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 protein, as deduced from its gene sequence, were used to devise a procedure 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-terminus. Consequently, the absorption coefficient at 280 nm, the isoelectric point, and the molecular mass of the immunity protein may be calculated to be, respectively, 8.2 x 10(3) M-1 cm-1, 10.2 and 11,163 Da from the amino acid sequence predicted from the nucleotide sequence. The immunity protein is a major cell protein component--one cell may contain (to an order of magnitude) 10(5) molecules--and it is in part associated with the cell membrane, as judged by immunoblot analysis of membrane vesicle-associated proteins. Exposing lactococcin-A-sensitive cells to an excess of the immunity protein did not affect the lactococcin-A-induced killing of the cells, indicating that the immunity protein does not protect cells by simply binding to lactococcin A, nor to externally exposed domains on the cell surface. Exposing immune-positive cells to antiserum against the immune protein did not sensitize the cells to lactococcin A, suggesting that the immunity protein in fact does not act extracellularly.
Laboratory of Microbial Gene Technology, NFR, Agricultural University of Norway, As.
J
J
Nissen-Meyer
L S
LS
Håvarstein
H
H
Holo
K
K
Sletten
I F
IF
Nes
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins; D001430:Bacteriocins / Q000378:metabolism; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000737:chemistry*; D013294:Lactococcus lactis / Q000737:chemistry*; D008565:Membrane Proteins; D008969:Molecular Sequence Data
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001430:Bacteriocins; D008565:Membrane Proteins; C083085:lactococcin A immunity factor, bacteria