A lysosomal fraction from polymorphonuclear leukocytes has been directly analyzed for basic proteins by zone electrophoresis. Evidence for the presence of bactericidal basic proteins in polymorphonuclear leukocyte lysosomes is presented.
A basic proteins fraction from guinea pig polymorphonuclear (PMN) granules was obtained by acid extraction and precipitation with 20% (v/v) ethyl alcohol. The fraction accounted for most of the antibacterial activity of the PMN granules and corresponded to the antibacterial cationic components of intact granules (bands I, II, and III) resolved by zone electrophoresis. Absence from the fraction of components identical to the enzymatic components of intact lysosomes showed that the fraction was essentially free from lysosomal enzymes. The amino acid analysis of proteins in the fraction gave a preponderance of basic amino acids (25 %), especially of arginine (16%). The comparative amino acid analysis showed that the lysosomal cationic proteins (LCP) fraction was markedly different from nuclear histones. The LCP fraction manifested antibacterial activity against certain gram-negative and gram-positive microorganisms, including Candida albicans, and exhibited stoichiometric relationship in its activity. Microorganisms treated with LCP fraction were agglutinated. Anionic substances such as nucleic acids, heparin, and endotoxin effectively blocked the antibacterial activity of the fraction. The LCP fraction caused suppression of oxygen uptake by bacterial cells and damaged the permeability barriers of cells as manifested by rapid release of P32 as well as ultraviolet-absorbing material at 260 m,u, in the supernatant fluid.
Cationic proteins of polymorphonuclear leukocyte lysosomes. I. Resolution of antibacterial and enzymatic activities. J. Bacteriol. 91:750-754. 1966.-A lysosomal fraction from polymorphonuclear (PMN) leukocytes of guinea pig peritoneal exudate was subjected directly to electrophoresis on cellulose acetate paper treated with cetyltrimethyl ammonium bromide. The lysosomal components resolved into seven bands moving towards the cathode. Assay of the eluted bands showed that the antibacterial activity was distinct from lysosomal enzymes and was associated with three cationic components (bands I, II, and III) which migrated most rapidly towards the cathode, ahead of lysozyme ribonuclease and deoxyribonuclease. Qualitatively, the antibacterial components appeared to be rich in arginine. The antibacterial components were absent in the pherograms of nuclear fractions of PMN leukocytes and in supernatant fractions that remained after lysosomes were removed from cell homogenates by centrifugation at 8,000 X g.
Previously we reported the isolation from polymorphonuclear leukocyte (PMN) granules of a group of highly basic proteins, the electrophorefie heterogeneity of which varies from species to species (1--4). These proteins as a group have antibacterial activity against a wide variety of microorganisms, and may also contribute to the tissue-damaging properties of PMN (5-11) as well as their pyrogenicity (12) and anticoagulant activities (13,14).During the past 80 yr, workers have repeatedly observed that cationic proteins may possess antimicrobial effects, and it has also been found repeatedly that antimicrobial extracts can be prepared from PMN (15). Observations by Skarnes and Watson (16), by Hirsch (17), and by Cohn and Hirsch (18) are especially pertinent to the results reported in this paper. Skames (16) isolated antimicrobial cationic proteins from autolyzed PMN that he believed were derived from nucleohistone; this material he caUed leukin. Hirsch (17) recovered antimicrobial activity in citric acid extracts of PMN suspensions. He attributed this to a cytoplasmic constituent of PMN which he named phagocytin. At first, both preparations were believed to show specific antimicrobial action, related to Gram reactivity of the microbial species tested. Although the concept of antimicrobial specificity was later abandoned, Cohn and Hirsch succeeded in showing that the cytoplasmic membrane-bonded granules--the lysosome fraction--of PMN contained, in addition to acid hydrolase activity, considerable antimicrobial phagocytin activity (18). Spitznagel and Chi (19) found that PMN lysosomes were rich in cationic protein and concluded that the antibacterial action of PMN lysosomes was associated with transfer of cationic proteins to ingested bacteria. Zeya and Spitznagel (1) showed that the antimicrobial action of PMN lysosomes is associated with their electrophoretically separable cationic proteins. They found that PMN lysosomes contain not one but several basic proteins that are different from nueleohistones and much *
Diflerences in antimicrobial specificities against Staphylococcusaureus, Streptococcus faecalis (groupD),and Proteus vulgaris exist amongthe electrophoretically separable components of lysosomal cationic proteins of polymorphonuclear leukocytes.
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