Abstract
A Pseudomonas aeruginosa exotoxin A (PE) binding glycoprotein was affinity purified from toxin sensitive mouse LM cells. The binding protein was solubilized with Triton X-100 or Nonidet P-40 and purified on a PE-Sepharose affinity column. Polyacrylamide gel electrophoresis yielded a single band with an estimated molecular mass of greater than 300,000 Da. N-Linked carbohydrate was present, accounting for approximately 10% of the total mass of the molecule. The purified protein specifically bound PE. Incubation of purified protein specifically bound PE. Incubation of purified PE binding protein with toxin reduced toxicity to LM cells. We speculate on the role of this toxin binding glycoprotein in the intoxication process.
Highlights
Electrophoresis yielded a single band with an estimated molecular massof greater than 300,000 Da
We have shown that biotinyl-Pseudomonas aeruginosaexotoxin A (PE) initially binds to sites randomly distributed on the LM cell surface [17]
Isolation of PE Binding Protein-PE binding protein was solubilized from mouse LM fibroblasts and purified by single step affinity chromatography on PE-Sepharose affinity columns
Summary
Glycosylation of Binding Protein-Commercially available glycan detection and differentiation kits were employed to identify carbohydrate and sialic acid constituents of purified binding protein (Boehringer Mannheim). Purified binding protein was electrophoresed as described above and transferred to nitrocellulose sheets. By using the glycan differentiation kit, glycan constituents were identified by a similar enzyme immunoassay, employing group-specificlectins. In some experiments, binding protein was incubated with Endo H or PNGase F prior to electrophoresis, electroblotting, and glycan detection to distinguish N-linked and 0-. Linked glycan structures.PNGaseF was incubated with binding protein in 50 mM EDTA, 0.05% sodium azide, 20 mM potassium phosphate buffer, pH 7.2. Endo Hwas incubated with binding protein in PBS, pH 7.2. Receptor purified from Triton X-100 extracts was chromatographed in PBS buffer, pH 7.1, containing 0.01% Triton X-100. Individual fractions were either assayed directly for receptor by ELISA, by directly staining SDS-
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