Antagonistic mechanism of sulfhydryl compounds on cellocidin activity.

Cellocidin is an antibacterial antibiotic discovered by SUZUKI et al.1) Although the antibiotic shows an excellent preventive effect against rice bacterial leaf blight, its use is restricted because of its phytotoxicity to rice plants. OKIMOTO and MISATO2) demonstrated cellocidin inhibited the metabolism of a-keto-glutarate to succinate in Xanthomonas oryzae. TANAKA et al.3) also found that cellocidin inhibited the incorporation of 14C-thymidine into the DNA of Bacillus subtilis. On the other hand, antibacterial activity of cellocidin is known to be blocked by sulfhydryl compounds such as cysteine, glutathione, etc.2) This paper describes the antagonistic mechanism of sulfhydryl compounds on cellocidin activity. Effect of cellocidin on the thiol-disulfide exchange reaction between 5,5-dithio bis (2-nitrobenzoic acid) [DTNB] and reduced glutathione was examined. The reaction mixture consisted of 2.0 ml of 0.1 M phosphate buffer pH 7.6 containing 0.3 mm ethylenediaminetetraacetate, 0.2 ml each of 4 mm DTNB and 0.5 or 1.0 mM reduced glutathione, 0.5 ml of cellocidin solution and water in a final volume of 4.0 ml. The reaction was initiated by the addition of DTNB in a cuvette of 10-mm light path. After 10-minute incubation, absorbance increase was measured at 412 nm by a Shimadzu UV-200 spectrometer. Thiol-disulfide exchange reaction between glutathione and DTNB was markedly blocked at a 101ag/ml of cellocidin as shown in Table 1. This result suggested that cellocidin reacted to sulfhydryl compounds chemically. The reaction of cysteine to cellocidin was made by stirring the mixture of 10 mmol cysteineHCl, 10 mmol cellocidin and 0.01 N NaOH in a volume of 1.0 liter at 25°C for 5 hours. The reactants were chromatographed on a silica gel TLC by n-butanol acetic acid water (4 : 1 : 1, v/v), and were detected with ninhydrin reagent. Besides cysteine and cystine spots, two ninhydrinpositive spots were observed, and their Rf values were 0.64 (designated compound 1) and 0.45 (designated compound II). In order to isolate the two products, the reaction mixture was concentrated and applied to an Avicel column. The column was eluted with n-butanol acetic acid water (2 : 1 : 1, v/v), and the eluate was fractionated in each 15 ml. Two ninhydrin-positive fractions other than cysteine and cystine were collected and concentrated, and each product was purified by crystallization. Compounds I and II obtained as needle crystals were watersoluble under acid or alkaline conditions. Antibacterial activities were not observed for these compounds even at a concentration of 100,/(g/ml in vitro. Melting points of compounds I and II were 193°C (decomp.) and 205~207°C (decomp.), respectively. The UV absorption spectra were measured in 0.01 N NaOH according to SUZUKI et al.1), and the maximum absorptions were 290 nm for compound I and 285 nm for compound II. The IR spectra of both compounds showed the broad absorptions for amino acids at 3400~ 2400cm 1, and the specific absorptions for -NH3+ -COOand RCONH2 , but not the absorptions for R-SH with cysteine. These results suggested that sulfhydryl group of cysteine might be bound to cellocidin. Elemental analytic data of compounds I and II gave the composition formulae of C14H24O9N6S2 and C10H20O7N4S2, respectively. Considering these elemental analyses on the basis of NMR analytic data described below, the molecular formulae were estimated as C7H11O4N3S•JH2O for compound I and C10H18O6N4S2 • H2O for compound II. The NMR spectrum was recorded on a Nihondenshi C-60 HL spectrometer by using D2O solution containing DCl. The NMR spectra of