Lipid A binding sites in membranes of macrophage tumor cells

Journal Article · Sat Oct 15 00:00:00 EDT 1988 · J. Biol. Chem.; (United States)

OSTI ID:6559189

Hampton, R Y; Golenbock, D T; Raetz, C R

Lipopolysaccharide affects a variety of eukaryotic cells and mammalian organisms. These actions are involved in the pathogenesis of Gram-negative septicemia. Many of the actions of lipopolysaccharide are believed to be caused by its active moiety, lipid A. Our laboratory has previously identified a bioactive lipid A precursor, termed lipid IVA, which can be labeled with 32P of high specific activity and purified. In this work we have used the labeled probe, 4'-32P-lipid IVA, to develop a novel assay for the specific binding of lipid IVA to whole cells. We have also demonstrated its use in a ligand blotting assay of immobilized cellular proteins. Using the whole cell assay, we show that 4'-32P-lipid IVA specifically binds to RAW 264.7 macrophage-like cultured cells. The binding is saturable, is inhibited with excess unlabeled lipid IVA, and is proteinase K-sensitive. It displays cellular and pharmacological specificity. Using the ligand blotting assay, we show that several RAW 264.7 cell proteins can bind 4'-32P-lipid IVA. The two principal binding proteins have Mr values of 31 and 95 kDa, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractionation studies indicate that the 31-kDa protein is enriched in the nuclear fraction and may be a histone, whereas the 95-kDa protein is enriched in the membrane fraction. The binding assays that we have developed should lead to a clearer understanding of lipid A/animal cell interactions.

• Research Organization: Univ. of Wisconsin-Madison (USA)
• OSTI ID: 6559189
• Journal Information: J. Biol. Chem.; (United States), Vol. 263:29
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
LIPIDS
RECEPTORS
BIOCHEMICAL REACTION KINETICS
CELL MEMBRANES
ELECTROPHORESIS
ENDOTOXINS
FRACTIONATION
LIGANDS
MACROPHAGES
MEMBRANE PROTEINS
MOLECULAR WEIGHT
PHOSPHORUS 32
RADIOASSAY
TUMOR CELLS
ANIMAL CELLS
ANTIGENS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CELL CONSTITUENTS
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
ISOTOPES
KINETICS
LIGHT NUCLEI
MATERIALS
MEMBRANES
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHAGOCYTES
PHOSPHORUS ISOTOPES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
SEPARATION PROCESSES
SOMATIC CELLS
TOXIC MATERIALS
TOXINS
550201* - Biochemistry- Tracer Techniques