ASSOCIATION OF THE LACTOCOCCIN-A IMMUNITY FACTOR WITH THE CELL-MEMBRANE - PURIFICATION AND CHARACTERIZATION OF THE IMMUNITY FACTOR

The physicochemical characteristics of the lactococcin A immunity prot ein, as deduced from its gene sequence, were used to devise a procedur e for its purification. The protein was purified from cell extracts by cation-exchange and reverse-phase chromatography. As judged from the amino acid composition and amino acid sequencing, the immunity protein is not post-translationally processed by cleavage at its N- or C-term inus. Consequently, the absorption coefficient at 280 nm, the isoelect ric point, and the molecular mass of the immunity protein may be calcu lated to be, respectively, 8.2 x 10(3) M-1 cm-1, 10.2 and 11163 Da fro m the amino acid sequence predicted from the nucleotide sequence. The immunity protein is a major cell protein component - one cell may cont ain (to an order of magnitude) 10(5) molecules - and it is in part ass ociated with the cell membrane, as judged by immunoblot analysis of me mbrane vesicle-associated proteins. Exposing lactococcin-A-sensitive c ells to an excess of the immunity protein did not affect the lactococc in-A-induced killing of the cells, indicating that the immunity protei n does not protect cells by simply binding to lactococcin A, nor to ex ternally exposed domains on the cell surface. Exposing immune-positive cells to antiserum against the immune protein did not sensitize the c ells to lactococcin A, suggesting that the immunity protein in fact do es not act extracellularly.