Differential effects of abscisic acid and methyl jasmonate on endoproteinases in senescing barley leaves

Endoproteinase activity was analyzed in chloroplasts isolated from barley leaf segments incubated in the dark with various hormonal senescence effectors. As a control, the endoproteinase activity of the supernatant fraction obtained during chloroplast preparation was also analyzed. Measured against azocaseine as substrate, the endoproteinase activity in chloroplasts increased 18 fold during the induction of senescence. This rise in activity was inhibited by kinetin (the activity increased only 10 fold) and very strongly stimulated by abscisic acid (ABA) (117 fold) and methyl jasmonate (Me-JA) (57 fold). Although less so, the endoproteinase activity of the supernatant fraction, mainly vacuolar and with acid pH optimum, was affected in the same way by all three effectors. Among the five endoproteinases (EC) found in chloroplasts, EC2 and EC4 were induced after incubation in water. ABA increased the levels of EC2 and EC4 (5 fold), and induced the development of EC3 and EC5, while Me-JA totally inhibited EC2 and EC4, and induced the development of EC1. At least one of the endoproteinases, EC2, is synthesized in chloroplasts. Among the six endoproteinases found in the supernatant fraction (E), E1, E2, E3 and E5, which are very probably extrachloroplastic endoproteinases, are stimulated by ABA to varying degrees. However, Me-JA stimulates E1 to a greater extent and totally inhibits E3. The differential effects of ABA and Me-JA on chloroplast and supernatant fraction endoproteinases suggest different action mechanisms for both senescence promotors.Abbreviations ABA abscisic acid - DTT dithiothreitol - E supernatant fraction endoproteinase - EC chloroplast endoproteinase - Me-JA methyl jasmonate - PNP p-nitrophenol - SDS-PAGE polyacrylamide gel electrophoresis containing sodium dodecyl sulphate - TCA trichloroacetic acid     

Partial purification and characterization of peroxidases from the leaves of Sapindus mukorossi

Peroxidases were isolated from Sapindus mukorossi (Reetha) and partially purified using acetone precipitation, ion-exchange chromatography with a 14-fold purification, 22% recovery and a specific activity of 266?×?10³ units/mg protein. Sapindus peroxidases (SPases) showed six bands after acetone precipitation and one distinct band after ion exchange chromatography on Native-PAGE after zymography. Enzymes purified by ion exchange chromatography were loaded on Sepahdex G-50 superfine column and their molecular weight was reported to be 25?kDa. They showed temperature optima at 50°C and pH optima at 5.0.?km for SPases was reported to be 1.05?mM and 0.186?mM for guaiacol and H₂O₂ respectively. The V_max/K_m value for o-dianisidine was 449 while for H₂O₂ it was 5?×?10⁵. Protocatechuic acid acts as a potent inhibitor for SPases (6.0% relative activity at 4.5???M) but ferulic acid inhibits its activity at a much lower concentration (0.02???M). Enzymes were stimulated by metal cations like Cu²⁺, Ca²⁺ (145, 168; percentage relative activity respectively) and showed mild inhibition (up to 20%) with Mn²⁺ and Mg²⁺. Alanine stimulated the enzyme activity (up to 33%; at 0?C100???M) while other amino acids like cysteine, methionine, tryptophan and tyrosine inhibited the SPases (13?C57% at 0?C100???M).     

Partial purification and characterization of aspartate aminotransferases from seedling oat leaves.

As relatively little information is available on the properties of aspartate aminotransferase from photosynthetic tissue, isolation and characterization of the two major electrophoretically distinct forms of this enzyme from seedling oat leaf homogenates were undertaken. These two forms are designated I for the more anionic form and II for the less anionic form. Form I, 80 to 90% of the total activity, has been purified to a specific activity of 120 mumol/min/mg of protein (1100-fold) and is estimated to be 90 to 95% homogeneous, as judged by analytical polyacrylamide gel electrophoresis. Form II, 10 to 20% of the total activity, has been purified to a specific activity of approximately 6 mumol/min/mg of protein (300-fold). Both forms exhibit optimal activity at pH 7.5. Michaelis constants do not differ greatly between forms I and II and are similar to those reported for the pig heart cytosolic enzyme as well as aspartate aminotransferase from other plant sources. A molecular weight of 130,000 for the purified aspartate aminotransferase I was estimated by sedimentation equilibrium centrifugation; molecular weights of the two forms are similar as estimated by sucrose density gradient centrifugation. No activation by pyridoxal phosphate has been observed during purification.     

Partial purification and characterization of the mRNA for alpha-amylase from barley aleurone layers

The poly(A)-containing mRNA from barley aleurone layers pretreated with gibberellic acid has been purified by phenol-chloroform extraction and repeated oligo[d(pT)]-cellulose chromatography. This RNA has been translated in both the wheat germ and reticulocyte lysate in vitro translation systems with greater than 50% of the synthesized protein being α-amylase. The mRNA for α-amylase has been further purified by dimethylsulfoxide-formamide-sucrose density gradient centrifugation and by gel electrophoresis. By these methods, its molecular weight has been determined to be 580,000.     

Partial purification and biochemical characterization of alkaline 5'-phosphodiesterase from barley malt sprouts

An alkaline 5-phosphodiesterase (5-PDE) from barley (Hordeum distichum) malt sprouts was partially purified by thermal treatment and acetone precipitation to diminish phosphomonoesterase (PME) activity. 5-PDE was purified 40-fold to a specific activity of 30 U mg^–1 protein with a final yield of about 32%. With synthetic substrate, the enzyme had an optimum pH of 8.9, maximum activity at 70 °C over 10 min, and a Km of 0.26 mM. The partially purified enzyme was activated by 10 mM Mg²⁺ up to 168% of the original activity, while Zn²⁺, Mn²⁺ and Cu²⁺ ions, chelating agent (EDTA) and NaN₃ (1–10 mM), and 5-ribonucleotides (1–5 mM) were inhibitory. Final enzyme preparation was stable over 8 d at 4 °C), at 70 °C for up to 120 min and without loss of activity over 90 d at –18 °C.     

Glycerate kinase from spinach leaves: Partial purification, characterization and subcellular localization

Spinach ( Spinacia oleracea L. cv. Melody hybrid) leaf glycerate kinase (EC 2.7.1.31) was partially purified and characterized. The enzyme did not exhibit any absolute stereospecificity towards the two enantiomers of glycerate, but the affinity for the D-isomer was 15-fold greater. The enzyme exhibited a broad pH optimum of 7.5–9.0 and a requirement for divalent cation, satisfied by Mg₂₊. The reaction product was identified as 3-phosphoglyceric acid. The observed high glycerate kinase activity together with its strategic localization exclusively in the chloroplast stroma are considered adequate for an efficient coupling of photosynthetic and photorespiratory carbon pathways.     

Partial purification of gibberellin oxidases from spinach leaves

Four enzyme activities catalyzing the following oxidative steps in the gibberellin (GA) biosynthetic pathway have been extracted from spinach (Spinacia oleracea L.) leaves after exposure to 8 long days: GA₁₂ → GA₅₃ → GA₄₄ → GA₁₉ → GA₂₀. Two of these, GA₅₃ oxidase and GA₁₉ oxidase, were separable from the other two, GA₄₄ oxidase and GA₁₂ 13-hydroxylase, by anion exchange high performance liquid chromatography (HPLC). Apparent molecular weights of the four enzymes as determined by gel filtration HPLC are: GA₁₂ 13-hydroxylase, 28,400; GA₅₃ oxidase, 42,500; GA₄₄ oxidase, 38,100; GA₁₉ oxidase, 39,500. GA₄₄ oxidase was purified approximately 100-fold in 0.3% yield by a combination of ammonium sulfate fractionation, anion exchange HPLC, phenyl-Sepharose chromatography and gel filtration HPLC.     

Partial purification and properties of nuclease 1 from barley malt

A sugar-unspecific nuclease has been purified 260-fold from barley malt diastase. The enzyme, a glycoprotein of 37 000 MW, is highly active on single-stranded polynucleotides at pH 5–6. The nuclease is inhibited by several adenine nucleotides, and it binds weakly to NADP-agarose and ATP-agarose.     

Partial purification and characterization of the major endoamylase of mature pea leaves

An endoamylase from leaves of pea (Pisum sativum) was purified to near homogeneity by affinity chromatography and ultrafiltration with a yield of about 20%. The purified protein had a specific activity of 686 to 1300 units per milligram protein. Molecular weights of 45 and 41 kilodalton were determined by SDS-PAGE and molecular sieve chromatography, respectively. The purified protein exhibited an action pattern commensurate with that of an endoamylase and exhibited properties indicating it to be very similar to cereal grain α-amylases (calcium requirement, stability to heat, lability to low pH-values, insensitivity to sulfhydryl reagents). Leaf frationation studies indicated that the enzyme was not primarily located in assimilatory mesophyll cells. Chloroplasts isolated from the leaves were found to contain endoamylases, but their activities represented only a small proportion of the total amylolytic potential of the leaf and reflected for the most part properties quite different from those exhibited by the purified enzyme.     

Xanthophyll cycle activity in detached barley leaves senescing under dark and light

Senescence-induced changes in the xanthophyll cycle activity and chlorophyll (Chl) fluorescence parameters were compared in detached barley (Hordeum vulgare L.) leaf segments kept for 6 d in darkness or under continuous white light (90 mol m^–2 s^–1). Before detachment of the leaf segments, the plants were grown at periodic regime [12 h light (90 mol m^–2 s^–1)/12 h dark]. The de-epoxidation state of the xanthophyll cycle pigments (DEPS) in the leaf samples was determined immediately (the actual DEPS), after 1 h of dark-adaptation (the residual DEPS), and during 14 min of a high-irradiance (HI) exposure (500 mol m^–2 s^–1) (HI-induced DEPS). In the light-senescing segments, senescence was delayed pronouncedly compared to dark-senescing ones as the Chl content, the photosystem 2 photochemistry, and electron transport processes were highly maintained. Further, the actual DEPS increased, probably due to the increased mean photon dose. The HI-induced increase in the DEPS was stimulated in the light-senescing segments, whereas it was slowed down in the dark-senescing ones. However, after the 14 min HI-exposure of the dark-senescing segments the HI-induced DEPS was not markedly lower than in the mature leaves, which indicated the maintenance of the xanthophyll cycle operation.     

Partial purification and characterization of an ascorbate-reducible b-type cytochrome from the plasma membrane of Arabidopsis thaliana leaves

Summary.  The plant plasma membrane (PM) contains more than one b-type cytochrome. One of these proteins has a rather high redox potential (can be fully reduced by ascorbate) and is capable of transporting electrons through the PM. Four genes encoding proteins with considerable homology to the sequences of cytochrome b ₅₆₁ proteins in the animal chromaffin granule membrane have recently been identified in the genome of Arabidopsis thaliana. In order to characterize the cytochrome b ₅₆₁ located in the Arabidopsis PM, first PM vesicles were purified by aqueous polymer two-phase partitioning from the leaves of 9-week-old A. thaliana. PM proteins were solubilized by nonionic detergent, and the fully ascorbate-reducible b-type cytochrome was partially purified by anion-exchange chromatography steps. Potentiometric redox titration of the fraction, containing the fully ascorbate-reducible b-type cytochrome after the first anion-exchange chromatography step, revealed the presence of two hemes with redox potentials of 135 mV and 180 mV, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions containing the fully ascorbate-reducible b-type cytochrome after the second anion-exchange chromatography step revealed the presence of a single polypeptide band at about 120 kDa. However, heat treatment (15 min, 90 °C) before electrophoresis was able to split the 120 kDa band into two bands with molecular masses of about 23 and 28 kDa. These values are lower than the apparent molecular mass for the fully ascorbate-reducible b-type cytochrome purified from Phaseolus vulgaris hypocotyls (about 52 kDa) but are in good agreement with those characteristic for the cytochrome b ₅₆₁ proteins purified from chromaffin granule membranes (about 28 kDa) and the four polypeptides predicted from the Arabidopsis genome (24–31 kDa). Received May 4, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Institute of Biophysics, BRC, Hungarian Academy of Sciences, POB 521, 6701 Szeged, Hungary.     

Partial purification and characterization of bacteriocin from Yersinia kristensenii

A raw milk bacterial isolate, identified as Yersinia kristensenii was found to produce a bacteriocin which was inhibitory to Yersinia enterocolitica but not to other selected species of Yersinia or Gram-negative bacteria. Maximum production of bacteriocin was obtained when the organism was grown in shake culture at 28°C. Mitomycin C at a concentration of 0.5 μg ml^-1 induced bacteriocin production. The bacteriocin was partially purified and characterized by ammonium sulphate fractionation and gel filtration. The bacteriocin was completely inactivated when treated with proteolytic enzymes (trypsin and chymotrypsin). Bacteriocin activity was heat-resistant and it retained some of its activity after 5 min at boiling temperature. A total of 15 bacteriocin sensitive-suspected food isolates were further identified biochemically as Yersinia enterocolitica and a non-sensitive isolate was identified as Yersinia intermedia.     

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis

Brettanomyces is the major microbial cause for wine spoilage worldwide and causes significant economic losses. The reasons are the production of ethylphenols that lead to an unpleasant taint described as 'phenolic odour'. Despite its economic importance, Brettanomyces has remained poorly studied at the metabolic level. The origin of the ethylphenol results from the conversion of vinylphenols in ethylphenol by Brettanomyces hydroxycinnamate decarboxylase. However, no information is available on the vinylphenol reductase responsible for the conversion of vinylphenols in ethylphenols. In this study, a vinylphenol reductase was partially purified from Brettanomyces bruxellensis that was active towards 4-vinylguaiacol and 4-vinylphenol only among the substrates tested. First, a vinylphenol reductase activity assay was designed that allowed us to show that the enzyme was NADH dependent. The vinylphenol reductase was purified 152-fold with a recovery yield of 1.77%. The apparent K(m) and V(max) values for the hydrolysis of 4-vinylguaiacol were, respectively, 0.14 mM and 1900 U mg(-1). The optimal pH and temperature for vinylphenol reductase were pH 5-6 and 30 degrees C, respectively. The molecular weight of the enzyme was 26 kDa. Trypsic digest of the protein was performed and the peptides were sequenced, which allowed us to identify in Brettanomyces genome an ORF coding for a 210 amino acid protein.     

Partial purification, characterization and properties of two isoforms of glutamine synthetase from Pennisetum glaucum L. leaves

Two isozymes of glutamine synthetase GS1 and GS2 were partially purified from Pennisetum glaucum leaves by ion-exchange and gel filtration chromatography and their kinetic and regulatory properties were studied using semisynthetase assay of GS. Mg2+ was the most effective cation for activity of both the isozymes; however, it could be efficiently replaced by Co2+. The pH optima for GS1 and GS2 were 7.0 and 8.0, respectively. GS1 exhibited maximum activity at 42 degrees C, with activation energy of 18 KJ mol(-1) and a Q10 of 3.0, whereas GS2 showed maximum activity at 50 degrees C, with activation energy of 40 KJ mol(-1) and Q10 of 2.25. GS1 was more thermostable than GS2. The Km value for Mg2+ of GS1 was 2-fold higher than GS2; however, these isozymes did not differ much in their affinity for other substrates. Alanine, serine and glycine lowered GS1 and GS2 activities, whereas cysteine enhanced their activities with a more pronounced effect on GS2. Serine inhibited the activity of both the isoforms in a competitive-manner, whereas alanine was a non-competitive inhibitor, with respect to glutamate. AMP and ADP were competitive inhibitor with respect to ATP for both the isozymes.     

Partial purification and characterization of the endotoxin from Aspergillus fumigatus

Summary The endotoxin fromAspergillus fumigatus was studied and appears to be protein in character. Attempts to purify the material by fractional precipitation with acetone resulted in increase of hemolytic and toxic potencies. The hemolytic activity of the toxin is stable from pH 3.6 to 8.4.Electrophoretic analysis enabled the assignment of hemolytic activity to a definite electrophoretic component.Chromatography with DEAE cellulose using gradient elution effected increase in hemolytic activity. The bioassay for toxic activity is less quantitative than the assay for the hemolytic activity.Column electrophoresis confirmed the assignment of hemolytic potency to an electrophoretic component. Recovery of activity was low in this method.Both the hemolytic and toxic activities occur in the same electrophoretic component; however, there is evidence that they may be separate components.Adapted from a Thesis submitted byEvelyn M. Rau to Northwestern University in partial fulfillment of the M. S. degree in Biochemistry, June, 1960.Supported by Grant E-2435 from the National Institute of Allergy and Infectious Diseases.