Interaction of indole derivatives and tryptophan peptides with interfaces of sodium dodecyl sulfate micelles.

The free energies of transfer for indole and tryptophan derivatives and pentapeptides having single tryptophan residues from aqueous to sodium dodecyl sulfate (SDS) micellar phases have been systematically studied using the conventional method of ultraviolet absorption spectrophotometry. The free energies for the position isomers of methyl indoles varied depending on the substitution positions. Thus, the contribution of the methyl group to the binding affinity of the 4-methyl indole to the micelle was about twice that of the 2- and 7-methyl indoles. The free energy changes with the introduction of halogen groups to the indole rings were correlated to the nonpolar water-accessible surface area (DeltaA(np)) of the halogen moieties, which were regarded as hydrophobic. The relationships followed straight lines passing through the origins. Position dependence having tendencies similar to the methyl indoles was observed among the magnitudes of the slopes of the straight lines. These results strongly suggest that the indole rings of the derivatives residing in the micellar interface regions direct their imino moieties --NH-- toward the micellar surfaces. Experiments using model tryptophan pentapeptides showed that the magnitude of free energy change per methylene unit of an alkyl amino acid residue in the pentapeptide increased with elongation of the alkyl moiety and was not a constant value as reported for various alkyl compounds. When the peptides distribute to the SDS micelles, the peptide backbones are anchored in aqueous phases and the amino acid side chains in the interfaces extend their alkyl groups toward the micellar centers. Thus, the free energy changes can be connected to the positions of the alkyl groups of the amino acid residues in the micelles.     

Microenvironment of tryptophan residues in beta-lactoglobulin derivative polypeptide-sodium dodecyl sulfate complexes.

The changes of microenvironment of tryptophan residues in -lactoglobulin A and its cyanogen bromide (CNBr) fragments with the binding of sodium dodecyl sulfate (SDS) were studied with measurements of the rates of N-bromosuccinimide (NBS) modification reactions by stopped-flow photometry. Two tryptophan residues of carboxyamidomethylated (RCM) -lactoglobulin A in the states of their complexes with SDS were clearly distinguishable by their differences in NBS modification rates. We confirmed by experiments with CNBr fragments containing tryptophan residue. The modification rates of Trp 19 in RCM -lactoglobulin A-SDS complexes were about 10-fold smaller than those expected for tryptophan residues exposed entirely to the aqueous solvent. The Trp 61 was hardly changed. The change of rate constants for Trp 19 was virtually consistent with those observed when N-acetyl-l-tryptophan ethylester was dissolved in SDS micelles. For various species of polypeptide-SDS complexes, all tryptophan residues were reactive to NBS and also, for some of them, the differences in NBS modification rates were observed between tryptophan residues on a common polypeptide chain. These results suggest micellar and heterogeneous bindings of SDS to polypeptides.     

Adsorption studies for the separation ofl-tryptophan froml-serine and indole in a bioconversion medium

l-tryptophan was produced froml-serine and indole by immobilized Escherichia coli cells in organic-aqueous systems. Selective adsorption was the method chosen to enable both product separation andl-serine reutilization. Amongst various adsorbents tested activated carbons and neutral polymeric resins (XAD-4 and XAD-7) showed good performance. The neutral resins could selectively concentrate thel-tryptophan from dilute aqueous solutions and adsorbed only 5% of the unconvertedl-serine. High separation factors (l-tryptophan/l-serine and indole/l-tryptophan) were obtained with these adsorbents. Despite a lower capacity, the XAD-7 resin had the advantage of desorbingl-tryptophan with basic or acidic solutions, while organic solvents were required to desorb, at the same concentration levels, this compound from XAD-4.In a packed bed column filled with XAD-4 resin or activated carbon, totall-tryptophan adsorption and recovery were achieved at linear velocities up to 5.0 cm/min and 3.2 cm/min respectively. Successive sorbent reutilization, following continuous sorption and elution steps, was carried out in packed bed columns with the neutral resins and activated carbon.Thel-form of tryptophan, after crystallization, was identified by HPTLC.List of Symbols HPLC High Performance Liquid Chromatography - HPTLC High Performance Thin Layer Chromatography - Trp tryptophan - Ser Serine - A amount of sorbent(g) - c equilibrium solute concentration in the aqueous phase (g/dm³) - c _i initial (before adding the sorbent) liquid phase concentration (g/dm³) - C _T tryptophan concentration in the inlet solution (g/dm³) - C _To tryptophan concentration in the outlet solution (g/dm³) - E _z axial dispersion coefficient (m²/s) - k experimental constant (Eq. 1, 2 and 3) - K ₁ rate constant of adsorption (min^–1) - L column length(m) - n experimental constant (eq. 1, 2 and 3) - q equilibrium solid phase concentration (g solute/g sorbent) - q _max maximum capacity of sorbent (g solute/g sorbent) - t time(s) - v liquid velocity (m/s) - V volume of liquid phase(dm³) - V _e eluted volume(dm³) - V _r volume needed to saturate the column (dm³)     

Effect of transmembrane helix packing on tryptophan and tyrosine environments in detergent-solubilized bacterio-opsin

Bacterio-opsin (bO) is folded in a nearly native conformation in mixed micelles of dimyristoyl phosphatidyl choline (DMPC) and 3-[(3-cholamidopropyl)-dimehtylamonio]-1-propane sulfonic acid (CHAPS), but bO is partially unfolded in sodium dodecyl sulfate (SDS). UV difference spectroscopy was used to study the changes in environment of bO aromatic amino acid side chains that occur upon partial unfolding. The UV difference spectra of peptides in CHAPS/DMPC minus peptides in SDS were measured for bO and the following subfragments of bO: C1 (residues 72–248), C2 (1–71), V1 (1–166), V2 (167–248), CB7 (119–145), CB9 (164–209), and CB10 (72–118). The spectra show that, in partially unfolded bO in SDS, the Tyr and Trp absorbance is blue-shifted. The difference spectra were compared to solvent perturbation difference spectra of N-acetyl-l-tyrosine ethyl ester and N-acetyl-l-tryptophanamide. The exposure change calculated from the difference spectra was found to correlate with the change in the number of van der Waals contacting atoms upon partial unfolding, and also with the number of transmembrane helical segments. This result suggests a simple experimental method of testing helix packing arrangements derived from hydropathy plots and model building.Abbreviations bO bacterio-opsin - bR bacteriorhodopsin - SDS sodium dodecyl sulfate - CHAPS 3-[(3-cholamidopropyl)-dimethylamonio]-1-propane sulfonic acid - C1 bacteriorhodopsin residues 72–248 - C2 1-71 - VI 1-166 - V2 167-248 - CB7 119-145 - CB9 164-209 - CB10 72-118 - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine - EDTA di-sodium ethylenediamine tetra-aceticacid     

Microenvironment of tryptophan residues in β-lactoglobulin derivative polypeptide-sodium dodecyl sulfate complexes

The changes of microenvironment of tryptophan residues in β-lactoglobulin A and its cyanogen bromide (CNBr) fragments with the binding of sodium dodecyl sulfate (SDS) were studied with measurements of the rates of N-bromosuccinimide (NBS) modification reactions by stopped-flow photometry. Two tryptophan residues of carboxyamidomethylated (RCM) β-lactoglobulin A in the states of their complexes with SDS were clearly distinguishable by their differences in NBS modification rates. We confirmed by experiments with CNBr fragments containing tryptophan residue. The modification rates of Trp 19 in RCM β-lactoglobulin A-SDS complexes were about 10-fold smaller than those expected for tryptophan residues exposed entirely to the aqueous solvent. The Trp 61 was hardly changed. The change of rate constants for Trp 19 was virtually consistent with those observed when N-acetyl-l-tryptophan ethylester was dissolved in SDS micelles. For various species of polypeptide-SDS complexes, all tryptophan residues were reactive to NBS and also, for some of them, the differences in NBS modification rates were observed between tryptophan residues on a common polypeptide chain. These results suggest micellar and heterogeneous bindings of SDS to polypeptides.     

Interaction of apocytochrome c and derived polypeptide fragments with sodium dodecyl sulfate micelles monitored by photochemically induced dynamic nuclear polarization 1H NMR and fluorescence spectroscopy.

The topology of apocytochrome c, the heme-free precursor of the mitochondrial protein cytochrome c, was investigated in a lipid-associated form. For this purpose photochemically induced dynamic nuclear polarization 1H nuclear magnetic resonance (CIDNP 1H NMR) spectroscopy and quenching of tryptophan and tyrosine fluorescence by acrylamide were applied to an apocytochrome c-sodium dodecyl sulfate (SDS) micellar system. A pH titration of the chemical shifts of the histidine C2 proton resonances of apocytochrome c, using conventional 1H NMR, yielded pK(a)'s of 5.9 +/- 0.1 and 6.2 +/- 0.1, which were assigned to histidine-18 and -33 and histidine-26, respectively. In the presence of SDS micelles an average pK(a) of 8.1 +/- 0.1 was obtained for all histidine C2 protons. Photo-CIDNP enhancements of the histidine, tryptophan, and tyrosine residues, contained in the intact apocytochrome c and in chemically and enzymatically prepared fragments of the precursor, were reduced in the presence of SDS micelles. Similarly, the quenching of the tryptophan fluorescence of the polypeptides by acrylamide was diminished in the presence of SDS. These results indicate the aromatic residues studied are localized in the interface of the SDS micelle.     

Red-edge excitation fluorescence spectroscopy of proteins in reversed micelles

The dependence of fluorescence emission maxima ofl-tryptophan and single-tryptophan-containing proteins (ribonuclease T1, melittin, and parvalbumin) on excitation wavelength has been studied in reversed micelle systems of sodium bis(2-ethyl-1-oxyl) sulfosuccinate (AOT). No effect of fluorescence maximum shift for different excitation wavelengths is observed for ribonuclease T1, in which a single tryptophan residue is located in the nonrelaxating, nonpolar protein interior.l-Tryptophan and the rest of the studied proteins, which contain single tryptophan residues exposed to the solvent, exhibit the dipolar relaxational processes of partly immobilized water molecules in micelles. This effect depends on the molar H₂O/AOT ratio. Circular dichroism measurements prove that there have been no structural changes of the studied proteins in micellar systems. The results provide information about dynamic relaxational processes in proteins.     

Content of indoleacetic acid and its metabolism in root nodules ofMelilotus alba

The root nodules ofMelilotus alba, a leguminous fodder herb, contain a high amount of indoleacetic acid (IAA). The tryptophan pool present in the nodule might serve as a source for the IAA production. Metabolism of IAA in the nodules was evidenced by the presence of IAA-metabolizing enzymes, IAA oxidase and peroxidase. A high amount of IAA was produced by the symbiont isolated from the nodules in culture, when supplemented with tryptophan. For IAA production, the bacteria preferred thel-isomer over thedl- ord-isomer of tryptophan. The possible role of nodular IAA production on the legume-Rhizobium symbiosis is discussed.     

Hyper-production of l-trytophan via fermentation with crystallization

A stable and fast l-tryptophan producer, AGX1757, was isolated from Escherichia coli W3110 trpAE1 trpR tnaA, which carried pSC101-trpI15·14. Cells of AGX1757 did not lose the composite plasmid during fermentation. Whenever a fed-batch culture of AGX1757 attained an l-tryptophan concentration of about 30 g/l, indole began to appear in the broth. The emergence of indole was caused by inhibition of tryptophan synthase due to accumulated l-tryptophan. Hence, the production rate of l-tryptophan sharply decreased. A higher solubility of l-tryptophan in the supernatant of culture broth (about 32 g/l) than that in the initial medium (about 22 g/l) was attributed to some unknown interaction between l-tryptophan and certain macromolecular material(s) coming from the bacterial cells. An addition of non-ionic detergents into the supernatant was effective for decreasing the solubility of l-tryptophan, hence causing crystallization of l-tryptophan. Pluronic L-61 was supplied from outside to an extent of 0.5% in terms of wt% concentration at around 45 h of fermentation when the l-tryptophan accumulated reached about 25 g/l. This addition actually caused crystallization of l-tryptophan and, as a result, the inhibitory effect of tryptophan synthase by l-tryptophan accumulated in the broth could be alleviated. Thus far, further fermentation became possible. l-Tryptophan of more than 50 g/l was finally produced by feeding solutions of both glucose and anthranilic acid. Correspondence to: H. Tsunekawa     

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene inEscherichia coli

Summary The application of an inducible regulation system using the trytophanase operon promoter (TPase promoter; P_tna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of P_tna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, l-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-P_tnaTS was increased 500-fold compared to that of the cultured host strain. About 1 mol l-tryptophan/l reaction mixture was formed from indole and l-serine at 37° C for 3.5 h. Offprint requests to: H. Yukawa     

Enzymatic production of l-phenylalanine from the racemic mixture of d,l-phenyllactate

Summary The production of l-phenylalanine from the racemate d,l-phenyllactate in an enzyme membrane reactor has been examined. In a first step the racemate is dehydrogenated to the prochiral intermediate phenylpyruvate by the enzymes d-and l-hydroxyisocaproate dehydrogenase. In a second step phenylpyruvate is reductively aminated to l-phenylalanine by l-phenylalanine dehydrogenase. Both steps are dependent on coenzyme, the first one requires NAD, the second one NADH in stoichiometric amounts; in this way the coenzyme is regenerated and only required catalytically. The coenzyme is covalently bound to polyethylene glyco-20 000 and can thus be retained in the reactor analogously to the three enzymes. In order to optimize the continuous production of l-phenylalanine from d,l-phenyllactate, models of the reaction kinetics and of the reactor system have been set up. By means of the reactor model, we can calculate the optimum ratio of the three enzymes, the optimum coenzyme concentration and the optimum phenylpyruvate concentration in the feed.In this process, at a substrate concentration of 50 mM d,l-phenyllactate we reached a spacetime-yield of 28 g l-Phe/(l*d).Abbreviations PEG polyethylene glycol - d-HicDH d-hydroxyisocaproate dehydrogenase - l-HicDH l-hydroxyisocaproate dehydrogenase - PheDH l-phenylalanine dehydrogenase - V _max maximum velocity - K _M Michaelis-Menten constant - K _l inhibition constant - R₁ reaction rate of the d-HicDH forward reaction - R₂ reaction rate of the d-HicDH reverse reaction - R₃ reaction rate of the l-HicDH forward reaction - R₄ reaction rate of the l-HicDH reverse reaction - R₅ reaction rate of the PheDH forward reaction - R₆ reaction rate of the PheDH reverse reaction - d-PLac d-phenyllactate - l-PLac l-phenyllactate - PPy phenylpyruvate - l-Phe l-phenylalanine - NH₄ ammonium - residence time     

Amino acid metabolism in the thermophilic phototroph,Chloroflexus aurantiacus: properties and metabolic role of two l-threonine (l-serine) dehydratases

Two l-threonine (l-serine) dehydratases (EC 4.2.1.16) of the thermophilic phototrophic bacterium Chloroflexus aurantiacus Ok-70-fl were purified to electrophoretic homogeneity by procedures involving anion exchange and hydrophobic interaction chromatography. Only one of the two enzymes was sensitive to inhibition by l-isoleucine (K _i=2 M) and activation by l-valine. The isoleucine-insensitive dehydratase was active with l-threonine (K _m=20 mM) as well as with l-serine (K _m=10 mM) whereas the other enzyme, which displayed much higher affinity to l-threonine (K _m=1.3 mM), was inactivated when acting on l-serine. Both dehydratases contained pyridoxal-5-phosphate as cofactor. When assayed by gel filtration techniques at 20 to 25° C, the molecular weights of both enzymes were found to be 106,000±6,000. In sodium dodecylsulfate-polyacrylamide gel electrophoresis, the two dehydratases yielded only one type of subunit with a molecular weight of 55,000±3,000. The isoleucine-insensitive enzyme was subject to a glucose-mediated catabolite repression.Abbreviations A absorbance - ile isoleucine - PLP pyridoxal-5-phosphate - SDS sodium dodecyl sulfate - TDH threonine dehydratase - U unit     

An investigation into the mechanism ofl-asparaginase resistance in L5178Y murine leukemia cells

Summary Resistance of leukemia cells tol-asparaginase is presumed to be due to increased expression of asparagine synthetase activity by resistant cells, so they are no longer dependent on an exogenous source ofl-asparagine for growth. The mechanism by which cells acquire the ability for increased enzyme expression, however, has not been clearly defined. Evidence presented here indicates that genomic alterations in the form of translocations, gene amplification, or increased P-glycoprotein expression, do not account for the phenotypic transformation froml-asparaginase sensitivity tol-asparaginase resistance. Instead, both sensitive and resistant L5178Y cells contain immunoreactive material detected by Western blotting with an antiserum prepared against bovine pancreatic asparagine synthetase. This suggests that the mechanism of resistance might involve modification of asparagine synthetase inl-asparaginase-resistant cells by an as-yet-unidentified mechanism or by inhibition of enzyme activity in thel-asparaginase-sensitive cells.Abbreviations CHO Chinese hamster ovary - DMEM Dulbecco's modified Eagle medium - EtBr ethidium bromide - I.U. international unit - PBS 0.14 M NaCl, 0.01 M KCl, 0.02 M phosphate, pH 7.4 - SDS sodium dodecyl sulfate     

Culture growth and IAA production by a microbial diazotropic symbiont of stem-nodules of the legumeAeschynomene aspera

The stem nodules of the legumeAeschynomene aspera contain indoleacetic acid and a high amount of tryptophan. TheAzorhizobium caulinodans isolated from the stem nodules of the leguminous emergent hydrophyte produced a high amount of IAA (14.8 mg/L) inl-tryptophan-supplemented basal medium. The IAA yields paralleled the culture growth rate and increased up to 52 h. No separate growth and production phase was observed. The IAA production was increased 344% when the medium was supplemented withl-tryptophan, sucrose, FeSO₄·7H₂O, NaNO₃, ascorbic acid and sodium dodecyl sulfate. The possible role of the IAA production in the legume-bacterium symbiosis is discussed.     

Isolation of a crustaceann-acetyl-d-glucosamine-1-phosphate transferase and its activation by phospholipids

Summary Then-acetyl-d-glucosamine-1-phosphate: dolichol phosphate transferase fromArtemia has been partially purified and characterized. The enzyme is solubilized from crude microsomes using Triton X-100, and after detergent removal appears to be associated with phospholipids. Using dolichol phosphate and UDP-n-acetyl-d-glucosamine as substrates, the enzyme catalyzes the formation of dolichol-pyrophosphate-n-acetyl-d-glucosamine. the product identity has been verified by TLC and paper chromatography following mild acid hydrolysis. Under the incubation conditions used only one product is made, i.e., Dol-p-p-GlcNAc. The formation of product is linear with increasing amounts of added protein and with time of incubation. The enzyme requires magnesium ions for activity. Activity of the enzyme is stimulated 6-fold by exogenous dolichol phosphate and is also stimulated by added phospholipids, with optimal activity being obtained in the presence of mixtures of phosphatidylcholine and phosphatidylglycerol. Enzymatic activity is not increased upon addition of GDP-mannose or dolichol phosphate mannose. The enzyme is rapidly inactivated by exposure to several detergents, including Triton X-100 and deoxycholate. The activity is inhibited by tunicamycin and by the purified B₂ homologue of this antibiotic. Other antibiotic inhibitors such as diumycin and polyoxin D have little effect on the enzyme. Both the microsomal and solubilized enzyme preparations are inactivated by 70% upon treatment with phospholipase A₂; activity may be restored by addition of phospholipids. Following hydrophobic interaction chromatography on Phenyl Sepharose, gel filtration chromatography on Sepharose CL-4B indicated that the enzyme, purified 81-fold, contained phophatidylcholine and phosphatidyl-ethanolamine.Abbreviations SDS sodium dodecyl sulfate - PMSF phenyl methanesulfonylfluoride - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - GlcNAc N-acetyl-d-glucosamine - Dol-PP-GlcNAc dolichol pyrophosphate N-acetyl-d-glucosamine - Dol-P-man dolichol-phosphate-mannose - Dol-PP- (GlcNAc)₂ dolichol-pyrophosphate-di-N- acetylchitobiose - DMSO dimethylsulfoxide - C:M (2:1) chloroform:methanol (2:1) - C:M:W (10:10:3) chloroform:methanol:water (10:10:3) - GlcNAc-1-P N-acetyl-d-glucosamine-1-phosphate - Dol-P dolichol phosphate - EGTA ethylene glycol bis (b-aminoethyl ether)-NNNN tetraacetic acid     

Production of diprenylated indole derivatives by tandem incubation of two recombinant dimethylallyltryptophan synthases

Two dimethylallyltryptophan synthases, FgaPT2 and 7-DMATS, which catalysed the prenylation of l-tryptophan at positions C4 and C7, respectively, have been recently identified in Aspergillus fumigatus and proven biochemically. These enzymes were successfully used for the production of monoprenylated indole derivatives. In this study, we showed that C4,C7-diprenylated indole derivatives, e.g. 4,7-di-(dimethylallyl)-l-tryptophan, 4,7-di-(dimethylallyl)-l-abrine and 4,7-di-(dimethylallyl)-11-methyltryptophan, could be conveniently produced by tandem incubation of both enzymes. The structures of the isolated enzymatic products were elucidated by NMR and MS analyses. High conversion yields of up to 93% were achieved by an incubation sequence of FgaPT2 followed by 7-DMATS. The results reported in this study demonstrated the potential of secondary metabolite enzymes as promising tools for the production of designed compounds.     

Preliminary studies on the growth of Pseudomonas sp. BA2 and the production of L-aminoacylase

Summary The bacterial strain Pseudomonas sp. BA2 did not develop l-aminoacylase activity in the absence of N-acetyl-dl-alanine. The maximum growth rate and enzyme production were obtained when the acetylated amino acid was used as the sole carbon and nitrogen source. A maximum biomass of A₆₆₀=1.543, after 24 h of cultivation, was obtained. The l-aminoacylase activity reached the maximum value (5.6 U ml^–1 broth) in the stationary growth phase.     

Anisotropy decays of single tryptophan proteins measured by GHz frequency-domain fluorometry with collisional quenching

We used harmonic-content frequency-domain fluorometry to determine the anisotropy decays of a variety of single tryptophan peptides and proteins. Resolution of the rapid and complex anisotropy decays was enhanced by global analysis of the data measured in the presence of quenching by either oxygen or acrylamide. For each protein, and for each quencher, data were obtained at four to six quencher concentrations, and the data analyzed globally to recover the anisotropy decay. The decrease in decay times produced by quenching allows measurements to an upper frequency limit of 2 GHz. The chosen proteins provided a range of exposures of the tryptophan residues to the aqueous phase, these being ACTH, monellin, Staphylococcus nuclease and ribonuclease T ₁, in order of decreasing exposure. Examination of indole and several small peptides demonstrates the resolution limitations of the measurements; a correlation time of 12 ps was measured for indole in methanol at 40°C. Comparison of the anisotropy decays of gly-trp-gly with leu-trp-leu revealed stearic effects of the larger leucine side chains on the indole ring. The anisotropy decay of gly-trp-gly revealed a 40 ps component for the indole side chain, which was resolved from the overall 150 ps correlation time of the tripeptide. Only the longer correlation time was observed for leu-trp-leu. With the exception of ribonuclease T ₁, each of the proteins displayed a subnanosecond component in the anisotropy decay which we assign to independent motions of the tryptophan residues. For example, Staphylococcus nuclease and monellin displayed segmental tryptophan motions with correlation times of 80 and 275 ps, respectively. The amplitudes of the rapid components increased with increasing exposure to the aqueous phase. These highly resolved anisotropy decays for proteins of known structure are suitable for comparison with molecular dynamic simulations.Abbreviations Ac acrylamide - ACTH adrenocorticotropin hormone (1–24) - BPTI bovine pancreatic trypsin inhibitor - NATA N-acetyl-L-tryptophanamide - RNase T ₁ ribonuclease T ₁ - S. Nuclease staphylococcus aureus nuclease Supported by grants DMB-8804931 and DIR-8710401 from the National Science Foundation, and GM-39617 from the National Institutes of Health. J. R. Lakowicz acknowledges support from the Medical Biotechnology Center at the University of Maryland. I. Gryczynski was on leave from University of Gáansk, Institute of Experimental Physics, Gdansk, Poland, with partial support from CPBP 01.06.2.01 (Poland). H. Cherek was on leave from Nicholas Copernicus University, Torun, Poland, with partial support from CPBP 01.06.2.03 Offprint requests to: J. R. Lakowicz     

Primary structure of the xylose-containingN-linked carbohydrate moiety from ascorbic acid oxidase ofCucurbita pepo medullosa

Ascorbic acid oxidase (E.C.1.10.3.3) from the green zucchini squash (Cucurbita pepo medullosa) is a copper-containing glycoprotein which catalyzes the reaction:l-ascorbic acid +1/2 O₂l-dehydroascorbic acid + H₂O. The carbohydrate content of the purified plant glycoprotein amounted to 3% (w/w), and monosaccharide analysis revealed the carbohydrate moiety to be of theN-glycosidic type. The carbohydrate chains were released from the apoenzyme by digestion with PNGase-F immobilized on Sepharose 4B. After fractionation on Bio-Gel P-2 and purification on Mono-Q, the neutral oligosaccharide was investigated by 500-MHz¹H-NMR spectroscopy. The primary structure of theN-linked carbohydrate chain was established to be: Abbreviations AAO ascorbic acid oxidase - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - Man mannose - Xyl xylose - GLC gas-liquid chromatography - FPLC fast protein liquid chromatography - NMR nuclear magnetic resonance - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The properties of l-fucose binding agglutinin associated with the cell wall of Rhizoctonia solani

The adherence of Escherichia coli B cells to cell wall associated-agglutinin of the soil borne plant pathogen Rhizoctonia solani, was inhibited by l-fucose, l-galactose, trypsin, SDS, cycloheximide and Na₂-EDTA. The coiling of the biocontrol agent Trichoderma harzianum around Rhizoctonia hyphae was prevented by SDS, cycloheximide, Na₂-EDTA and methyl--l-fucoside — an inhibitor of Rhizoctonia agglutinin not metabolized by both fungi. The possible role of the agglutinin in Trichoderma-Rhizoctonia interaction is discussed.