Characterization of Pesticin

Pesticin was prepared from cell-free extracts of Yersinia pestis strain All22 by a procedure involving fractionation with (NHa)nS04 and chromatography with Sephadex G-200, diethylaminoethylcelulose, and calcium hydroxylapatite. A single antibacterial fraction (a-pesticin) was recovered from extracts of uninduced yersiniae, whereas extracts of mitomycin C-induced cells yielded an additional active fraction (/3-pesticin) at the final step of purification. Antibacterial specificity and specific activity of the two fractions were identical; their absorption spectra were typical of simple proteins and neither contained significant carbohydrate (measured as hexose or hexosamine). Both pesticins were monomers with a s20,W of 4.4 and molecular weight of about 65,000 as determined by equilibrium sedimentation and gel filtration. in guanidine hydrochloride. In addition, both preparations exhibited similar ratios of amino acids (both lacked detectable cysteine) and possessed common primary structure as judged by peptide analysis of tryptic digests. Isoelectric points of 5.49 and 5.87 were determined for (Yand P-pesticin, respectively. Although cyand P-pesticin were interconvertable in Vitro, with equilibrium favoring formation of cr-pesticin, both forms became rapidly altered to a third form, termed y-pesticin. The latter was eluted on calcium hydroxylapatite after /3-pesticin and was without significant biological activity. The three forms of pesticin were antigenically identical as judged by immunodiffusion. The results suggested that pesticin, like certain cysteinedeficient colicins, can exist in conformer states.