The pH-induced glycosylation of secreted phosphatases is mediated in Aspergillus nidulans by the regulatory gene pacC-dependent pathway

In this communication, we show that the pacC(c)14 mutation drastically reduced the mannose and N-acetylglycosamine content of the pacA-encoded acid phosphatase secreted by the fungus Aspergillus nidulans when grown at 22 degrees C, pH 5.0, compared to a control strain. The staining after PAGE was not observed for the pacA-encoded acid phosphatase, while the palD-encoded Pi-repressible alkaline phosphatase had an altered electrophoretic mobility. In addition, the secreted acid phosphatase also had a reduced number of isoforms visualized by staining after IEF and glycosylation had a protective effect against its heat inactivation. We also show that a full-length version of gene pacC-1 cloned from Neurospora crassa complemented the pacC(c)14 mutation of A. nidulans, including the remediation of both the acid and alkaline Pi-repressible phosphatases secreted at pH 5.0, which indicates that glycosylation of secreted phosphatases is mediated in A. nidulans by the conserved PacC pathway that governs pH-responsive gene expression.     

Induction of chalcone synthase in cell suspension cultures of carrot (Daucus carota L. spp. sativus) by ultraviolet light: evidence for two different forms of chalcone synthase

Two cell lines of carrot (Daucus carota L. spp. sativus), grown as cell-suspension cultures in the dark, were irradiated with ultraviolet light (315–420 nm) 10 d after the onset of cultivation. Chalcone synthase (CHS) enzyme activity was induced in both cell lines. Anthocyanin synthesis was only stimulated in the anthocyanin-containing cell line DCb. Parallel to the increase in CHS activity there was an increase with time in the amount of one CHS form with an isoelectric point of 6.5 and a molecular weight of 40 kilodaltons (kDa) per subunit. Whereas the anthocyanin-free cell line DCs failed to accumulate anthocyanin, it did stimulate another CHS form with an isoelectric point at pH 5.5 and a molecular weight of 43 kDa per subunit. Both enzyme activities could be separated by isoelectric focusing and stabilized using sodium hydrosulfite as an oxidation protectant. In carrot plants, CHS was restricted to the dark purple petals of the inflorescence (40 kDa) and to the leaves (43 kDa).Abbreviations BSA bovine serum albumin - CHS chalcone synthase - IEF isoelectric focusing - kDa kilodaltons - KP_i potassium phosphate buffer - PAL phenylalanine ammonialyase - pI isoelectric point - UV ultraviolet     

Molecular analysis of the packaging signal in bacteriophage CP-T1 of Vibrio cholerae

Summary We have previously identified a unique site, pac, from which packaging of precursor concatameric viral DNA into proheads starts during the maturation process of bacteriophage CP-T1. The direction of this packaging was determined from restriction enzyme cleavage patterns of CP-T1 DNA. A restriction enzyme generated fragment containing pac was cloned and the surrounding DNA region sequenced. Analysis of the nucleotide sequence revealed numerous repeat regions related to the consensus sequence PuagttGAT.AAT.aa.t. Within the sequenced region an open reading frame encoding a 12260 M_r protein was also identified. This protein appears to share homology with the binding domains of known DNA binding proteins and may represent a putative Pac terminase possessing the specific endonuclease activity required for cleavage at the pac site. Minicell analysis of deletion derivatives of the pac-containing clone revealed a protein of approximately 12900 M_r encoded within this same region, confirming that this Pac protein is phage encoded.     

Allotypes of mouse complement component C6 in inbred strains and some wild populations

This collaborative work was undertaken to resolve discrepancies in reports of the number of forms of complement component C6 present in the circulation of mice from various inbred strains. Plasma C6 was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by isoelectric focusing (IEF), and C6 band patterns were developed by electroblotting and immunoprobing. Results of C6 allotyping of mice from 36 strains confirmed that while 20 strains (prototype strain BALB/c) possessed only one relative mass (M _r) form which typed C6A1 on IEF, the other 16 strains all possessed more than one C6 M _r form. Moreover, IEF analysis demonstrated additional polymorphic differences; among these 16 strains, 11 typed C6A l B l like the prototype strain CBA, the AKR and RF/J strains typed C6A2B2, and the Japanese MOM strain as well as the C57BR/cdJ and C57L/J strains possessed two forms with IEF mobilities intermediate between C6A1B1 and C6A2B2. These will now be referred to as C6A3B3. Thus, a total of four different mouse C6 haplotypes have been identified.Testing C6 allotypes in a limited number of wild mice revealed that haplotypes found in inbred strains of Western or Eastern origin tend to reflect haplotypes of the wild mice from Europe or Japan, respectively.     

Analysis of cytoplasmic membrane from wild type and mutant Paracoccus denitrificans containing excess nitrate reductase protein and cytochrome b

Cytoplasmic membranes were isolated from wild type and mutants strain M-1 of Paracoccus denitrificans grown with low aeration to promote synthesis of nitrate reductase protein and cytochrome b. The presence of 10-100-fold excess of nitrate reductase in the wild type or the corresponding enzymically inactive protein in the mutant did not significantly affect respiratory oxidase activities with NADH, succinate or TMPD-ascorbate as electron donor. A cytochrome b-nitrate reductase complex was resolved by isoelectric focussing of Triton X-100 solubilized membranes from the wild type grown with azide and from the mutant, whereas the enzyme complex from nitrate-grown wild type was not resolved from cytochrome c. Preparations from azideinduced wild type or from the mutant could be a suitable source of the cytochrome b associated with nitrate reductase for more detailed studies.Non standard abbreviations IEF isoelectric focussing - TMPD N, N, N, N-tetramethylphenylenediamine - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Purification and characterization of a NAD+-dependent secondary alcohol dehydrogenase from propane-grown Rhodococcus rhodochrous PNKb1

NAD⁺-linked primary and secondary alcohol dehydrogenase activity was detected in cell-free extracts of propane-grown Rhodococcus rhodochrous PNKb1. One enzyme was purified to homogeneity using a two-step procedure involving DEAE-cellulose and NAD-agarose chromatography and this exhibited both primary and secondary NAD⁺-linked alcohol dehydrogenase activity. The Mr of the enzyme was approximately 86,000 with subunits of Mr 42,000. The enzyme exhibited broad substrate specificity, oxidizing a range of short-chain primary and secondary alcohols (C₂–C₈) and representative cyclic and aromatic alcohols. The pH optimum was 10. At pH 6.5, in the presence of NADH, the enzyme catalysed the reduction of ketones to alcohols. The K _m values for propan-1-ol, propan-2-ol and NAD were 12 mM, 18 mM and 0.057 mM respectively. The enzyme was inhibited by metal-complexing agents and iodoacetate. The properties of this enzyme were compared with similar enzymes in the current literature, and were found to be significantly different from those thus far described. It is likely that this enzyme plays a major role in the assimilation of propane by R. rhodochrous PNKb1.Abbreviations HPLC high performance liquid chromatography - DEAE diethyl amino ethyl - IEF isoelectrofocusing - NTG nitrosoguanidine - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - pI isoelectric point     

Detection of elastase activity with a zymogram method after isoelectric focusing in polyacrylamide gel

A zymogram method for detecting elastase activity following isoelectric focusing in polyacrylamide gel is described. After enzyme activity has been visualized, the gel itself is available for protein staining and for analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in second dimension. The zymogram method is suitable for detecting microgram amounts of elastase and has one step only. It can be used with the purified enzyme as well as with crude extracts of tissue containing elastases showing activity toward succinyl-(Ala)₃-p-nitroanilide. By this method a major component of elastase in both porcine and rat pancreas was detected. In addition, two forms of elastase with isoelectric points of 8.2 and 8.8, respectively, were identified in rat leukocyte extracts.     

Purification and characterization of sialidase fromClostridium sordellii G12

Sialidase secreted by the urease-positiveClostridium sordellii strain G12 was isolated from culture medium and purified to apparent homogeneity as estimated by Fast Protein Liquid Chromatography (FPLC) and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). For this purpose, ion-exchange chromatography, gel filtration, isoelectric focusing, and FPLC on ion-exchange resin and gel filtration materials were used. The sialidase was purified 159 300-fold from 5 l of culture medium, yielding 9 g of enzyme protein with a specific activity of 480 U/mg. For the denatured (SDS-PAGE) and native (FPLC) sialidase relative molecular masses of 40 000 and 38 500 Da, respectively, were estimated. The substrate specificity, kinetic data, and pH-optimum of the enzyme are similar to those of other bacterial sialidases. The influences of salt or serum proteins on enzyme activity are of interest.Abbreviations MU-Neu5Ac 4-methylumbelliferyl -d-N-acetylneuraminic acid - Ganglioside GD1a IV³NeuAc, ll³NeuAc-GgOse₄Cer - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Glutamine synthetase oligomers and isoforms in sugarbeet (Beta vulgaris L.)

The -amino-N compounds that accumulate in the thickening storage root of sugarbeet (Beta vulgaris L.) were synthesized in the leaves (NO ₃ ^– nutrition) and also in the lateral roots (NH ₄ ⁺ nutrition). Ammonium stimulated glutamine synthetase (GS, EC 6.3.1.2) activity, especially in the lateral roots. With non-denaturing polyacrylamide-gel isoelectric focussing, simultaneously active charge-isomers of GS were separated in both leaves and roots. The leaf isoforms were active in an octameric and also in a tetrameric form. In the root only octameric isoforms were found. The tetramer was more active than the octamer in the leaf blade and vice versa in the leaf stem. Only the tetramer needed -mercaptoethanol for activity stabilization in vitro. A reactivation, however, of an inactive tetramer by the addition of thiol/thioredoxin was not possible. The same isoforms of GS were separated in different organs of sugarbeet but with different patterns of relative activity. The activity pattern depended also on the N-source of the plant. With increasing age of the plant the number of active GS isoforms declined in both leaves and roots although the in-vitro activity remained unchanged (NO ₃ ^– -fed plants) or even increased (NH ₄ ⁺ -fed plants).Abbreviations GS glutamine synthetase (E.C. 6.3.1.2.) - IEF isoelectric focussing - PAGE polyacrylamide gel electrophoresis This work was supported by a grant from Bundesministerium für Forschung und Technologie and by Kleinwanzlebener Saatzucht AG, Einbeck.     

Effect of aPMR1 disruption on the processing of heterologous glycoproteins secreted in the yeastSaccharomyces cerevisiae

TheSaccharomyces cerevisiae PMR1 gene encodes a Ca²⁺-ATPase localized in the Golgi. We have investigated the effects ofPMR1 disruption inS. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human α₁-antitrypsin (α₁-AT), human antithrombin III (ATHIII), andAspergillus niger glucose oxidase (GOD). Thepmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite a lower growth rate of thepmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from thepmr1 mutant compared to that of the wild-type strain. Thepmr1 mutant strain secreted α₁-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the wild-type strain. Furthermore, the core-glycosylated forms secreted in thepmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in themnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-α1,3-mannose antibody revealed that GOD secreted in thepmr1 mutant did not have terminal α1,3-linked mannoses unlike those secreted in themnn9 mutant and the wild type strains. The present results indicate that thepmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose outer chains.     

Purification and properties of a carboxylesterase from germinated finger millet (Eleusine coracana Gaertn.)

A carboxylesterase (EC 3.1.1.1) was purified from germinated finger millet by ammonium sulphate fractionation, diethylaminoethyl-cellulose chromatography and Sephadex G-200 filtration. The homogeneity of the enzyme was established by Polyacrylamide gel electrophoresis, isoelectric focussing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme has a single polypeptide chain with a molecular weight of 70,000. The amino acid analysis of the purified enzyme revealed that it contained a greater number of neutral and acidic, compared to, basic amino acid residues. The isoelectric pH of the enzyme was found to be 5·1. Studies with different organophosphate and carbamate inhibitors showed that this enzyme was more sensitive to organophosphate inhibitors than carbamates. The rate constantsk _i andl ₅₀ for different inhibitors were calculated. The product inhibition studies with this enzyme showed linear competitive inhibition with acetate and linear noncompetitive inhibition with 1-naphthol     

Purification and characterization of a cellulase-free xylanase of a moderate thermophile Bacillus licheniformis A99

Two cellulase-free xylanases were secreted by a thermophile, Bacillus licheniformis A99. Of the two, the predominant one was purified to homogeneity. The enzyme was optimally active at 60 °C, pH 6–7.5, and had a molecular weight of about 45 KDa and isoelectric point of 7.0 ± 0.2. The K _m (for birchwood xylan) and V _max were 3.33 mg/ml and 1.111 mmols mg^–1 protein min^–1 respectively. The half-life of the enzyme was 5 h at 60 °C. All cations except Hg²⁺ and Ag⁺ as well as EDTA were well tolerated and did not adversely affect xylanase activity. However, SDS inhibited the enzyme activity. The release of reducing sugars from unbleached commercial pulp sample on treatment with the enzyme indicated its potential in prebleaching of paper pulp. The enzyme caused saccharification of lignocellulosics such as wheat bran, wheat straw and sawdust. This is the first report on purification and characterization of cellulase-free xylanase from a moderate thermophile Bacillus licheniformis.     

Isolation and biochemical characterization of grape malic enzyme

Malic enzyme (ME=L-malate: NADP oxidoreductase; E.C. 1.1.1.40) was extracted by Triton X-100-induced resolubilization of enzyme proteins which denaturize spontaneously upon homogenization of grape berry material. The purification procedure included fractionating with (NH₄)₂SO₄, preparative IEF, and Sephadex G-100 chromatography. ME was identified by TLC of the radioactive product after supplementing the assay mixture with [¹⁴C]malate. Cofactor dependence, pH-optimum and affinities for substrates and cosubstrates were determined. Enzymic pI was found to be 5.8, the Hill coefficients range from 1 to 3. In malate decarboxylating direction at pH 7.4, grape ME displayed positive cooperativity toward the substrate, the curve approaching normal Michaelis-Menten-kinetics at pH 7.0. Substituting Mn²⁺ for Mg²⁺ not only increased maximal turnover rates, but also enzymic affinity for malate. These features were considered indicative of the regulatory properties of the enzyme. Their relevance for grape malate metabolism and fruit ripening is discussed.Abbreviations EDTA ethylenediaminetetraacetic acid - IFF isoelectric focusing - MDH malate dehydrogenase - ME malic enzyme - OAA oxaloacetic acid - PAG polyacrylamide gel - TCA trichloroacetic acid - TLC thin layer chromatography     

Investigations on the antioxidative defence responses to NaCl stress in a mangrove, Bruguiera parviflora: Differential regulations of isoforms of some antioxidative enzymes

Two-month-old healthy seedlings of a true mangrove, Bruguiera parviflora, raised from propagules in normal nursery conditions were subjected to varying concentrations of NaCl for 45 d under hydroponic culture conditions to investigate the defence potentials of antioxidative enzymes against NaCl stress imposed oxidative stress. Changes in the activities of the antioxidative enzymes catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POX), glutathione reductase (GR) and superoxide dismutase (SOD) were assayed in leaves to monitor the temporal regulation. Among the oxidative stress triggered chemicals, the level of H₂O₂ was significantly increased while total ascorbate and total glutathione content decreased. The ratio of reduced to oxidized glutathiones, however, increased due to decreased levels of oxidized glutathione in the leaf tissue. Among the five antioxidative enzymes monitored, the APX, POX, GR and SOD specific activities were significantly enhanced at high concentration (400 mM NaCl), while the catalase activities declined, suggesting both up and downregulations of antioxidative enzymes occurred due to NaCl imposed osmotic and ionic stress. Analysis of the stress induced alterations in the isoforms of CAT, APX, POX, GR and SOD revealed differential regulations of the isoforms of these enzymes. In B. parviflora one isoform of each of Mn-SOD and Cu/Zn-SOD while three isoforms of Fe-SOD were observed by activity staining gel. Of these, only Mn-SOD and Fe-SOD2 content was preferentially elevated by NaCl treatment, whereas isoforms of Cu/Zn-SOD, Fe-SOD1 and Fe-SOD3 remained unchanged. Similarly, out of the six isoforms of POX, the POX-1,-2,-3 and -6 were enhanced due to salt stress but the levels of POX-4 and -5 remained same as in control plants suggesting preferential upregulation of selective POX isoforms. Activity staining gel revealed only one prominent band of APX and this band increased with increased salt concentration. Similarly, two isoforms of GR (GR1 and GR2) were visualized on activity staining gel and both these isoforms increased upon salt stress. In this mangrove four CAT-isoforms were identified, among which the prominent CAT-2 isoform level was maximally reduced again suggesting differential downregulation of CAT isoforms by NaCl stress. The results presented in this communication are the first report on the resolutions of isoforms APX, POX and GR out of five antioxidative enzymes studied in the leaf tissue of a true mangrove. The differential changes in the levels of the isoforms due to NaCl stress may be useful as markers for recognizing salt tolerance in mangroves. Further, detailed analysis of the isoforms of these antioxidative enzymes is required for using the various isoforms as salt stress markers. Our results indicate that the overproduction of H₂O₂ by NaCl treatment functions as a signal of salt stress and causes upregulation of APX, POX, GR and deactivations of CAT in B. parviflora. The concentrations of malondialdehyde, a product of lipid peroxidation and lipoxygenase activity remained unchanged in leaves treated with different concentrations of NaCl, which again suggests that the elevated levels of the antioxidant enzymes protect the plants against the activated oxygen species thus avoiding lipid peroxidation during salt stress.     

A cytochrome cd 1-type nitrite reductase mediates the first step of denitrification in Alcaligenes eutrophus

Respiratory nitrite reductase (NIR) has been purified from the soluble extract of denitrifying cells of Alcaligenes eutrophus strain H16 to apparent electrophoretic homogeneity. The enzyme was induced under anoxic conditions in the presence of nitrite. Purified NIR showed typical features of a cytochrome cd ₁-type nitrite reductase. It appeared to be a dimer of 60 kDa subunits, its activity was only weakly inhibited by the copper chelator diethyldithiocarbamate, and spectral analysis revealed absorption maxima which were characteristic for the presence of heme c and heme d ₁. The isoelectric point of 8.6 was considerably higher than the pI determined for cd ₁ nitrite reductases from pseudomonads. Eighteen amino acids at the N-terminus of the A. eutrophus NIR, obtained by protein sequencing, showed no significant homology to the N-terminal region of nitrite reductases from Pseudomonas stutzeri and Pseudomonas aeruginosa.     

Occurrence of two isoforms of glutathione reductase in the green alga Chlamydomonas reinhardtii

Two isoforms (isoenzymes) of glutathione reductase (NADPH: oxidized glutathione oxidoreductase, EC 1.6.4.2; GR) were clearly resolved when enzyme preparations partially purified from the unicellular alga Chlamydomonas reinhardtii were subjected to column chromatofocusing in the pH range from 8 to 4. One isoform (GR I) exhibited an almost electroneutral isoelectric point (pI, 6.9–7.1) and the other (GR II) was a very acidic protein (pI, 4.7–4.9). Both GRs are, however, homodimeric flavoproteins with similar molecular masses of approx. 127 kDa. Cross-reaction with an antibody against the cyanobacterial GR allowed determination of their subunit molecular masses by Western blotting after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a value of 66 kDa being estimated in both cases. The two algal GR isoforms showed similar K _m values for the oxidized form of glutathione (approx. 50 M). However, the K _m values for NADPH were different, being 7 M and 28 M for GR I and GR II, respectively. The two isoforms also differed in their optimum pH. Thus, whereas GR I showed a clear maximum at neutral pH, GR II exhibited a broader optimum around pH 8.5 and was more active in the alkaline range. The relative contribution of the two isoforms to the total activity in enzyme preparations of cells disrupted by two different methods indicates that GR I should be a cytoplasmic isoform and GR II a plastidic isoform. The physiological roles of the GR isoenzymes found in Chlamydomonas are discussed and some of their properties compared with those of GRs isolated from other photosynthetic organisms.Abbreviations GSSG glutathione, oxidized form - GR NAD-PH-glutathione reductase (EC 1.6.4.2) - G3P glyceraldehyde-3-phosphate - pI isoelectric point - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate This work was supported in part by grants NO. PB 87–401, PB 90–99 and BIO 91–1078 of the DGICYT (Ministerio de Educatión y Ciencia, Spain) and the Autonomous Government of Andalusia (Spain). Postdoctoral aid from the Alexander von Humboldt Foundation (Bonn, FRG) to A.S. is also acknowledged.     

Isolation and characterization of Pichia stipitis mutants with enhanced xylanase activity

Pichia stipitis strain NRRL Y-11,543 was mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) to improve xylanolytic activity. A total of 20,000 mutants were screened for xylanase overproduction by observing the clear zones around the colonies on remazol-briliant-blue-xylan (RBB-xylan)-containing agar. Of 94 mutants isolated 11 of them were found to have enhanced xylanase activity compared to the parental strain. The most active mutant NP54376 had superior properties to the wild type which included: double the enzyme activity of wild type, a shorter generation time of 2.22 h compared to 3.13 h when grown on xylan, and an enhanced growth and yield of xylanase when low levels of xylose were added to the medium. Zymogram analysis of the crude enzyme preparations from both NP54376 and the wild type by isoelectric focusing showed multiple bands ranging between pI 4.2 and 7.4. No significant difference was observed in the K _m and V _max values of the parental strain and NP54376. K _m and V _max values of xylanase for birchwood xylan were 4.2 mg ml⁻¹ and 0.08 μmol min⁻¹ mg⁻¹ of protein, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation of <Emphasis Type="BoldItalic">Serratia marcescens</Emphasis> as a chondroitinase-producing bacterium and purification of a novel chondroitinase AC

A strain of Serratia marcescens that produced chondroitinase was isolated from soil. It produced a novel chondroitinase AC, which was purified to homogeneity. The enzyme was composed of two identical subunits of 35 kDa as revealed by SDS-PAGE and gel filtration. The isoelectric point for the chondroitinase AC was 7.19. Its optimal activity was at pH 7.5 and 40 °C. The purified enzyme was active on chondroitin sulfates A and C and hyaluronic acid, but was not with chondroitin sulfate B (dermatan sulfate), heparin or heparan sulfate. The apparent K_m and V_max of the chondroitinase AC for chondroitin sulfate A were 0.4 mg ml^–1 and 85 mmol min^–1 mg^–1, respectively, and for chondroitin sulfate C, 0.5 mg ml^–1 and 103 mmol min^–1 mg^–1, respectively.     

Cytosolic and cell-wall-bound acid invertases from leaves of Urtica dioica L.: a comparison

Two different forms of acid invertase (EC 3.2.1.26) were extracted from expanding leaves of the stinging nettle (Urtica dioica L.). One form was soluble and could be localized within the cytosol, whereas the other was ionically bound to the cell-wall and could not be detected in protoplasts. Both forms were purified, the latter to homogeneity. Western blotting with antibodies against the pure enzyme from cell walls was positive with the cell-wall enzyme but negative with the soluble form of acid invertase. Both forms are glycoproteins with identical molecular weights of 58 kDa. The K_m values for sucrose (raffinose) are 5 mM (4.8 mM) for the soluble and 1.2 mM (3.6 mM) for the cell-wall-bound enzyme. The pH optimum of the latter is slightly more acidic (4.5) than that of the soluble invertase (5.5). Both forms could easily be distinguished by their isoelectric points which were determined at pH 4.6 for the soluble and pH 9.3 for the wall-bound enzyme. When extraction and purification were carried out in the absence of protease inhibitors, both acid invertases showed microheterogeneity (multiple forms). However, with benzamidine and phenylmethylsulfonylfluoride as protease inhibitors each invertase produced only one protein band upon isoelectric focusing and gel electrophoresis, respectively.Abbreviations B benzamidine - Con A concanavalin A - FPLC fast protein liquid chromatography - IEF isoelectric focusing - kDa kilodalton - pI isoelectric point - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate This work was supported by the Deutsche Forschungsgemeinschaft within the scope of the Sonderforschungsbereich 137.     

Profile of peroxidase isoforms influenced by spongy tissue disorder in Alphonso mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L.) fruits

This paper describes the profile of peroxidase (POX) isoenzymes induced due to the natural infection of Staphylococcus xylosus in spongy Alphonso mango fruits. Very low levels of protein and POX activity was observed in non-spongy unripe fruits, and when these fruits turned table-ripe, the levels of both the protein content and POX activity increased several fold. The spongy fruits, however, showed further 2-fold increase in POX activity; although drastic decrease in protein content was observed. Anionic and cationic PAGE, and isoelectric focusing (IEF), resulted in separation of various isoenzymes of POX. Both, anionic and cationic PAGE indicated that, at unripe stage, only basic isoforms were present in trace amounts. In non-spongy ripe fruits, increased levels of both anionic and cationic isoforms were observed after staining the gel with o-dianisidine, the POX substrate. In spongy fruits, however, an anionic PAGE showed appearance of four acidic isoforms with relative electrophoretic mobility (REM) of 0.52, 0.73, 0.78, and 0.84 and an isoenzyme (REM 0.52), showed further activation, as indicated by the intense dark color formation. Cationic PAGE also indicated higher levels of two basic isoforms (REM 0.56 and 0.62), in the spongy fruits. Isoelectric focusing resolved these isoenzymes in acidic, neutral, and basic isoforms. Two acidic isoforms in the pI range of 2–3.5 were detected toward the anode region and two cationic isoforms of pI 7.8 and 8.7, toward the cathode, giving visible indication of increased levels of these isoforms. The increased intensities of the POX bands observed in anionic and cationic PAGE, and IEF, gave confirmatory evidence for the up regulation of anionic and cationic isoforms in spongy fruits. These isoenzymes could have been overexpressed as a defense response of the spongy fruits against the Staphylococcus infection.