The influence of pH on the inhibition of DNA cleavages induced by pyrogallol

Abstract

Induction of DNA damage by pyrogallol has been shown at physiological pH, but mutagenesis data also suggest there is inhibition in acidic media. In the present work, the plasmid pBSK was incubated with pyrogallol, under aerobic conditions at 37°C, at pH 7.4, 4.5 or 3.5, for 1, 3 or 5 h, in the absence or presence of Cu²⁺. Cleavage of the supercoiled DNA form was analyzed through topology modifications by agarose gel electrophoresis and quantified by densitometry. Independently of the presence of Cu²⁺ , DNA cleavage at pH 7.4 was significantly (P < 0.001) induced and occurred extensively after 1-h incubation. At pH 4.5, the cleavage was significantly (P < 0.05) induced only after 5 h incubation in the absence of Cu²⁺ , but was extensive (P < 0.001) after 1-h incubation when the metal ion was present. At pH 3.5, DNA cleavage was inhibited (P > 0.05), after 5-h incubation, even in the presence of Cu²⁺. Our results provide evidence that DNA cleavage by pyrogallol is pH-dependent, catalyzed by Cu²⁺ , and extensively decreased in acidic pH. Due to the abundant presence of the pyrogallate ion in physiological media, we suggest that this conjugate base form is responsible for DNA cleavage.     

Purification and characterization of an alpha-amylase of Pichia burtonii isolated from the traditional starter "murcha" in Nepal

Among more than 20 yeast strains isolated from the traditional starter "murcha" in Nepal, we characterized a yeast that might be involved in saccharification. This strain, identified as Pichia burtonii, produced an extracellular amylolytic enzyme when cultured in the presence of starch in the medium. Since no amylase secreted by P. burtonii has yet been reported, we purified the enzyme and determined its N-terminal amino acid sequence. Together with the results of a hydrolyzing activity assay toward various substrates, it was found to be an alpha-amylase. The purified enzyme, named Pichia burtonii alpha-amylase (PBA), was a glycoprotein with an apparent molecular mass of 51 kDa. Enzyme activity was optimal at pH 5.0 at 40 degrees C. The enzyme retained 80% of its original activity after incubation under the optimal pH condition at 50 degrees C for 30 min. The activity was inhibited by metal ions such as Cd(2+), Cu(2+), Hg(2+), Al(3+), and Zn(2+).     

Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions

We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me(2+)) ions, with the level of activity decreasing in the order Mn(2+)> or =Mg(2+)>Ca(2+)> or =Cu(2+)>Co(2+)> or =Ni(2+)> or =Zn(2+), whereas Fe(2+) did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me(2+) ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me(2+) ions in the 0.1-12 mM range, depending on the particular metal ion. In the presence of all Me(2+) ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu(2+), Zn(2+), or Ca(2+) inhibited the Mg(2+)-dependent hydrolysis of scDNA, while Ni(2+), Co(2+), and Mn(2+) activated this reaction. The Mn(2+)-dependent hydrolysis of scDNA was activated by Ca(2+), Ni(2+), Co(2+), and Mg(2+) ions but was inhibited by Cu(2+) and Zn(2+). After addition of the second metal ion, only in the case of Mg(2+) and Ca(2+) or Mn(2+) ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0-100%) in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.     

Identification and characterization of a divalent metal ion-dependent cleavage site in the hammerhead ribozyme.

We describe a new RNA cleavage motif, found in the hammerhead ribozyme. Cleavage occurs between nucleotides G8 and A9, yielding a free 5'-hydroxyl group and a 2',3'-cyclic phosphate. This cleavage is dependent upon divalent metal ions and is the first evidence for a metalloribozyme known to show preference for Zn(2+). Cleavage is also observed in the presence of Ni(2+), Co(2+), Mn(2+), Cd(2+), and Pb(2+), while negligible cleavage was detected in the presence of the alkaline-earth metal ions Mg(2+), Ca(2+), Sr(2+), and Ba(2+). A linear relationship between the logarithm of the rate and pH was observed for the Zn(2+)-dependent cleavage, which is indicative of proton loss in the cleavage mechanism, either prior to or in the rate-determining step. We postulate that a zinc hydroxide complex, bound to the known A9/G10.1 metal ion binding site, abstracts the proton from the 2'-hydroxyl group of G8, which attacks the A9 phosphate and initiates cleavage. This hypothesis is supported by a previously reported crystal structure [Murray, J. B., Terwey, D. P., Maloney, L., Karpeisky, A., Usman, N., Beigelman, L., and Scott, W. G. (1998) Cell 92, 665-673], which shows the conformation required for RNA cleavage and proximity of the 2'-hydroxyl group to the metal ion complex.     

The requirement for bivalent cations in formation of nicotinamide–adenine dinucleotide by nicotinamide mononucleotide adenylyltransferase of pig-liver nuclei

1. The requirement for bivalent cations in catalysis of NAD formation from ATP and NMN in the presence of NMN adenylyltransferase of pig-liver nuclei was studied. Rates of NAD formation in the presence of the activating cations Cd(2+), Mn(2+), Mg(2+), Zn(2+), Co(2+) and Ni(2+) were approximately a linear function of heats of hydration of the corresponding ions. Ba(2+), Sr(2+), Ca(2+), Cu(2+) and Be(2+) did not activate the enzyme; Be(2+) inhibited the reaction in the presence of Mg(2+) and, to a greater extent, in the presence of Ni(2+). 2. Michaelis constants for NAD formation, measured in a coupled assay with NMN adenylyltransferase and alcohol dehydrogenase at pH8.0 and 25 degrees , in the presence of 3mm concentrations of the unvaried reactants, were 88+/-7mum-ATP, 42+/-4mum-NMN and 85+/-4mum-Mg(2+). The results at this pH and at pH7.5 were consistent with mechanisms in which Mg(2+)-ATP complex is a reactant and free ATP a competitive inhibitor. 3. Formation of nicotinamide-hypoxanthine dinucleotide from NMN and ITP in the presence of the transferase was also more rapid with Ni(2+) and Co(2+) than with Mg(2+).     

The role of chitin in uranium adsorption by R. arrhizus

In order to further refine and support the uranium biosorption mechanism hypothesis proposed for Rhizopus arrhizus, uranium competitive equilibrium uptake isotherms by chitin were determined at two different solution pH levels and in the presence of different concentrations of competing ions, namely, Cu(2+), Zn(2+), and Fe(2+). The co-ion effect became more poronounced as the co-ion concentration in solution and pH increased. Obtained equilibrium data are in agreement with uranium biosorption data reported earlier. Infrared, mass, and electron paramagnetic resonance (EPR) spectra of chitin before and after uranium uptake in the presence of the competing ions Cu(2+), Zn(2+), and Fe(2+) were recorded. The combination of the spectral data and the information from equilibrium studies supported the hypothesis advanced earlier on the mechanism of uranium uptake by R.arrhizus. In addition, the data suggested the participation of a free radical in uranium coordination by the cell wall chitin. The mechanism of reduction of the uranium uptake capacity of the biomass in the presence of competing ions was also elucidated further.     

Characterization of the metal-substituted dipeptidyl peptidase III (rat liver)

Dipeptidyl peptidase III (DPP III) (EC 3.4.14.4), which has a HELLGH-E (residues 450-455, 508) motif as the zinc binding site, is classified as a zinc metallopeptidase. The zinc dissociation constants of the wild type, Leu(453)-deleted, and E508D mutant of DPP III at pH 7.4 were 4.5 (+/-0.7) x 10(-13), 5.8 (+/-0.7) x 10(-12), and 3.2 (+/-0.9) x 10(-10) M, respectively. The recoveries of the enzyme activities by the addition of various metal ions to apo-DPP III were also measured, and Co(2+), Ni(2+), and Cu(2+) ions completely recovered the enzyme activities as did Zn(2+). The dissociation constants of Co(2+), Ni(2+), and Cu(2+) ions for apo-DPP III at pH 7.4 were 8.2 (+/-0.9) x 10(-13), 2.7 (+/-0.3) x 10(-12), and 1.1 (+/-0.1) x 10(-14) M, respectively. The shape of the absorption spectrum of Co(2+)-DPP III was very similar to that of Co(2+)-carboxypeptidase A or Co(2+)-thermolysin, in which the Co(2+) is bound to two histidyl nitrogens, a water molecule, and a glutamate residue. The absorption spectrum of Cu(2+)-DPP III is also very similar to that of Cu(2+)-thermolysin. The EPR spectrum and the EPR parameters of Cu(2+)-DPP III were very similar to those of Cu(2+)-thermolysin but slightly different from those of Cu(2+)-carboxypeptidase A. The five lines of the superfine structure in the perpendicular region of the EPR spectrum in Cu(2+)-DPP III suggest that nitrogen atoms should coordinate to the cupric ion in Cu(2+)-DPP III. All of these data suggest that the donor set and the coordination geometry of the metal ions in DPP III, which has the HExxxH motif as the metal binding site, are very similar to those of the metal ions in thermolysin, which has the HExxH motif.     

Characterization of nucleotide binding sites of the isolated H(+)-ATPase from spinach chloroplasts, CF(0)F(1)

Soluble purified CF(0)F(1) from chloroplasts was either oxidized or reduced and then incubated with [alpha-(32)P]ATP in the presence or in the absence of Mg(2+). Depending on the conditions of incubation, the enzyme showed different tight-nucleotide binding sites. In the presence of EDTA, two sites bind [alpha-(32)P]ATP from the reaction medium at different rates. Both sites promote ATP hydrolysis, since equimolar amounts of [alpha-(32)P]ATP and [alpha-(32)P]ADP are bound to the enzyme. In the presence of Mg(2+), only one site appears during the first hour of incubation, with characteristics similar to those described in the absence of Mg(2+). However, after this time a third site appears also permitting binding of ATP from the reaction medium, but in this case the bound ATP is not hydrolyzed. Covalent derivatization by 2-azido-[alpha-(32)P]ATP was used to distinguish between catalytic and noncatalytic sites. In the presence of Mg(2+), there are at least three distinct nucleotide binding sites that bind nucleotide tightly from the reaction medium: two of them are catalytic and one is noncatalytic.     

Copper(II) inhibits in vitro conformational conversion of ovine prion protein triggered by low pH

To gain insight into the conformational conversion of ovine prion protein (OvPrP(C)) at different pH values and/or in the presence of CuCl(2), the secondary structure of OvPrP(C) was analysed by circular dichroism (CD) spectroscopy. Copper treatment of OvPrP(C) under moderately acidic conditions (pH approximately 5.0-6.0) as well as physiological conditions (pH 7.4) also makes OvPrP(C) adopt protease-resistant and beta-sheet-rich conformation. However, under lower pH conditions (2.0-4.5) with copper treatment, OvPrP(C) gained higher alpha-helix structure. This study demonstrated that Cu(2+) can significantly modulate conformational conversion triggered by acidic pH, and this will provide therapeutic intervention approaches for prion diseases.     

Improved efficiency of site-specific copper(II) ion-catalysed protein cleavage effected by mutagenesis of cleavage site

The peptide sequence (N)DKTH(C) was previously investigated as a site for efficient, specific cleavage of a fusion protein by cupric ions using a humanized gamma1 Fab' as a model protein. Here we show that conservative mutations to three of the residues in the introduced cleavage site resulted in cleavage sites that were significantly improved. They were cleaved more efficiently by Cu(2+), such that cleavage reactions could be shorter, of lower pH or at a lower temperature. Some were even found to be measurably cleaved by Ni(2+). Use of these new cleavage sequences along with cupric ions may provide a more rapid and less harsh method for cost-effective, large-scale proteolytic cleavage of fusion proteins and peptides.     

Mechanism of non-enzymic transamination reaction between histidine and α-oxoglutaric acid

Non-enzymic transamination reactions at 85 degrees between various amino acids and alpha-oxoglutaric acid are catalysed by metal ions, e.g. Al(3+), Fe(2+), Cu(2+) and Fe(3+). The reaction is optimum at pH4.0. Of the 14 amino acids studied histidine is the most active. In the presence of Al(3+) histidine transaminates with alpha-oxoglutaric acid, forming glutamic acid and Al(3+)-imidazolylpyruvic acid complex as the end products. However, in the presence of Fe(2+) or Cu(2+) the products are glutamic acid and a 1:2 metal ion-imidazolylpyruvic acid chelate. The greater effectiveness of histidine in these reactions is attributed to the presence of the tertiary imidazole nitrogen atom, which is involved in the formation of stable sparingly soluble metal ion-imidazolylpyruvic acid complexes or chelates as end products of these reactions. Of the metal ions studied only Al(3+), Fe(2+), Fe(3+) and Cu(2+) are effective catalysts for the transamination reactions, and EDTA addition completely inhibits the catalytic effect of the Al(3+). Spectrophotometric evidence is presented to demonstrate the presence of metal ion complexes of Schiff bases of histidine as intermediates in the transamination reactions. These results may contribute to understanding the role of histidine in enzyme catalysis.     

Metal ions,pH, and cholesterol regulate the interactions of Alzheimer's disease amyloid-beta peptide with membrane lipid

The interaction of A beta peptides with the lipid matrix of neuronal cell membranes plays an important role in the pathogenesis of Alzheimer's disease. By using EPR and CD spectroscopy, we found that in the presence of Cu(2+) or Zn(2+), pH, cholesterol, and the length of the peptide chain influenced the interaction of these peptides with lipid bilayers. In the presence of Zn(2+), A beta 40 and A beta 42 both inserted into the bilayer over the pH range 5.5-7.5, as did A beta 42 in the presence of Cu(2+). However, A beta 40 only penetrated the lipid bilayer in the presence of Cu(2+) at pH 5.5-6.5; at higher pH there was a change in the Cu(2+) coordination sphere that inhibited membrane insertion. In the absence of the metals, insertion of both peptides only occurred at pH < 5.5. Raising cholesterol to 0.2 mol fraction of the total lipid inhibited insertion of both peptides under all conditions investigated. Membrane insertion was accompanied by the formation of alpha-helical structures. The nature of these structures was the same irrespective of the conditions used, indicating a single low energy structure for A beta in membranes. Peptides that did not insert into the membrane formed beta-sheet structures on the surface of the lipid.     

Kinetic analyses of divalent cation-dependent EcoRV digestions on a DNA-immobilized quartz crystal microbalance

Enzymatic digestion with a type IIP restriction endonuclease EcoRV was investigated on a DNA-immobilized 27-MHz quartz crystal microbalance (QCM). Real-time observations of both the enzyme binding process and the DNA cleavage process of EcoRV were followed by frequency (mass) changes on the QCM, which were dependent on divalent cations such as Ca(2+) or Mg(2+). In the presence of Ca(2+), the site-specific binding of EcoRV to DNA could be observed, without the catalytic process. On the other hand, in the presence of Mg(2+), both the binding of the enzyme to the specific DNA (mass increase) and the site-specific cleavage reaction (mass decrease) could be observed continuously from QCM frequency changes. From time courses of frequency (mass) changes, each kinetic parameter, namely binding rate constants (k(on)), dissociation rate constants (k(off)), dissociation constants (K(d)) of EcoRV to DNA, and catalytic rate constant (k(cat)) of the cleavage reaction, could be determined. The binding kinetic parameters of EcoRV in the presence of Ca(2+) were consistent with those of the binding process followed by the cleavage process in the presence of Mg(2+). The k(cat) value obtained by the QCM method was also consistent with that obtained by other methods. This study is the first to simultaneously determine k(on), k(off), and k(cat) for a type IIP restriction endonuclease on one device.     

Extracellular nuclease from Rhizopus stolonifer: purification and characteristics of - single strand preferential - deoxyribonuclease activity

An extracellular nuclease from Rhizopus stolonifer (designated as nuclease Rsn) was purified to homogeneity by chromatography on DEAE-cellulose followed by Blue Sepharose. The M(r) of the purified enzyme determined by native PAGE was 67? omitted?000 and it is a tetramer and each protomer consists of two unidentical subunits of M(r) 21? omitted?000 and 13? omitted?000. It is an acidic protein with a pI of 4.2 and is not a glycoprotein. The purified enzyme showed an obligate requirement of divalent cations like Mg(2+), Mn(2+) and Co(2+) for its activity but is not a metalloprotein. The optimum pH of the enzyme was 7.0 and was not influenced by the type of metal ion used. Although, the optimum temperature of the enzyme for single stranded (ss) DNA hydrolysis in presence of all three metal ions and for double stranded (ds) DNA hydrolysis in presence of Mg(2+) was 40 degrees C, it showed higher optimum temperature (45 degrees C) for dsDNA hydrolysis in presence of Mn(2+) and Co(2+). Nuclease Rsn was inhibited by divalent cations like Zn(2+), Cu(2+) and Hg(2+), inorganic phosphate and pyrophosphate, low concentrations of SDS, guanidine hydrochloride and urea, organic solvents like dimethyl sulphoxide, dimethyl formamide and formamide but not by 3'- or 5'-mononucleotides. The studies on mode and mechanism of action showed that nuclease Rsn is an endonuclease and cleaves dsDNA through a single hit mechanism. The end products of both ssDNA and dsDNA hydrolysis were predominantly oligonucleotides ending in 3'-hydroxyl and 5'-phosphoryl termini. Moreover, the type of metal ion used did not influence the mode and mechanism of action of the enzyme.     

Characterisation of aminopeptidase activity in scab mites (Psoroptes spp.)

Soluble and membrane-bound aminopeptidase activities were demonstrated in extracts of P. cuniculi (Delafond). Leucine aminopeptidase (LAP) activity in the soluble fraction of P. cuniculi extracts displayed substrate preference for amino acid derivatives with terminal leucine and methionine over those with acidic, basic or heterocyclic groups. P. cuniculi LAP was inhibited by leucinethiol (IC(50) = 1.4 +/- 0.4 nM), bestatin (IC(50) = 3.9 +/- 1.7 microM), Arphamenine A (IC(50) = 0.37 +/- 0.03 mM) the chelating agent 1,10-phenanthroline (IC(50) = 2.3 +/- 0.5 mM), Zn(2+), Cu(2+) Ni(2+), and Co(2+), and activated by Mn(2+) and Mg(2+). The LAP activity was visualised as a single major band after electrophoresis on native gels and eluted from a size exclusion column as a single major peak representing a molecular mass range of 85-116 kDa. Degenerate oligonucleotide primers were used to amplify short fragments of genomic DNA containing nucleotide sequence coding for the cation-binding motifs of the co-catalytic Zn(2+) binding domains of dizinc leucine aminopeptidases in both P. cuniculi and P.ovis (Hering). The major soluble aminopeptidase from these mites therefore displays most of the characteristics associated with typical cytosolic leucine aminopeptidases belonging to the M17 family of metalloproteinases.     

Degradation of DNA into 5'-monodeoxyribonucleotides in the presence of Mn(2+) ions

DNA is known to be aggregated by metal ions including Mn(2+) ions, but analysis of the aggregation process from a chemical viewpoint, which means identification of the product yielded during the process, has not been performed yet. On examination of the kinds of degraded materials that were in the supernatant obtained on centrifugation of a DNA mixture aggregated under conditions of 10 mM Mn(2+) ions ([Mn]/[P] = 46.3) at 70 degrees C for 1 h, the degradation products were found to be dAMP, dCMP, dGMP, and TMP. These dNMPs were purified by HPLC on TSKgel ODS-80Ts and identified by LC-TOF/MS. The degradation activity was lost on pretreatment of the DNA with a phenol-chloroform mixture, and the activity was recovered by pretreatment with a mixture of DMSO and a buffer containing surfactants. Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), and Cd(2+), as transition element metal ions, were effective as to the degradation into dNMP. Mg(2+), Ca(2+), Sr(2+), and Ba(2+), as alkali earth element metal ions, were not effective as to the degradation. Monovalent anions such as Cl(-), CH(3)OO(-), and NO(3)(-) were found to increase the degradation rate. Sixty mug of the 120 mug of the starting DNA in 450 mul was degraded into dNMP on reaction for 1 h in the presence of 100 mM NaCl and 10 mM Mn(2+) ions. In this process, aggregation did not occur, and thus was not considered to be necessary for degradation. The degradation was found not to occur at pH 7.0, and to be very sensitive to pH. The OH(-) ion should have a critical role in cleavage of the phosphodiester linkages in this case. The dNMP obtained in the degradation process was found to be only 5'-NMP, based on the H(1)NMR spectra. This prosess should prove to be a new process for the production of 5'-dNMP in addtion to the exonuclease.     

Influence of the physiologically widespread substances on the oxidative activity of copper(II) complexes with sinefungin, a nucleoside antibiotic

The oxidation-promoting reactivity of the Cu(II)-sinefungin complex in the presence of hydrogen peroxide was studied at pH 7.4, using N,N-dimethyl-p-nitrosoaniline (NDMA), as well as plasmid DNA as target molecules. Mixture of the complex with H(2)O(2) was found to be an efficient oxidant, bleaching NDMA solution, and generating single- and double-strand breaks in DNA. The oxidative DNA damage was investigated also in the presence of varying amounts of glutathione, histidine, Gly-Gly-His peptide, H2A histone, and ascorbic acid, showing diverse influence of those substances on the cleavage extension.     

Low-pH association of proteins with the membranes of intact red blood cells. II. Studies of the mechanism

The low-pH interaction of proteins with erythrocyte membranes has been found to be correlated with pH-induced changes in the erythrocyte membrane. Using a 90 degree lightscattering method it was shown that red blood cell hemolysis was slow between pH 5.8 and 5 (t1/2 above 1 h) but became fast at and below pH 4.7 (t1/2 less than 20 min). At pH 4.7, the presence of glycophorin in the incubation medium inhibited the hemolysis of erythrocytes and this protective effect was found to be dependent on the glycophorin concentration. Electron microscope experiments showed the presence of membrane defects after 10 s incubation at pH 4.6 in the absence of glycophorin in the incubation medium. These defects could further develop into openings with average widths of 14 nm after 1.5 min incubation under the acidic conditions. Fluorescence and flow cytometry studies showed that at pH 4.7, but not at pH 7.4, glycophorin tightly associates with phosphatidylcholine liposomes, and that liposome associated glycophorin molecules are recognized by anti-glycophorin monoclonal antibodies.     

Peptide nucleic acid-metal complex conjugates: facile modulation of PNA-DNA duplex stability

Conjugates of peptide nucleic acids (PNA) and metal binding ligands were prepared using solid-phase synthesis. Stability of duplexes of bis-picolylamine-PNA conjugates and DNA was found to be modulated by equimolar concentrations of bioavailable metal ions: Ni(2+), Zn(2+)>Cu(2+). Sequence specificity of PNA was not compromised in the presence of these metal ions.     

Purification and properties of two chitinolytic enzymes of Serratia plymuthica HRO-C48

The chitinolytic rhizobacterium Serratia plymuthica HRO-C48 was previously selected as a biocontrol agent of phytopathogenic fungi. One endochitinase (E.C. 3.2.1.14), CHIT60, and one N-acetyl-beta-1,4- D-hexosaminidase (E.C. 3.2.1.52), CHIT100, were purified and characterized. The endochitinase CHIT60, with an apparent molecular mass of 60.5 kDa, had a N-terminal amino acid sequence highly similar to that of chitinases A from Serratia liquefaciens and Serratia marcescens. The enzyme activity had its peak at 55 degrees C and pH 5.4, and increased by more than 20% in the presence of 10 mM Ca(2+), Co(2+) or Mn(2+). Activity was inhibited by 80% in the presence of 10 mM Cu(2+). CHIT100 appeared to be a monomeric enzyme with a molecular mass of 95.6 kDa and a pI of 6.8. Optimal activity was obtained at 43 degrees C and pH 6.6, and decreased by more than 90 % in the presence of 10 mM Co(2+) or Cu(2+). CHIT100 (100 microg ml(-1)) inhibited spore germination and germ tube elongation of the phytopathogenic fungus Botrytis cinerea by 28 % and 31.6 %, respectively. With CHIT60 (100 microg ml(-1)), the effect was more pronounced: 78 % inhibition of of germination and 63.9 % inhibition of germ tube elongation.