partially purified megagametophytic isoenzymes T(0) and T(90) digested the megagametophytic granules better than those isolated from embryos. Studies of in vitro sequential digestion of starch granules with these isoenzymes corroborated their specificity. The isoenzyme T(90) digested starch granules previously digested by the isoenzyme T(0). This suggests that in vivo these two isoenzymes may act sequentially in starch granule digestion.     

Separation and partial characterization of multiple forms of rat liver alcohol dehydrogenase

Rat liver alcohol dehydrogenase was purified and four isoenzyme forms, demonstrated by starch gel electrophoresis, were separated by O-(carboxymethyl)-cellulose chromatography. Each of the isoenzymes had a distinct isoelectric point. All isoenzymes were active with both ethanol (or acetaldehyde) and steroid substrates, and had similar Michaelis-Menten constants for each of the substrates and coenzymes studied. The three isoenzymes with the lowest migration toward the cathode exhibited the same pH optimum of 10.7 for ethanol oxidation, a greater activity with 5 beta-androstan-3 beta-ol-17-one than with ethanol as a substrate, and an unchanged electrophoretic mobility following storage in the presence of 100 microM dithiothreitol. By contrast the isoenzyme with the highest mobility toward the cathode exhibited a pH optimum of 9.5 for ethanol oxidation, a low steroid/ethanol ratio of activity, and converted to the migrating pattern of the two isoenzymes with intermediate mobility when stored. The similarities between the isoenzymes of rat liver alcohol dehydrogenase differ considerably from differences in substrate specificity exhibited by isoenzymes of horse liver alcohol dehydrogenase.     

Selective carboxymethylation of cysteine-174 of the beta 2 beta 2 and beta 1 beta 1 human liver alcohol dehydrogenase isoenzymes by iodoacetate

The beta 1 beta 1 and beta 2 beta 2 human liver alcohol dehydrogenase isoenzymes differ by only one residue at the coenzyme-binding site; Arg-47 in beta 1 is replaced by His in the beta 2 subunit. Since Arg-47 is thought to facilitate the carboxymethylation of Cys-46 in horse liver alcohol dehydrogenase by binding halo acids in a Michaelis-Menten complex prior to inactivation, the specificity and kinetics of modification of the two human liver beta beta isoenzymes with iodoacetate were compared. Both of the beta beta isoenzymes were inactivated by treatment with iodo[14C]acetate, and one Cys per subunit was carboxymethylated. Cys-174, which is a ligand to the active-site zinc atom in horse liver alcohol dehydrogenase, was selectively carboxymethylated in each of the human beta beta isoenzymes; less than 15% of the iodo[14C]acetate incorporated into the enzyme appeared in Cys-46. Therefore, the three-dimensional structure of the basic amino acids in the anion-binding site of the human beta beta isoenzymes appears to be different from that of horse liver alcohol dehydrogenase. The kinetics of alkylation are consistent with the formation of a Michaelis-Menten complex before inactivation of the isoenzymes. The average Ki values for iodoacetate were 10 and 16 mM for beta 1 beta 1 and beta 2 beta 2, respectively, and maximal rate constants for inactivation were 0.22 and 0.17 min-1, respectively. From these data, it can be concluded that there is a relatively minor effect of the substitution of His for Arg at position 47 on the kinetics of inactivation.     

Interaction of galactoglucomannan oligosaccharides with auxin in mung bean primary root

In the present paper timing of galactoglucomannan oligosaccharides (GGMOs) with exogenously added indole-3-butyric acid (IBA) action on early germination stage (24 h) and primary root elongation of mung bean (Vigna radiata (L.) Wilczek) has been studied. GGMOs inhibited primary root elongation induced by low concentration (10^?8 M) of IBA. This inhibition was considerably higher after preincubation with GGMOs compared with other timing experiments. The most intensive inhibition of elongation has been ascertained at the 10^?8 M concentration of GGMOs. On the other hand GGMOs stimulated this elongation inhibited by high IBA concentration (10^?4 M). This stimulation was the most intensive by simultaneous addition of IBA and GGMOs at the beginning of the experiment and subsequent seeds incubation in distilled water. Our results indicate competition between GGMOs and auxin. The root growth inhibition, induced by GGMOs and/or IBA, was accompanied by the increase of cell wall-associated peroxidase activity and by a higher number of peroxidase isoenzymes. The presence of different peroxidase isoenzymes in experiments with distinct treatment of GGMOs and IBA could indicate variations in the mechanism of interaction between GGMOs and IBA.     

Comparison of tryptic peptide maps of two isoenzymes of 6-phosphogluconate dehydrogenase from tobacco tissue cultures

In a previous study by the authors, two isoenzymes of 6-phosphogluconate dehydrogenase were isolated from cultures of tobacco tissue Nicotiana tabacum W-38 and shown to be similar in their pH optima and MWs and in their affinities toward 6-phosphogluconate or NADP⁺. In an attempt to clarify the structural relationships between these two isoenzymes, peptide mapping of trypsin digests of the purified isoenzymes was performed. The maps were found to be similar, with at least 29 peptide groups from the trypsin digestion of each isoenzyme being alike. There were, however, definite minor differences in the peptide maps of the two isoenzymes.     

Molecular Properties of the ATP:D-Fructose-6-Phosphate 1-Phosphotransferase Isoenzymes from Cucumis sativus

The ATP:D-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.11 [EC] )isoenzymes from cucumber seeds were separated and purified.The calculated molecular weights of the two isoenzymes (approximately180,000) are similar and the isoenzymes are probably hetro-tetramers.The purified isoenzymes contained three polypeptides of 53.3,41.5 and 39.0 kDa for the plastid and 47.2, 42.4 and 40.4 forthe cytosolic isoenzyme, respectively. The purified phosphofructokinaseisoenzymes were used as the antigen for the production of polyclonalantibodies in rabbits. The obtained antisera clearly indicatedthat there is no immunological similarity between the two isoenzymes.The results also show that the phosphofructokinase isoenzymesin cucumber are not merely different stages of association ofthe same protein. (Received June 29, 1987; Accepted October 21, 1987)     

Transformation of Industrial Dyes by Manganese Peroxidases from Bjerkandera adusta and Pleurotus eryngii in a Manganese-Independent Reaction

We investigated the transformation of six industrial azo and phthalocyanine dyes by ligninolytic peroxidases from Bjerkandera adusta and other white rot fungi. The dyes were not oxidized or were oxidized very little by Phanerochaete chrysosporium manganese peroxidase (MnP) or by a chemically generated Mn³⁺-lactate complex. Lignin peroxidase (LiP) from B. adusta also showed low activity with most of the dyes, but the specific activities increased 8- to 100-fold when veratryl alcohol was included in the reaction mixture, reaching levels of 3.9 to 9.6 U/mg. The B. adusta and Pleurotus eryngii MnP isoenzymes are unusual because of their ability to oxidize aromatic compounds like 2,6-dimethoxyphenol and veratryl alcohol in the absence of Mn²⁺. These MnP isoenzymes also decolorized the azo dyes and the phthalocyanine complexes in an Mn²⁺-independent manner. The reactions with the dyes were characterized by apparent K_m values ranging from 4 to 16 μM and specific activities ranging from 3.2 to 10.9 U/mg. Dye oxidation by these peroxidases was not increased by adding veratryl alcohol as it was in LiP reactions. Moreover, the reaction was inhibited by the presence of Mn²⁺, which in the case of Reactive Black 5, an azo dye which is not oxidized by the Mn³⁺-lactate complex, was found to act as a noncompetitive inhibitor of dye oxidation by B. adusta MnP1.     

Effect of Cadmium on Activities of Some Enzymes of Glycolysis and Pentose Phosphate Pathway in Pea

Activities of alcohol dehydrogenase, hexokinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were significantly inhibited by cadmium in germinating pea (Pisum sativum L. cv. Bonneville) seeds. The effect was concentration dependent in the range of 0.25 to 1.0 mM CdCl₂. The magnitude of detrimental effect on these enzymes was reduced during later stage of germination (9 d) largely because of fall in the activities of these enzymes in the control seeds germinated in water. In vitro, activities of hexokinase, glucose-6-phosphate dehydrogenase, and alcohol dehydrogenase were inhibited at 0.5 mM Cd²⁺ in the reaction mixture by 62, 67, and 36 %, respectively, however, 6-phosphogluconate dehydrogenase was insensitive to Cd²⁺. This revised version was published online in June 2006 with corrections to the Cover Date.     

Wounding Induces One of Two Isoenzymes of 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase in Solanum tuberosum L

Potato (Solanum tuberosum L.) tubers contain two isoenzymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15), the enzyme that catalyzes the first step of aromatic amino acid biosynthesis. One of the isoenzymes is specifically activated by Mn²⁺, and the other requires Co²⁺, Mg²⁺, or another divalent cation for activity. Monospecific polyclonal antibodies against the Mn²⁺-activated isoenzyme do not cross-react with the other isoenzyme. Wounding of potato tubers induces the Mn²⁺-activated form but not the other. We conclude that two different genes encode two different isoenzymes that catalyze the first step in the shikimate pathway.     

Isoenzymatic variability in seeds of some spanish common beans (Phaseolus vulgaris L. Leguminosae): Relation to their domestication centers

Phaseolus vulgaris is an important crop species. The cultivated common bean derives from wild forms in two independent domestication centers in Mesoamerica and South America. We report a study of electrophoretic patterns of seven isoenzymatic systems in 29 samples of P. vulgaris seeds. Nineteen of them are from northern Spain, four are from Mesoamerican and six are South American forms. The isoenzymatic activity of esterases, cytochrome c oxidase, acid phosphatase, alkaline phosphatase, alcohol dehydrogenase, shikimate dehydrogenase and glutamate oxaloacetic transaminase were studied. On the basis of electrophoretic patterns of seed isoenzymes, four groups can be recognized using clustering procedures (UPGMA). Comparing this biochemical information with previous morpho-agronomic studies, the possible primary domestication centers of the cultivars are discussed.     

Acyl transfer strategy for the biocatalytical characterisation of Candida rugosa lipases in organic solvents

The yield obtained in a Candida rugosa lipase-catalysed heptyl acetate synthesis via transesterification of 1-heptanol with vinyl acetate depends linearly on the concentration of lipases of each crude lyophilised powder. Thus, it is possible to characterise the amount of lipases present in the biocatalyst by calculating the parameter called Catalytic Performance (CP), defined as (% final yield) × (mg crude biocatalyst)⁻¹. On the other hand, the relative yield obtained in the C. rugosa lipase-catalysed transesterifications of different alcohols (1-heptanol, geraniol, nerol and cyclohexanol) with vinyl acetate as acyl donor depends on the proportion of isoenzymes of each crude biocatalyst. Therefore, it is possible to qualitatively evaluate the proportion of isoenzymes in those crude preparations and to predict the biocatalytical behaviour of each isoenzyme according to the alcohol employed. The methodology described is successfully used in two non-conventional asymmetric syntheses in organic media.