Synthesis and Posttranslational Activation of Sulfhydryl-Endopeptidase in Cotyledons of Germinating Vigna mungo Seeds

A sulfhydryl-endopeptidase was purified as a 33 kilodalton (kD) mass polypeptide from cotyledons of Vigna mungo seedlings. Immunoblot analysis with antiserum made against the purified enzyme showed that the sulfhydryl-endopeptidase was synthesized only in the cotyledons during germination and that the amount of the enzyme increased until 4 days after imbibition and decreased thereafter. Next, an RNA fraction was prepared from cotyledons of 3 day old seedlings and translated in a wheat germ system. The synthesis of a 45 kD polypeptide was shown by the analysis of its translation products by immunoprecipitation with the antiserum to the endopeptidase and gel electrophoresis. When the RNA fraction was translated in the presence of canine microsomal membranes, a smaller polypeptide, having a 43 kD molecular mass, was detected as the translation product. When membrane-bound polysomes, but not free polysomes, prepared from cotyledons were used for translation in the wheat germ system, both the 43 and 45 kD polypeptides were synthesized. By incubation of a crude enzyme extract from cotyledons at 5 ± 1°C at neutral pH, the 43 kD polypeptide was sequentially cleaved to the 33 kD polypeptide via 39 and 36 kD intermediate polypeptides. The endopeptidase was activated simultaneously with the processing. Two-dimensional polyacrylamide gel electrophoresis showed that the 33 kD polypeptide was the fully activated form of the enzyme, whereas little or no activity was detected in other forms. From the present results, we postulate that the sulfhydryl-endopeptidase is first synthesized as the 45 kD precursor with a 2 kD signal peptide being cleaved, and that the 43 kD polypeptide is further cleaved to give the 33kD mature enzyme.     

Purification and characterization of a cysteine endopeptidase in cotyledons of germinated mung bean seeds

A cysteine endopeptidase (EC 3.4.22.-) present in cotyledons of mung bean (Vigna radiata) seedlings was purified to homogeneity, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This proteinase has an apparent molecular mass of 33 kilodaltons as estimated by SDS-PAGE and belongs to the class of cysteine proteinases as judged by the effects of various proteinase inhibitors on the activity of the enzyme. When proangiotensin is used as a substrate, the enzyme preferentially hydrolyzes the peptide bonds formed by the amino group of Leu or lle in this oligopeptide chain; for the enzyme to cleave those bonds, peptide sequences consisting of at least three amino acid residues on the amino side of Leu or lle must be present. The proteinase readily digests globulin present in mung bean cotyledons to smaller polypeptides.     

Effect of growth temperature on reserve mobilization and hydrolytic enzyme activities in Vigna mungo cotyledons

Seeds of Vigna mungo were allowed to germinate at 27, 18 and15°C, and time-course changes of hydrolytic enzyme activitiesand the mobilization rate of reserve components in cotyledonswere studied. The seeds germinated at 27 and 18°C grew normally,whereas the growth at 15°C was markedly retarded. In cotyledonsof seedlings grown at 27 and 18°C, amylolytic and proteolyticenzyme activities increased at early stages of growth and therates of starch and protein mobilization changed correspondingto the hydrolytic enzyme activities. At 15°C the enzymeactivities increased gradually during the experimental periodof 16 days, but the reserves in cotyledons remained almost unchangeduntil the end of the experimental period. Changes of zymogram patterns of amylolytic and proteolytic activitiesin cotyledons of seedlings grownat 27, 18 and 15°C wereexamined using polyacrylamide gel electrophoresis. The intensitiesof a main band of a-amylase and at least two bands of protease(gelatin-hydrolyzing activity) increased concurrently with invitro activities of amylolytic and proteolytic enzymes. At leastthree bands of starch phosphorylase were present in cotyledonsat early stages of germination and their intensities decreasedduring the growth of seedlings at 27, 18 and 15°C. (Received June 4, 1980; )     

Serological evidence confirming the assignment of phaseolus aureus and P. mungo to the genus vigna

Endopeptidase activity was detected in extracts of cotyledons of 11 species of Vigna and Phaseolus Antibodies against the purified protease isolated from the cotyledons of 5-day-old P.aureus seedlings inhibited the activity of that enzyme in crude extracts of cotyledons. A similar inhibition was obtained with P. mungo, P. adenanthus and 4 species of Vigna, while there was no inhibition of endopeptidase activity in extracts of cotyledons of 4 species of Phaseolus. Immunodiffusion tests proved that the protease of Vigna is distinct from that of Phaseolus. The evidence supports the reassignment of P. aureus and P. mungo to the genus Vigna and indicates that the names Vigna radiata and Vigna mungo are more appropriate than P. aureus and P. mungo for green gram and black gram respectively.     

Multiple Forms of Acid Phosphatase in Cotyledons of Vigna mungo Seedlings: Immunological Detection and Quantitation

Using a combination of column chromatography and gel electrophoresis,we have found that acid phosphatase in cotyledons of Vigna mungoseedlings is composed of at least six forms (Ia₁, Ia₂, Ib₁,Ib₂, IIa and IIb). We purified one of the major forms, Ia₁,as a polypeptide of 53 kDa. Using an antiserum raised againstthe enzyme Ia₁, we examined the immunological relationshipsbetween the multiple forms from cotyledons and the distributionof the enzyme in organs of maturing and germinating seeds. (Received December 25, 1989; Accepted July 11, 1990)     

In Vivo Synthesis and Turnover of alpha-Amylase in Attached and Detached Cotyledons of Vigna mungo Seeds

α-Amylase activity increased in attached cotyledons of germinated Vigna mungo seeds until the 5th day after imbibition and decreased thereafter, whereas in detached and incubated cotyledons the activity continuously increased and, at the 6th day, reached the value more than three times that of the maximum activity of attached cotyledons. Zymograms of the activities and Ouchterlony double immunodiffusion test on the activities of attached and detached cotyledons showed that the increase of activity in detached cotyledons was due to the identical enzyme as in attached tissues. α-Amylase contents, determined by single radial immunodiffusion method, changed in parallel with enzyme activity in both attached and detached cotyledons, which also suggested the de novo synthesis of α-amylase in V. mungo cotyledons.

The rate of incorporation of the label from [³H]leucine into α-amylase and the ratios of dpm in α-amylase/dpm in trichloroacetic acid-insoluble fraction did not show significant difference between attached and detached cotyledons. The results indicated that in attached cotyledons fluctuation of α-amylase activity was regulated by both synthesis and degradation of the enzyme, whereas in detached cotyledons α-amylase was synthesized and accumulated, because of low degrading activity during incubation.

     

Properties and substrate specificity of proteinase from buckwheat seeds]

A thiol proteinase was isolated from buckwheat seeds and purified 300-fold, using ammonium sulfate, acetone fractionation ion-exchange chromatography on Sephadex CM-50 and electrofocussing. The proteinase preparation obtained was found homogenous after polyacrylamide gel electrophoresis at pH 4.5. The molecular weight of the enzyme (75.000) was determined by gel-filtration through Sephadex G-100. The activation of proteinase by cysteine, 2-mercaptoethanol and dithiothreitol, its inhibition by p-chloromercurybenzoate and the absence of inhibition by diisopropyl fluorophosphate and EDTA suggest that the enzyme isolated is a thiol proteinase. The enzyme hydrolyzed many peptide bonds in the B-chain of insulin, showing high substrate specificity. The glutelin and globulin fractions of buckwheat seed proteins were hydrolyzed by the enzyme. It is assumed that the hydrolysis of reserve proteins of buckwheat seeds is the main function of the proteinase isolated.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: III. alpha-Amylase Isozymes

The formation of amylase isozymes in germinating rice (Oryza sativa) seeds was studied by isoelectric focusing on polyacrylamide gel disc electrophoresis. Time sequence comparisons of the amylase zymogram were made between extracts from gibberellic acid-treated embryoless and embryo-attached half-endosperm of rice seeds. In both cases, 4 major and 9 to 10 minor isozyme bands were detectable at the maximal stage of the enzyme induction. However, in the embryo-attached half-seeds, bands started to diminish after the 5th day of incubation, in agreement with the results of time sequence analyses of enzyme activities. Nearly identical patterns of amylase isozyme bands on a polyacrylamide gel disc electrophoresis in combination with isoelectric focusing indicate the intrinsic role of gibberellic acid in the starch breakdown in germinating rice seeds. We tentatively assign the newly synthesized enzymes to be α-amylases based on experimental results concerning the lability of the preparation on a prolonged treatment at pH 3.3 and the stability on heat treatment for 15 minutes at 70 C.     

Biochemical and immunocytochemical localization of a BAPAase in developing and mature lupin cotyledons

Summary Activity measurements and specific antibodies were used to detect and localize in developing and mature cotyledons ofLupinus albus seeds an endopeptidase, active on BAPA, previously isolated from the same seeds. Total activity and enzyme amount were highest at full seed maturation and then declined during germination. Protein bodies were isolated from mature dry cotyledons under anhydrous conditions with a yield of intact organelles of about 80% as assessed by dot blotting with antibodies to lupin legumin-like storage globulin. Activity assays on the isolated protein bodies indicated that 72% of BAPAase activity was associated with these organelles. Quantitative immunocytolocalization with antibodies to the enzyme on thin sections of mature lupin cotyledons confirmed that 75% of the enzyme was located inside the protein bodies. The possible involvement of the BAPAase in the proteolytic processing of the storage proteins during seed ontogeny is discussed.Abbreviations BAPA N-benzoyl-L-arginine-4-p-nitroanilide - DAF days after flowering - EM electron microscopy - NaPi sodium phosphate buffer - LRW London resin white - SDS sodium dodecylsulphate - PAGE polyacrylamide gel electrophoresis     

Vicilin variants and the resistance of cowpea (Vigna unguiculata) seeds to the cowpea weevil (Callosobruchus maculatus)

1. A globulin fraction prepared from the meal of Callosobruchus maculatus-resistant cowpea (Vigna wiguiculata) seeds was shown to be detrimental to this bruchid when incorporated in artificial seeds.2. The performance of C. maculatus was also shown to be strongly hindered by vicilins from resistant seeds when these storage proteins were incorporated in artificial seeds at the level of 2%.3. The purified vicilins from seeds of both resistant and susceptible cowpea varieties were shown to have the same SDS-PAGE pattern but different mobilities in non-denaturing polyacrylamide gel electrophoresis.4. These results and previous ones obtained by us (Silva and Xavier-Filho, 1991; Sales et al., 1992) strongly suggest that the resistance of cowpea seeds from the cultivar TVu 2027 and from others bred from it is associated with the presence of vicilin molecules which are refractory to digestion by bruchid midgut proteinases.     

Untersuchungen über rnasen in kotyledonen von nachgereiften und dormantenagrostemma githago-samen

Activities of RNasea were studied in cotyledons of dormant and afterripenedAgrostemma githago seeds. Activity of RNase increases during imbibition and germination. This increase in activity cannot be observed in variants which are not able to germinate (dormant seeds and seeds blocked by higher temperature). The development of RNase activities during germination cannot be inhibited by concentrations of cycloheximide or actinomycine D completely preventing phosphatase synthesis. These results may be indicative for the assumption that the increase of RNase during germination is caused by enzyme activation and not by enzyme synthesis. Cytokinins and a combination of cycloheximide and gibberellic acid stimulate the activity of RNase in dormant cotyledons, whereas neither cycloheximide nor gibberellic acid, applicated by themselves, show any effect. Cytokinins and gibberellic acid do not influence the activity of RNase of afterripened cotyledons, abscisic acid inhibits the increase of enzyme activity. There are characteristic changes in the pattern of RNases during germination revealed by polyacrylamide gel electrophoresis. The increase in RNase activity of dormant cotyledons caused by cytokinins is accompanied by obvious changes in the RNase pattern on polyacrylamide gel. Treating dormant cotyledons with cytokinins dormancy is partially overcome. In consequence of the application of cytokinins the differences in the electrophoretic RNase pattern between dormant and afterripened cotyledons can be nearly balanced.     

Influence of Axis Removal on Amino-, Carboxy- and Endopeptidase Activities in Cotyledons of Germinating Vigna mungo Seeds

Endopeptidase (azocaseolytic enzyme) and carboxypeptidase activitiesin cotyledons of germinating Vigna mungo seeds increased until3 days after the onset of imbibition and decreased thereafter.In detached and incubated cotyledons, the endopeptidase activityincreased only a little while the carboxypeptidase activitycontinued increasing even after 3 days of incubation. The activitiesof leucine-aminopeptidase and alanine-aminopeptidase, exceptfor that of one leucine-aminopeptidase isoenzyme relativelyabundantly present in unimbibed dry cotyledons, increased slightlyon the first day and declined during germination. In detachedcotyledons, the activities maintained their initial levels throughoutthe incubation period. When cotyledons were detached from germinatingseedlings on days 2 and 4 then incubated, the endopeptidaseactivity started to decrease just after removal of the axisbut the carboxypeptidase activity increased more markedly thanwhen the axis remained attached. Exogenously supplied GA₃, kinetin,IAA, or their combinations, showed no significant effect onthe developmental patterns of the endopeptidase and carboxypeptidaseactivities in cotyledons. These results are discussed in relationto the role of the axis in controlling peptidase formation incotyledons of germinating V. mungo seeds. (Received November 18, 1983; Accepted February 28, 1984)     

Association of phosphoenolpyruvate phosphatase activity with the cytosolic pyruvate kinase of germinating mung beans

The procedure of Malhotra and Kayastha ([1990] Plant Physiology 93: 194-200) for the purification to homogeneity of a phosphoenolpyruvate-specific alkaline phosphatase (PEP phosphatase) from germinating mung beans (Vigna radiata) was followed. Although a higher specific activity of 1.4 micromoles pyruvate produced per minute per milligram protein was obtained, the final preparation was less than 10% pure as judged by polyacrylamide gel electrophoresis. Attempts to further purify the enzyme resulted in loss of activity. The partially purified enzyme contained significant pyruvate kinase activity (0.13 micromole pyruvate produced per minute per milligram protein) when assayed at pH 7.2, but not at pH 8.5. The PEP phosphatase activity of the final preparation exhibited hysteresis; a lag time of 5 to 6 minutes was required before a steady-state reaction rate was attained. A western blot of the final preparation revealed an immunoreactive 57 kilodalton polypeptide when probed with monospecific rabbit polyclonal antibodies prepared against germinating castor bean cytosolic pyruvate kinase. No antigenic cross-reaction of the final preparation was observed with antibodies against castor bean leucoplast pyruvate kinase, or black mustard PEP-specific acid phosphatase. Nondenaturing polyacrylamide gel electrophoresis of the final preparation resulted in a single PEP phosphatase activity band; when this band was excised and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting, a 57 kilodalton silver-staining polypeptide was obtained that strongly cross-reacted with the anti-(cytosolic pyruvate kinase) immunoglobulin G. It is suggested that mung bean PEP-specific alkaline phosphatase activity is due to cytosolic pyruvate kinase, in which pyruvate and ortho-phosphate are formed in the absence of ADP.     

Imunohistochemical Localization of alpha-Amylase in Cotyledons of Vigna mungo Seedlings

We studied the localization of α-amylase with indirect fluorescence microscopy in transversely sectioned cotyledons of Vigna mungo seedlings. Tissue sections were fixed in periodate-lysine-paraformaldehyde and treated with anti-α-amylase immunoglobulin G followed by fluorescein isothiocyanate labeled goat anti-rabbit immunoglobulin G. α-Amylase appeared in the cells farthest from vascular bundles on the second day of growth and appeared gradually closer to the vascular bundles as growth progressed. The pattern of α-amylase appearance was similar in detached cotyledons, indicating that attachment of the embryonic axis has no effect on this pattern. However, in attached cotyledons, α-amylase disappeared from the regions where starch grains had been digested, but in detached cotyledons there was no disappearance of α-amylase, and digestion was slower than in intact cotyledons.     

Isolation and Partial Characterization of the Major Amide-Linked Conjugate of Indole-3-Acetic Acid from Phaseolus vulgaris L

A major indole-3-acetic acid conjugate from Phaseolus vulgaris seed has been isolated and partially characterized. It is a 3 kilodalton peptide with apparently 2 indole-3-acetyl moieties in amide linkage per peptide. The indole-3-acetic acid component was identified by gas chromatography-mass spectrometry and the peptide characterized by polyacrylamide gel electrophoresis, by amino acid analysis using dabsyl derivatives and by its Fourier transform-infrared spectrum. This is the first higher molecular weight amide-linked indole-3-acetic acid conjugate to be characterized from higher plants.     

Structure and Expression of a-Amylase Gene from Vigna mungo

A single copy of the a-amylase gene, composed of three intronsand four exons, was found in Vigna mungo. Examination of levelsof a-amylase and its mRNA in detached cotyledons indicated thatattachment of the embryonic axis is not required for expressionof the gene in cotyledons of germinating seeds. (Received December 21, 1993; Accepted March 14, 1994)     

Promoter regions of cysteine endopeptidase genes from legumes confer germination-specific expression in transgenic tobacco seeds

Cysteine endopeptidases, SH-EP from Vigna mungo and EP-C1 from Phaseolus vulgaris, act to degrade seed storage protein during seed germination. Using transgenic tobacco plants, expression of SH-EP and promoter activity of the EP-C1 gene were analyzed in transgenic tobacco plants. The promoters of the two genes in tobacco seeds showed germination-specific activation, although post-translational processing of SH-EP and regulatory regions of promoter of the gene for EP-C1 were found to differ between leguminous seeds and transgenic tobacco seeds.     

Acid Phosphatase in Cotyledons of Germinating Vigna mungo Seeds

A marked increase in acid phosphatase activity took place incotyledons of germinating Vigna mungo seeds. The attachmentof axis organs was not required for this development of enzymeactivity in cotyledons. DEAE-cellulose column chromatographyrevealed that the phosphatase is composed of at least threeforms. (Received August 19, 1981; Accepted October 30, 1981)     

An inhibitor of phosphoinositol kinase from ungerminated mung bean seeds

A protein inhibitor of phosphoinositol kinase has been detected in the later stages of ripening of mung bean seeds. This has been isolated and purified from the ungerminated seeds. It migrated as a single protein band when subjected to polyacrylamide gel electrophoresis. The MW of the inhibitor is approx. 86 000. The phosphoinositol kinase inhibition has been found to be dependent on the protein concentration of the purified inhibitor. It seems that 1 molecule of the inhibitor is necessary to inhibit 1 molecule of enzyme. The nature of the inhibition has been found to be non-competitive, the K_i of which is around 1·47 × 10^?6 M. The enzyme inhibitor complex dissociates on gel electrophoresis without any loss of enzyme activity.     

Heterogeneity of catalase in maturing and germinated cotton seeds

To investigate possible charge and size heterogeneity of catalase (EC 1.11.1.6) in cotton (Gossypium hirsutum L. cv Deltapine 62), extracts of cotyledons from different developmental ages were subjected to nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing. Special precautions (e.g. fresh homogenates, reducing media) were necessary to prevent artefacts due to enzyme modification during extraction and storage. When the gels were stained for enzyme activity, two distinct electrophoretic forms of catalase were resolved in extracts of maturing and mature cotton seeds. In germinated seeds, three additional cathodic forms were detected revealing a total of five electrophoretic variants. In green cotyledons, the two anodic forms characteristic of ungerminated seeds were less active; whereas, the most cathodic form was predominant. All forms of catalase were found in isolated glyoxysomes. Corresponding electrophoretic patterns were found on Western blots probed with anticatalase serum; no immunoreactive, catalytically inactive forms were detected. Western blots of sodium dodecyl sulfate-polyacrylamide gels revealed only one immunoreactive (55 kilodaltons) polypeptide in cotton extracts of all developmental ages. Results from isoelectric focusing and Ferguson plots indicate that the electrophoretic variants of catalase are charge isomers with a molecular weight of approximately 230,000.