Electrophoretic characterization of six selected enzymes of peanut cultivars

The isozyme patterns (both anodic and cathodic) of esterase, catalase, leucine aminopeptidase, acid phosphatase, alcohol dehydrogenase and INT oxidase in individual seeds from several peanut cultivars (Arachis hypogaea) were characterized by polyacrylamide and starch gel electrophoresis in relation to the stages of seed development, maturity, and germination, geographic areas where grown and phylogenetic relationship. Of the six enzymes examined, only esterase contained cathodic isozymes, of which the patterns served to distinguish between the Spanish and the Virginia-type peanuts. Anodic esterase and acid phosphatase zymograms of early developing and germinating peanuts could be distinguished from those of predormant and mature seeds and the latter showed much intravarietal variation which was consistent among cultivars and the geographic areas where grown. Anodic isozymes of catalase, leucine aminopeptidase, alcohol dehydrogenase and INT oxidase were synthesized very early in peanut development and remained constant through maturity and to at least 24 hr germination; they were consistent within and between peanut cultivars and they were not influenced by the environmental conditions of the areas where the peanuts were grown. The consistency of the isozyme patterns within and between cultivars supports the suggestion that plant breeding programs used to develop superior cultivars have produced genetic uniformity in peanuts.     

Light or high (30°C) temperature effects on l-[U-C]leucine incorporation and protein patterns in embryos and endosperm during the early phase of germination of the negatively photoblastic and thermosensitive seeds of Phacelia tanacetifolia

The activities of l-[U-¹⁴C]leucine uptake and incorporation into proteins of embryos and endosperm of seeds of Phacelia tanacetifolia Benth. cv Bleu-Clair were analysed during the first 24 h of incubation under conditions optimal for germination (16°C in darkness) and in two inhibitory conditions: 16°C in the light and 30°C in darkness. Blocking germination induced by light or 30°C was accompanied by the inhibition of l-[U-¹⁴C]leucine uptake and incorporation in embryos. In the endosperm, the activation of l-[U-¹⁴C]leucine uptake was of the same magnitude for the non-inhibited and the light-inhibited seeds and much higher for the 30°C-inhibited seeds; the activation of l-[U-¹⁴C]leucine incorporation was quantitatively similar in all three conditions, with the patterns of newly synthesised proteins qualitatively different in the endosperm from light- or 30°C-inhibited seeds. The results showed that germination of P. tanacetifolia seeds is controlled by light or super-optimal temperature through the inhibition of the activation of transport and protein synthetic activities in embryo without effect on the endosperm. We suggest, on the basis of the translational activity, the possibility that in the inhibitory conditions the blockade of the embryo to operate as a sink affects the transition of the endosperm to operation as a source.     

Seed esterases,leucine aminopeptidases and catalases of species of the genus Gossypium

Summary Polyacrylamide and starch gel electrophoresis were used to analyze the isozyme makeup of three enzyme systems (esterases, leucine aminopeptidases and catalases) from the dormant seeds of twenty-nine species within the genus Gossypium.Isozyme variation was observed for all three enzymes between the species of the different genome groups. The within species polymorphism noted for the esterases was not observed for the leucine aminopeptidase and catalase patterns. In general, only minor qualitative banding pattern differences distinguished the A and B genome species, whereas, band variations were greatest between the more distantly related species in the C, D and E genomes. Gossypium longicalyx (F genome) showed an overall banding pattern unique to itself. The species of the genomes (C, D, E and F) removed from the postulated area of genetic origin (Southern Africa) also exhibited greater isozyme variability than that of the wild species of the A and B genomes, both located in Southern Africa.Synthetic mixtures of seed extracts from parent species of recently formed synthetic allopolyploids produced additive isozyme patterns for esterase, leucine aminopeptidase and catalase that were closely comparable to the zymograms produced by their hybrids. In contrast all three enzyme systems showed significant qualitative isozyme variations between the three natural allotetraploids, G. tomentosum, G. barbadense and G. hirsutum when compared to the zymograms of the synthetic mixtures of their alleged parental forms.This paper is part of a dissertation by the first author for the degree of Doctor of Philosophy in Genetics. Journal paper 1763 of the Arizona Agricultural Experiment Station.National Research Council Postdoctoral Research Associate.     

Purification and Identification of a Novel Leucine Aminopeptidase from <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>

A novel leucine aminopeptidase was purified from a Bacillus thuringiensis israelensis (Bti) culture. The purification stages included heating the concentrated supernatant to 65°C for 90 min, anion-exchange chromatography by DEAE cellulose, and hydrophobic chromatography by phenyl Sepharose. The specific activity of leucine aminopeptidase after the hydrophobic chromatography increased by 215.5-fold and the yield was 16%. The molecular weight of the active enzyme was 59 kDa. Mass spectrometry analysis of the 59-kDa leucine aminopeptidase revealed that this protein has at least 41% homology with the cytosol leucine aminopeptidase produced by Bacillus cereus. Maximal leucine aminopeptidase activity occurred at 65°C, pH 10 toward leucine as the amino acid terminus. The enzyme was strongly inhibited by bestatin, dithiothreitol, and 1,10-phenanthroline, indicating that the enzyme might be considered as a metallo-aminopeptidase that has disulfide bonds at the catalytic site or at a region that influences its configuration. Examination of the purified leucine aminopeptidase’s effect on the activation of the protoxin Cyt1Aa from Bti revealed that when it acts synergistically with Bti endogenous proteases, it has only a minor role in the processing of Cyt1Aa into an active toxin.     

Isolation and characterization of Brush border fragments from mosquito mesenterons

Brush border fragments were isolated from homogenates of mesenterons from the mosquito, Culex tarsalis, by a combination of Ca²⁺ precipitation and differential centrifugation. These preparations were routinely enriched seven- to eightfold for the brush border marker enzyme, leucine aminopeptidase. Alkaline phosphatase, a putative brush border marker for both vertebrate and invertebrate brush borders, was found to be unsuitable for Cx. tarsalis. Isoelectric focusing electrophoresis coupled with histochemical enzyme detection was used to enumerate isozymic species of nonspecific esterases [3], leucine aminopeptidase [1], and alkaline phosphatase [1] in isolated brush border fragments. Leucine aminopeptidase activity was solubilized by papain digestion, suggesting an extrinsic active site for this membrane-bound enzyme. The predominant nonspecific esterase isozyme remained membrane-bound. Conventional staining (ie, Coomassie Blue and silver) of proteins separated by isoelectric focusing, sodium dodecylsulfate, and two-dimensional electrophoresis indicated a simple pattern for brush border fragments, with two proteins predominating among the 11–14 routinely detected.     

Studies of esterase 6 in Drosophila melanogaster. I. The genetics of a posttranslational modification

A locus has been found, an allele of which causes a modification of some allozymes of the enzyme esterase 6 in Drosophila melanogaster. There are two alleles of this locus, one of which is dominant to the other and results in increased electrophoretic mobility of affected allozymes. The locus responsible has been mapped to 3-56.7 on the standard genetic map (Est-6 is at 3-36.8). Of 13 other enzyme systems analyzed, only leucine aminopeptidase is affected by the modifier locus. Neuraminidase incubations of homogenates altered the electrophoretic mobility of esterase 6 allozymes, but the mobility differences found are not large enough to conclude that esterase 6 is sialylated.This work was supported by NIH Grant No. GM23706 and PHS Grant SO7RR7031 to Rollin C. Richmond and by NIH Genetics Training Grant No. 82 to Indiana University.     

Rapporti Fra Endosperma ed Embrione Nella Germinazione di Pinus Pinea L.

Abstract

Endosperm embryo interrelationships during germination of PINUS PINEA L. (2nd Note). — Experiments have been performed with unripe fresh seeds paralleled with unripe stored seeds (50 days of storage).

The effect of storage is very similar to the effect of further permanence on the tree.

It is confirmed the importance of the endosperm in the germination process already put in evidence in the 1rst note.

The relationships existing between endosperm and embryo during the germination of the seed become more and more strict as the seed ripens, so that in a ripe seed the embryo develops in a normal seedling only in connection of its own endosperm.

In an unripe seed the embryo is uncompletely controlled by its endosperm.

Some remarks have been made about the degree of maturity of the different parts of the embryo.     

Effetto di Inibitori Della Sintesi Proteica Sull'aumento di Attività Enzimatiche e sul Risveglio del Metabolismo Nell'Endosperma di Semi di Ricino in Germinazione

Abstract

Effects of inhibitors of protein synthesis on the development of metabolic activity in the endosperm during the germination of castor bean seeds. — The effect of chloramphenicol, streptomycin and actinomycin-C on the increase of the activities of glyceroaldehyde-phosphate dehydrogenase, aldolase, glucose-6-phosphate dehydrogenase, fructose 1–6 diphosphate-1-phosphatase, phosphomonoesterase, in the endosperm of germinating castor bean seeds was investigated.

In all cases, the protein synthesis inhibitors depressed the activation of the enzymes tested: in particular, actinomycin (50 μg/ml) completely suppressed the increase of the activities.

The development of the rate of oxygen uptake and the conversion of fats to sugars was strongly affected by the inhibitors.

These data suggest that the increase of the activities of several enzymes in the germinating endosperm is dependent on enzyme synthesis rather than on the conversion from the inactive to the active form of the enzymes.     

Formation of enzymes required for the hydrolysis of plant cell wall polysaccharides byTrichoderma reesei

Summary Enzyme preparations fromTrichoderma reesei RUT-C30, in addition to cellulase, contained various glucanase and glucosidase, acetylxylan esterase, glucuronidase and xylanase activities. These preparations were able to hydrolyse endosperm cell walls of corn and wheat and commercially-available xylans and plant gums having stright chains, but lacked the ability to hydrolyse branched or substituted hemicelluloses.

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Formation d'enzymes requises pour l'hydrolyse des polysaccharides de la paroi de plantes chez Trichoderma reesei

Résumé Les préparations enzymatiques deTrichoderma reesei RUT-C30, contiennent, outre la cellulase, diverses glucanases et glucosidases, acétyl-xylane esterases, glucuronidases et xylanases. Ces préparations demeurent stables pour l'hydrolyse de parois cellulaires de l'endosperme de maïs et de froment ainsi que des xylanes et gommes de plantes à chaînes linéaires, disponibles dans le commerce, mais ne présentent pas le pouvoir d'hydrolyser les hémicelluloses branchées ou substituées.

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Issued as NRCC No. 29855     

Substrate specificity of a human-specific esterase

A human species-specific esterase has been identified in tissues, cell cultures, and urine. It is the most slowly migrating (i.e., cathodal) of the esterase isoenzymes in agarose electrophoresis; it is not a choline estrase, a pseudocholine esterase, an acetyl phenylalanine-3-naphthyl esterase or N-benzoyl-arginine-3-naphthyl esterase. Hydrolysis of N-methyl indoxyl acetate caused by this esterase is not inhibited by eserine, eserine sulfate, or EDTA. Phenylmethylsulfonyl fluoride, however, did inhibit hydrolysis. Furthermore, this cathodal esterase does not show any chymotrypsin, trypsin, or leucine aminopeptidase enzyme activity.     

Electrophoretic techniques for detection of Glypta fumiferanae, an endoparasitoid of western spruce budworm

Parasitoid-free western spruce budworms and western spruce budworm larvae containing the endoparasitoid Glypta fumiferanae (Vier.) (Hymenoptera: Ichneumonidae) were assayed electrophoretically to identify isozyme bands diagnostic for the parasitoid. Bands characteristic of G. fumiferanae were observed with 4 of 15 enzymes tested. These were aconitase, esterase, leucine aminopeptidase and 6-phosphoglucose dehydrogenase. Presence of these isozyme bands is a reliable indication of parasitism by G. fumiferanae. The efficiency of electrophoresis as a tool for routine detection of parasitoids is discussed.

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Zusammenfassung Raupen von Choristoneura occidentalis ohne und mit dem Endoparasitoiden Glypta fumiferanae (Vier.) (Hymenoptera: Ichneumonidae) wurden elektrophoretisch auf Isoenzymbänder untersucht zum Nachweis des Parasitoiden. Für G. fumiferanae typische Bänder wurden in 4 der untersuchten 15 Enzymen beobachtet. Dies waren Aconitase, Esterase, Leucinaminopeptidase und 6-Phosphoglucose-dehydrogenase. Das Vorkommen dieser Isoenzymbänder ist ein zuverlässiges Zeichen der Parasitierung durch G. fumiferanae. Die Brauchbarkeit der Elektrophorese als Mittel zum Routinenachweis von Parasitoiden wird diskutiert.

     

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.     

Über die genetischen Ursachen der Samenbildung

Zusammenfassung Innerhalb von Primula malacoides Franchet wurden zwischen verschiedenen Formen Valenzkreuzungen durchgeführt. Es traten 2x-, 3x-, 4x- und 5x-Pflanzen auf. An der Entstehung dieser Valenztypen sind sowohl reduzierte als auch unreduzierte Gameten beteiligt. Auf Grund der erzielten Kreuzungsergebnisse wird versucht, Rückschlüsse auf die genetischen Ursachen der Samenbildung zu ziehen.Es wird angenommen, daß der jeweiligen Konstitution des Endosperms eine vorrangige Bedeutung für die Entstehung von lebensfähigen Samen zukommt.

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On the genetic causes of seed formation

Summary New crossings were made between different diploid and polyploid forms of Primula malacoides Franchet. 2x, 3x, 4x and 5x plants appeared. Both reduced and unreduced gametes participated in the formation of these types. Based on the results conclusions are drawn about the genetic causes of seed development. It is suggested that the endosperm constitution has prime importance for the development of viable seeds.

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Herrn Prof. Dr. Hans Stubbe zum 65. Geburtstag gewidmet.

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Quedlinburger Beiträge zur Züchtungsforschung Nr. 78.     

Mechanical Resistance of the Seed Coat and Endosperm during Germination of Capsicum annuum at Low Temperature

Decoated pepper (Capsicum annuum L. cv Early Calwonder) seeds germinated earlier at 25°C, but not at 15°C, compared to coated seeds. The seed coat did not appear to impose a mechanical restriction on pepper seed germination. Scarification of the endosperm material directly in front of the radicle reduced the time to germination at both 15°C and 25°C.

The amount of mechanical resistance imposed by the endosperm on radicle emergence before germination was measured using the Instron Universal Testing Machine. Endosperm strength decreased as imbibition time increased. The puncture force decreased faster when seeds were imbibed at 25°C than at 15°C. The reduction in puncture force corresponded with the ability of pepper seeds to germinate. Most radicle emergence occurred at 15°C and 25°C after the puncture force was reduced to between 0.3 and 0.4 newtons.

Application of gibberellic acid₄₊₇ (100 microliters per liter) resulted in earlier germination at 15°C and 25°C and decreased endosperm strength sooner than in untreated seeds. Similarly, high O₂ concentrations had similar effects on germination earliness and endosperm strength decline as did gibberellic acid₄₊₇, but only at 25°C. At 15°C, high O₂ concentrations slowed germination and endosperm strength decline.

     

Rapporti Fra Endosperma ed Embrione Nella Germinazione di Pinus Pinea L.: Nota i: Effetti del Grado di Maturità del Seme

Abstract

Endosperm embryo interrelationships during germination of PINUS PINEA L. 1rst note. — Seeds and excised embryos have been cultured in vitro in the dark. Experiments with ripe seeds and embryos have been paralleled with exper. with unripe ones (6 months before natural seed dispersal). The results are as follows:

Both ripe and unripe seeds germinate in the dark into green seedlings.

In both types of seedlings the rootlet is the only part which rapidly increases in length, actually growing, but the growth speed of the root is greater in ripe seeds than in the unripe ones.

Both ripe and unripe excised embryos grow slowly, with greenles cotyledons and dwarf roots, showing a very slight geotropic curve.

The root stops growing more sharply in ripe embryos than in unripe ones.

The hypothesis is raised that the rootlet reaches its full growing power, under the endosperm control, and that this control encreases more and more, as the seed approaches its full maturity.     

Use of Isoenzymes to Differentiate Growth Categories of Pericopsis Mooniana Trees

Leaf isoenzymes of Pericopsis mooniana from twenty-one trees at forest plantation were evaluated for their use in identification of elite trees among heterogeneous population. Trees were grouped morphologically, before leaf extracts were separated by one-dimensional polyacrylamide gel electrophoresis. Isoenzyme analysis were carried out for peroxidase, esterase, alcohol dehydrogenase, formate dehydrogenase, acid phosphatase, aspartate aminotransferase, isocitrate dehydrogenase, malate dehydrogenase, leucine aminopeptidase, phosphoglucoisomerase, phosphogluconate dehydrogenase, phosphoglucomutase, and shikimate dehydrogenase. From the thirteen enzymes studied only four gave distinct banding patterns. Level of significance of appearing particular band for each enzyme of a given category was investigated using χ²-test, followed by cluster analysis for categorization. The isozyme type A of formate dehydrogenase showed promising results that could be used for differentiating trees of categories investigated. This revised version was published online in July 2006 with corrections to the Cover Date.     

Attività Respiratoria Livelli Enzimatici e Tenore in Acqua Durante la Maturazione del Seme di Ricino

Abstract

Respiratory activity, enzyme levels and water contents in the ripening castor beans seed endosperm. — In the endosperm of the developing castor bean seed oxygen uptake, water contents and the « in vitro » measurable activity of various enzymes parallely drop during the terminal strages of ripening. The present investigations shows that also the capacity of water uptake decreases (and that of water loss increases) during ripening.

When developing seeds at a stage close to ripeness are removed from the fruits and incubated under a condition of easy water availability, both respiration and enzyme activities rapidly rise; while this is not observed for seeds removed from the fruit at an earlier stage of development.

These results are interpreted as indicating that the dehydration of the seed during ripening is both a consequence and a cause of the inactivation of enzyme systems.     

Analysis of linkage between genes controlling enzymes in sunflower

The polymorphism of leucine aminopeptidase in 23 inbred lines of sunflower of mutant origin was studied. The isozyme spectrum of leucine aminopeptidase is presented as a single zone of enzyme activity controlled by one gene. This gene was shown to be inherited independently from genes of esterase and 6-phosphogluconate dehydrogenase.     

Analysis of linkage between genes controlling enzymes in sunflower

The polymorphism of leucine aminopeptidase in 23 inbred lines of sunflower of mutant origin was studied. The isozyme spectrum of leucine aminopeptidase is presented as a single zone of enzyme activity controlled by one gene. This gene was shown to be inherited independently from genes of esterase and 6-phosphogluconate dehydrogenase.