Department of Preventive Dentistry, Kyushu University Faculty of Dental Science, Fukuoka 812-8582, Japan1
Author for correspondence: Yoshio Nakano. Tel: +81 92 642 6423. Fax: +81 92 642 6354. e-mail: yosh{at}dent.kyushu-u.ac.jp
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ABSTRACT |
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Keywords: L-cysteine desulfhydrase, hydrogen sulfide
Abbreviations: RBC, sheep erythrocyte
The DDBJ accession number for the Streptococcus anginosus lcd gene sequence reported in this paper is AB084812.
a Deceased, October 14, 2001. This paper is in recognition of his fine work and is dedicated to his memory.
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INTRODUCTION |
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It has been reported that hydrogen sulfide is produced from L-cysteine by the enzymic action of L-cysteine desulfhydrase, which catalyses the ,ß-elimination of L-cysteine to hydrogen sulfide, pyruvate and ammonia (Guarneros & Ortega, 1970
). Since hydrogen sulfide is highly toxic for mammalian cells (Beauchamp et al., 1984
) and induces the modification and release of haemoglobin in erythrocytes (Kurzban et al., 1999
), enzymes participating in the production of hydrogen sulfide in the subgingival sulcus might contribute to the initiation and progression of periodontal diseases, and to abscess formation (Carlsson et al., 1993
; Claesson et al., 1990
; Persson et al., 1990
). Based on this hypothesis, investigations have focused on Gram-negative periodontopathogenic bacteria, as the main organisms capable of producing hydrogen sulfide (Ratcliff & Johnson, 1999
), while little attention has been paid to other bacteria, even though they also have the capacity to produce hydrogen sulfide (Persson et al., 1990
). Consequently, there have been few published reports on the enzymes associated with the production of hydrogen sulfide by oral bacteria other than periodontopathogenic bacteria.
Recently, we observed that the crude enzyme extract obtained from S. anginosus has a higher capacity to produce hydrogen sulfide from L-cysteine than extracts from other oral streptococci, including Streptococcus mutans, Streptococcus sobrinus, Streptococcus oralis, Streptococcus gordonii and Streptococcus salivarius. Based on this finding and clinical reports that S. anginosus is frequently isolated from several infectious sites (Fisher & Russell, 1993 ; Whiley et al., 1992
), the high capacity of S. anginosus to produce hydrogen sulfide might play an important role in the pathogenicity of this micro-organism. In this study, we cloned the gene encoding L-cysteine desulfhydrase in S. anginosus and characterized its product. In addition, the haemolytic activity of this enzyme resulting from the modification of haemoglobin by hydrogen sulfide was also determined.
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METHODS |
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DNA manipulation.
Standard DNA recombinant procedures, such as DNA isolation, endonuclease restriction digestion and ligation, were carried out as described by Sambrook et al. (1989) . Chromosomal DNA of S. anginosus was prepared as described by Perry et al. (1983)
.
Purification of L-cysteine desulfhydrase from S. anginosus FW73.
S. anginosus FW73 was grown to an OD550 value of about 1·0. The cells were harvested and then washed with ice-cold cell-suspension buffer (50 mM Tris/HCl, 10 mM PMSF, 10 mM EDTA, 10 µM pyridoxal 5'-phosphate, pH 7·5). A 1 ml aliquot of the cell suspension was transferred to a screw-top microcentrifuge tube containing 1·0 g glass beads (0·10·15 mm diameter; Biospec Products). The cells were vortexed with the glass beads for 45 s 10 times at 1 min intervals. After vortexing, the glass beads were allowed to settle before the supernatant was transferred to a clean tube. The supernatant was centrifuged at 18000 g for 30 min at 4 °C. To the supernatant, solid ammonium sulfate was slowly added to 70% saturation. After a 20 min incubation at 4 °C, the suspension was centrifuged at 10000 g for 10 min at 4 °C. The precipitate was then dissolved in ice-cold cell-suspension buffer.
Initial purification was performed on a 2·5x46 cm gel filtration column [TSKgel TOYOPEARL HW-55 (F); Tosoh], equilibrated with a buffer consisting of 50 mM Tris/HCl (pH 7·5), 0·1 M NaCl, 0·1 mM EDTA and 10 µM pyridoxal 5'-phosphate. The column was loaded with 4 ml of the cell extract obtained as described above. The column was run at 0·25 ml min-1 at 4 °C, and 2·0 ml fractions were collected. Fractions containing L-cysteine desulfhydrase were identified as described below and then concentrated using a Centricon YM-30 (Amicon) with a 30000 Da cut-off. The retentate was processed for HPLC with an anion-exchange column (TSKgel DEAE-5PW, 0·8x7·5 cm; Tosoh). A linear gradient (00·4 M) of NaCl in 50 mM Tris/HCl buffer (pH 7·0) was used at a flow rate of 1 ml min-1 at 25 °C, and 2·0 ml fractions were collected. Fractions containing L-cysteine desulfhydrase were subjected to HPLC with a hydrophobic column (TSKgel Phenyl-5PW, 0·75x7·5 cm; Tosoh). The protein was eluted with a linear gradient of 750% ammonium sulfate in 100 mM sodium phosphate buffer (pH 6·8). The column was run at 1·0 ml min-1 at 25 °C, and 2·0 ml fractions were collected. Fractions containing L-cysteine desulfhydrase were concentrated using a Centricon YM-30. The retentate was then subjected to HPLC with a hydroxyapatite column (Bio-Scale CHT-1; Bio-Rad) equilibrated with 10 mM sodium phosphate buffer (pH 6·8). After sample application, the column was washed with 10 ml of the same buffer at a flow rate of 1 ml min-1. A linear 0500 mM NaCl gradient in 30 ml of the same buffer was passed through the column, and fractions (2 ml) containing L-cysteine desulfhydrase were collected.
Visualization of enzyme activities.
The presence of L-cysteine desulfhydrase in the HPLC fractions was confirmed by visualizing enzyme activities (Claesson et al., 1990 ). Briefly, 30 µl of each HPLC fraction were added to 150 µl of the visualizing solution, which consisted of 100 mM triethanolamine/HCl (pH 7·6), 10 µM pyridoxal 5'-phosphate, 0·5 mM bismuth trichloride, 10 mM EDTA, 1% Triton X-100 and 5·0 mM L-cysteine. The reaction mixture was incubated at 37 °C to detect the black precipitate formed by the reaction of sulfide with bismuth. L-Cysteine desulfhydrase activity was also detected in non-denaturing polyacrylamide gels. The samples were electrophoresed at 20 mA per gel at 4 °C for 2 h on 12·5% resolving (pH 8·8) and 3% stacking (pH 6·5) polyacrylamide gels. After electrophoresis, the gel was incubated in 10 ml of the visualizing solution at 37 °C to detect a black band at the enzyme position.
N-terminal sequencing of L-cysteine desulfhydrase.
The HPLC fraction containing L-cysteine desulfhydrase was subjected to non-denaturing PAGE. After electrophoresis, proteins in the gel were transferred to a PVDF membrane (Millipore), as described by Towbin et al. (1979) , and stained with Coomassie brilliant blue. The protein band corresponding to L-cysteine desulfhydrase was excised from the membrane and destained with a solution containing 60% methanol and 10% acetic acid. The N-terminal amino acid sequence of L-cysteine desulfhydrase was determined by Edman degradation at the Takara Custom Service Center in Shiga, Japan. The FASTA program was used to perform a homology search of protein databases (Lipman & Pearson, 1985
).
DNA sequencing of lcd of S. anginosus.
Primers that amplified lcd from S. anginosus were constructed using the S. mutans genomic DNA database (forward primer, 5'-AAGGAGATGCTGTTTTAATTAAC-3'; reverse primer, 5'-AGCCAAACCAAATAAGTACCTT-3'). The 600 bp fragment amplified by PCR was cloned into the pGEM-T Easy vector (Promega). The nucleotide sequence was confirmed by the dideoxy chain termination technique of Sanger et al. (1977) with an ABI 310 Genetic Analyser (Perkin-Elmer). Inverse PCR was used to isolate flanking regions of the 600 bp fragment. To obtain the DNA sequence upstream of the 600 bp fragment, the genomic DNA of S. anginosus was digested with MspI and a dilution of the DNA sample was self-ligated for 12 h at 16 °C. The sample from the ligation reaction was then amplified under standard PCR conditions using a forward primer (LCD-1, 5'-AAAGGTTAAACAGGAGA-3') and a reverse primer (LCD-2, 5'-ATTGTCGAAAATAATGTGAA-3'), to obtain a 800 bp fragment that included 180 bp from the originally amplified 600 bp PCR product. Primers LCD-1 and LCD-2 were designed on the basis of the 180 bp sequence. To obtain the DNA sequence downstream of the original amplicon, the genomic DNA of S. anginosus was digested with BfaI, and a dilution of the DNA sample was self-ligated. The sample from the ligation reaction was then amplified using a forward primer (LCD-3, 5'-CTAACCAAGGTTTTCCATAG-3') and a reverse primer (LCD-4, 5'-GTTAAAAGTTATGAAACCAC-3'), to obtain a 600 bp fragment that included 130 bp from the original PCR product. Primers LCD-3 and LCD-4 were designed on the basis of the 130 bp sequence. The resulting amplicons were cloned into the pGEM-T Easy vector and sequenced.
Purification of recombinant L-cysteine desulfhydrase of S. anginosus.
The recombinant L-cysteine desulfhydrase was purified using the expression vector pGEX-6P-1 (Amersham). To construct a pGEX-6P-1 derivative, designated pMILCD110, the lcd gene was amplified by PCR (forward primer, 5'-TCCGGATCCCGCAAATACAATTTTCAAAC-3'; reverse primer, 5'-GACGTCGACTTATTGCGCCAAACAATGCA-3'). These primers were designed so that BamHI and SalI restriction sites (bold) were created in the PCR product. E. coli BL21 was transformed with pMILCD110 and grown in 50 ml of 2xTY broth with ampicillin at 37 °C until an OD550 value of 1·0 was attained. Expression was induced with 0·1 mM IPTG. The cells were harvested 1·5 h after induction and lysed by ultrasonication. The cell extract was obtained by centrifugation at 18000 g for 30 min at 4 °C. Binding to glutathione-Sepharose 4B medium (Amersham), cleavage of the fusion protein by using PreScission protease and elution of the product were performed according to the manufacturers instructions. The purified proteins were mixed with 50% (v/v) glycerol and stored at -20 °C until used. The purity of the protein was analysed by SDS-PAGE.
Identification of the degradation products from several substrates.
The recombinant L-cysteine desulfhydrase activity was assayed with a reaction mixture containing 50 mM potassium phosphate buffer (pH 8·0) with 10 µM pyridoxal 5'-phosphate and 4·5, 225 or 1125 µg l-1 of the recombinant L-cysteine desulfhydrase with various amounts of L-cysteine or the substrates listed in Table 1. The formation of the following compounds was then used to examine the L-cysteine desulfhydrase activity: (a) sulfur compounds, such as hydrogen sulfide, methyl mercaptan, homocysteine and cysteamine; (b) pyruvate; (c) ammonia.
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(b) Pyruvate formation was detected as described previously (Soda, 1968 ; Yoshimura et al., 2000
) (see enzyme assay below).
(c) The formation of ammonia as an end product was determined using Ammonia-testWako (Wako Pure Chemical Industries). The reaction mixture in the final volume of 100 µl contained 20 nmol of each substrate listed in Table 1 and 22·5 ng of the recombinant L-cysteine desulfhydrase.
Enzyme assay.
The general approach for determining the kinetic properties of L-cysteine desulfhydrase followed the method of Yoshimura et al. (2000) . The kinetic parameters (Km and Vmax) were computed from the LineweaverBurk transformation (V-1 versus S-1) of the MichaelisMenten equation, where V is the formation rate of pyruvate (µmol min-1 mg-1) and S is the concentration (M) of each substrate listed in Table 1
. The assay was carried out with 500 µl potassium phosphate buffer (pH 8·0) containing 5 nmol of pyridoxal 5'-phosphate, 111·5 ng of the purified L-cysteine desulfhydrase and various amounts of L-cysteine or the substrates listed in Table 1
. After 10 min incubation at 37 °C, the reaction was terminated by adding 250 µl of 4·5% trichloroacetic acid. The reaction mixture was centrifuged, and 250 µl of the supernatant was added to 750 µl of 0·33 M sodium acetate (pH 5·2) containing 0·017% 3-methyl-2-benzothiazolinone hydrazone. The reaction mixture was then incubated at 50 °C for 30 min (Soda, 1968
). The amount of pyruvate was determined at A335. The amount of protein was determined using the Bio-Rad protein assay reagent with bovine serum albumin as a standard.
Assay of haemoglobin release and modification.
Sheep erythrocytes (RBCs) were washed three times with PBS (0·12 M NaCl, 0·01 M Na2HPO4, 5 mM KH2PO4, pH 7·5), to remove soluble haemoglobin from them before they were used. To obtain isolated haemoglobin, the washed RBCs were centrifuged at 3000 g and then lysed with the same volume of distilled water. The reaction mixture consisted of 0·12 M NaCl, 0·01 M Na2HPO4, 5 mM KH2PO4 (pH 7·5), 10 µM pyridoxal 5'-phosphate, 25 ng purified L-cysteine desulfhydrase ml-1 and 4% (v/v) RBCs or 4% (v/v) isolated haemoglobin, with 0, 1, 3 or 6 mM L-cysteine. The reaction mixture of the positive control contained 70 µg sodium hydrosulfide n-hydrate ml-1 instead of both L-cysteine and the enzyme. The concentration of hydrogen sulfide produced by the degradation of sodium hydrosulfide n-hydrate was estimated to be 6 mM. The reaction mixture of the negative control contained 6 mM pyruvate or 6 mM L-cysteine without the enzyme. The mixture was incubated at 37 °C with slow rotation. Samples (1 ml) were withdrawn at 0, 1, 2, 3, 6, 7, 9 and 11 h, and then centrifuged at 15000 g for 3 min for spectrum analysis. The supernatants of the reaction mixtures including RBCs were used to observe the changes in the amounts of haemoglobin and modified forms of haemoglobin, such as methaemoglobin, sulfhaemoglobin, sulfmethaemoglobin and choleglobin, released from RBCs (Kurzban et al., 1999 ). The pellets of the mixtures including RBCs were washed with PBS and then lysed by the addition of distilled water (1 ml). These samples were then centrifuged at 15000 g for 3 min and used to show the haemoglobin modification in unbroken RBCs. The samples including isolated haemoglobin were used to demonstrate the changes in the total amounts of haemoglobin and modified haemoglobins. Spectral scans were made from 300 to 680 nm in 1 nm steps using an Ultraspec 2000 UV/Visible spectrophotometer (Amersham). The changes in absorbance at fixed wavelengths (540 and 620 nm) were recorded to measure the amounts of haemoglobin and modified forms of haemoglobin, respectively.
Nucleotide sequence accession number.
The sequence reported here was submitted to the EMBL and GenBank databases through the DDBJ and assigned accession no. AB084812.
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RESULTS |
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Cloning and characterization of the gene encoding L-cysteine desulfhydrase from S. anginosus
Using the gene sequences from S. pneumoniae and S. mutans, PCR primers were designed to clone the gene homologue from S. anginosus. The amplified fragment was cloned and sequenced. However, the entire ORF was not contained in the 600 bp fragment amplified by PCR. Therefore, inverse PCR was used to isolate DNA sequences upstream and downstream of the 600 bp fragment. The amplified fragments were cloned into the pGEM-T Easy vector and sequenced. The gene was 1164 bp long, and a possible ShineDalgarno sequence (Shine & Dalgarno, 1974 ) was identified just upstream of the potential start codon. We named this gene lcd. The amino acid sequence deduced from lcd showed 34·0 and 30·6% identity with the sequences of Treponema denticola cysteine desulfhydrase (Chu et al., 1995
) and E. coli ßC-S lyase (Zdych et al., 1995
), respectively (Fig. 2
).
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Enzymic characterization of the lcd gene product
The formation of hydrogen sulfide, ammonia and pyruvate from L-cysteine by the recombinant L-cysteine desulfhydrase was confirmed. To identify substrates other than L-cysteine for the recombinant L-cysteine desulfhydrase of S. anginosus, several compounds were assayed. As shown in Table 1, incubation of L-cystine with recombinant L-cysteine desulfhydrase resulted in the formation of the same end products as from L-cysteine, namely hydrogen sulfide, ammonia and pyruvate. Incubation of S-methyl-L-cysteine, S-(2-aminoethyl)-L-cysteine and DL-cystathionine with recombinant L-cysteine desulfhydrase resulted in the formation of ammonia and pyruvate, while no hydrogen sulfide formation was confirmed. GC analysis of the S-methyl-L-cysteine interaction with the recombinant L-cysteine desulfhydrase revealed that methyl mercaptan was produced instead of hydrogen sulfide. By contrast, cysteamine and homocysteine were detected as end products of S-(2-aminoethyl)-L-cysteine and DL-cystathionine, respectively, by reversed-phase HPLC. Incubation of 3-chloro-DL-alanine with recombinant L-cysteine desulfhydrase resulted in the formation of ammonia and pyruvate, although this amino acid lacks a ßC-S linkage. D-Cysteine, DL-homocysteine, DL-serine, DL-alanine, L-cysteine methyl ester, L-tryptophan, L-tyrosine, L-phenylalanine and L-methionine were not substrates for the recombinant L-cysteine desulfhydrase under the experimental conditions tested.
The breakdown of substrates by the recombinant enzyme was determined by assaying the production of pyruvate, as described by Soda (1968) . The Km and Vmax values of the recombinant L-cysteine desulfhydrase from S. anginosus FW73 are shown in Table 1
. The values for cystathionine and 3-chloro-alanine were calculated for the mixtures of D- and L-isomers.
Release and modification of haemoglobin
We examined the changes in the amounts of haemoglobin and modified haemoglobins in haemoglobin samples when they were incubated with L-cysteine and the purified recombinant L-cysteine desulfhydrase. Absorption spectra of the samples including isolated haemoglobin are shown in Fig. 3. The spectrum of the negative control (no L-cysteine, no hydrogen sulfide) was unchanged during incubation. The spectrophotometric assay using the sample containing either only pyruvate or L-cysteine as a negative control exhibited a similar pattern (data not shown). Incubation of the isolated haemoglobin with L-cysteine and the purified recombinant L-cysteine desulfhydrase resulted in spectral changes. Bands at 414 (Soret band), 540 and 576 nm dropped in intensity with the appearance of a band in the visible region at 620 nm. Isolated haemoglobin exposed to hydrogen sulfide as a positive control for 1 h underwent the same spectral changes as in incubation for 7 h with L-cysteine and the purified recombinant L-cysteine desulfhydrase. These findings indicated that haemoglobin in the reaction mixture was changed to modified types of haemoglobin (methaemoglobin, sulfhaemoglobin, sulfmethaemoglobin and choleglobin) (Kurzban et al., 1999
). The changes in the absorbance readings at fixed wavelength (540 and 620 nm) are shown in Fig. 4
. In the presence of both L-cysteine and L-cysteine desulfhydrase, the total amount of isolated haemoglobin decreased with time (Fig. 4a
). By contrast, the total amount of modified haemoglobins increased. More modified haemoglobins were observed as the concentration of added L-cysteine increased (Fig. 4b
).
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DISCUSSION |
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The amino acid sequence deduced from the lcd gene showed 34·0% identity with the T. denticola Hly protein (Chu et al., 1995 ) and 30·6% identity with the E. coli MalY protein (cystathionase) (Zdych et al., 1995
) (Fig. 2
). The Hly protein, a 46 kDa haemolysin, is capable of removing the thiol and amino groups from certain sulfur-containing compounds to produce ammonia and pyruvate (Chu et al., 1995
, 1997
); L-cysteine, L-cystine, S-(2-aminoethyl)-L-cysteine and cystathionine are substrates for the enzyme (Chu et al., 1997
). The MalY protein is a 4244 kDa pyridoxal-5'-phosphate-dependent enzyme with ßC-S lyase activity, which forms ammonia and pyruvate from amino acids containing a ßC-S linkage (Zdych et al., 1995
); L-cystine, S-(2-aminoethyl)-L-cysteine, L-cystathionine and lanthionine are substrates for the enzyme (Zdych et al., 1995
). The Km value of Hly for L-cysteine was reported to be 3·6 mM (Chu et al., 1997
), which is considerably higher than that of Lcd of S. anginosus (0·62 mM). By contrast, the Km value of MalY for L-cystine was reported to be 1·7 mM (Zdych et al., 1995
), which is also high compared with that of Lcd (1·29 mM). The lcd product also showed weak homology to the metC products in E. coli (Belfaiza et al., 1986
) and Lactococcus lactis (Fernández et al., 2000
) (17 and 14%, respectively). MetC can degrade cystathionine and is involved in methionine biosynthesis. The homology together with the low Km value for cystathionine (Table 1
) suggest that the lcd product may contribute to methionine biosynthesis in the cell.
Pyridoxal 5'-phosphate was identified as a cofactor of pyridoxal-5'-phosphate-dependent enzymes, forming a Schiff base with the -amino group of the active site. Pyridoxal-5'-phosphate-dependent enzymes have been classified into the
, ß and
families based on sequence alignments and the construction of protein profiles (Alexander et al., 1994
). All four amino acid residues that are invariant in the comprehensive alignment of aminotransferases belonging to the
family (Mehta & Christen, 1993
) are conserved in Lcd (Fig. 2
). Of these four residues, the lysine residue that binds to the cofactor pyridoxal 5'-phosphate is found in Lcd at position 234 (Fig. 2
), and is located between residues 209 and 256 in various members of the
family (Alexander et al., 1994
). In addition, six of the eight residues that are found in most aminotransferases in the
family are conserved (Mehta et al., 1989
; Mehta & Christen, 1993
; Alexander et al., 1994
) (Fig. 2
). These findings indicate that Lcd belongs to the
family of the pyridoxal-5'-phosphate-dependent enzymes.
Chemical analysis revealed that enzymic cleavage of L-cysteine occurred in a dose-dependent manner and produced hydrogen sulfide, ammonia and pyruvate. Besides L-cysteine, sulfur amino acids containing a ßC-S linkage, such as DL-cystathionine, L-cystine, S-(2-aminoethyl)-L-cysteine and S-methyl-L-cysteine, were cleaved at the C-N and ßC-S linkages, producing pyruvate, ammonia and other sulfur-containing molecules. In contrast, the enzyme failed to catalyse
C-S elimination reactions and also failed to cleave all of the tested amino acids that have no ßC-S linkages, with the single exception of 3-chloro-DL-alanine. Interestingly, incubation of 3-chloro-DL-alanine, which is an amino acid without a ßC-S linkage, with L-cysteine desulfhydrase resulted in the formation of ammonia and pyruvate. This might be ascribed to the structural identity of 3-chloro-alanine to cysteine; the difference is that the S- ion at the C-3 position is replaced with a Cl- ion.
Exposure of RBCs to both L-cysteine and L-cysteine desulfhydrase or to hydrogen sulfide resulted in the release of haemoglobin and the production of modified types of haemoglobin, while incubation of RBCs with any one of L-cysteine, L-cysteine desulfhydrase or pyruvate did not result in the same phenomena. Based on these findings, it is likely that the hydrogen sulfide produced from L-cysteine by the enzymic action of L-cysteine desulfhydrase plays an important role in the release and modification of haemoglobin. It is very interesting that the amounts of modified haemoglobins in the unbroken RBCs began to decrease after an initial 6 h increase when incubated with 6 mM L-cysteine (Fig. 5b), and that the release of haemoglobin and modified haemoglobins from RBCs into the supernatant started at the same time (Fig. 6a
, b
). In the case of the sample including 3 mM L-cysteine, these phenomena were also observed after a 9 h incubation. These results obviously show that haemoglobin was modified in RBCs, but was not released from RBCs for at least 6 or 9 h under the experimental conditions tested. Therefore, the formation of modified haemoglobin seems to play an important role in haemolysis. These findings are consistent with a general mechanism that connects sulfhaemoglobin formation to erythrocyte lysis (Moxness et al., 1996
). However, the effects of hydrogen sulfide on RBC lysis might not be due solely to the presence of modified haemoglobins, because hydrogen sulfide can be cytotoxic and can lyse other types of mammalian cells.
In conclusion, we cloned the lcd gene encoding L-cysteine desulfhydrase from S. anginosus and then purified and characterized the gene product. This enzyme cleaved substrates at C-N and ßC-S linkages, producing pyruvate, ammonia and sulfur-containing molecules. In addition, the hydrogen sulfide produced by this enzyme and L-cysteine affect the release and modification of haemoglobin in RBCs. Further studies are necessary to clarify why the enzyme obtained from S. anginosus has such a high capacity to produce hydrogen sulfide from L-cysteine in comparison with the enzymes from other oral streptococci, and how this high capacity to produce hydrogen sulfide is associated with the virulence of the organism.
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ACKNOWLEDGEMENTS |
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Received 20 May 2002;
revised 2 August 2002;
accepted 13 September 2002.
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