Related Experiment Videos
Necrosin: purification and properties of a cytotoxin derived from a murine macrophage-like cell line
Abstract:
An acidic proteinaceous cytotoxin was isolated from the serum-free, cell-free supernatants of J774.1 (a murine macrophage-like line) cells that had been treated with bacterial endotoxin. Cytotoxic activity was routinely monitored using the sensitive murine tumorigenic fibroblast line L-M. The toxin was purified 8000-fold by ion-exchange and electrophoretic procedures. It was purified to greater than 90% homogeneity, as assessed by photometric scanning of silver-stained NaDodSO4/polyacrylamide gels. The specific activity of the purified toxin was 32,000 units/microgram. The toxin was composed of self-aggregating non-sulfhydryl-linked multimers of a Mr 15,000 subunit. The pI of the monomer was 4.6. The active multimeric forms, as assessed by gel filtration and assayed using L-M cells, were of Mr 70,000 and 55,000. These forms were identical to those observed both in crude supernatants and in purified fractions that had not been subjected to denaturing agents. We call these forms "holotoxins" and conclude that they are aggregates of the Mr 15,000 protein. The purified toxin (1-1000 pM, 0.06-60 ng/ml) was active against a random assortment of tumorigenic and normal cell lines of murine, bovine, and human origin. For example, the diploid bovine endothelial line CPAE was nearly as sensitive as the L-M line. Similarities to other toxic macrophage products, lymphotoxin, and tumor necrosis factor are discussed.
Insights
Researchers isolated an acidic protein cytotoxin from endotoxin-treated macrophage cells. This potent toxin, active against various cell lines, exists as self-aggregating multimers of a 15,000 molecular weight subunit.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Macrophage cells, when stimulated by bacterial endotoxin, produce and release various bioactive molecules.
- Understanding these secreted factors is crucial for elucidating immune responses and host defense mechanisms.
Purpose of the Study:
- To isolate and characterize a novel acidic proteinaceous cytotoxin from J774.1 macrophage cell supernatants.
- To determine the purification, molecular properties, and cytotoxic activity of the isolated toxin.
Main Methods:
- Isolation and purification of the cytotoxin using ion-exchange chromatography and electrophoresis.
- Assessment of purity and molecular weight via NaDodSO4/polyacrylamide gel electrophoresis and gel filtration.
- Cytotoxicity assays using murine tumorigenic fibroblast L-M cells and other cell lines.
Main Results:
- An acidic protein cytotoxin was purified 8000-fold with a specific activity of 32,000 units/microgram.
- The toxin consists of self-aggregating multimers (Mr 70,000 and 55,000) of a 15,000 molecular weight subunit (pI 4.6).
- The purified toxin exhibited broad-spectrum cytotoxicity against murine, bovine, and human cell lines at picomolar concentrations.
Conclusions:
- The study successfully isolated and characterized a novel macrophage-derived cytotoxin, termed 'holotoxin'.
- The holotoxin's active forms are multimeric aggregates of a 15,000 molecular weight subunit.
- This cytotoxin demonstrates significant activity against diverse cell types, suggesting a role in cellular interactions and immune modulation.