Lipid A binding proteins in macrophages detected by ligand blotting
Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with /sup 32/P/sub i/ (10/sup 9/ dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with (/sup 32/P)-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessed using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS.
- Research Organization:
- Univ. of Wisconsin, Madison
- OSTI ID:
- 6326591
- Report Number(s):
- CONF-870644-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
HEMIC DISEASES
PATHOGENESIS
LIPIDS
BIOCHEMICAL REACTION KINETICS
AUTORADIOGRAPHY
ELECTROPHORESIS
ENDOTOXINS
LIGANDS
MACROPHAGES
PHOSPHORUS 32
RESPONSE MODIFYING FACTORS
SCINTILLATION COUNTING
ANIMAL CELLS
ANTIGENS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CONNECTIVE TISSUE CELLS
COUNTING TECHNIQUES
DAYS LIVING RADIOISOTOPES
DISEASES
ISOTOPES
KINETICS
LIGHT NUCLEI
MATERIALS
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHAGOCYTES
PHOSPHORUS ISOTOPES
RADIOISOTOPES
REACTION KINETICS
SOMATIC CELLS
TOXIC MATERIALS
TOXINS
550201* - Biochemistry- Tracer Techniques
550901 - Pathology- Tracer Techniques