Biochemical purification and partial characterization of a murine macrophage surface receptor possessing specificity for small aromatic moieties including fluorescein.
Binding properties and requirements for internalization of hapten-protein antigens, such as fluorescein-polyderivatized bovine serum albumin (FITC-BSA) and poly-D-lysine (FITC-PDL), by murine macrophage was consistent with surface receptor recognition of fluorescyl moieties (Cherukuri et al., Mol. Immunol. 34, 21-32, 1997; Cherukuri et al., Cytometry 31, 110-124, 1998). Ligand binding properties of the putative macrophage receptor pointed toward specificity for various aromatic moieties including phenylalanine, phenyloxazolone and fluorescein (Cherukuri and Voss, Mol. Immunol. 35, 115-125, 1998). Purification of the hapten-recognizing receptor from J774 macrophage cells involved subcellular fractionation of plasma membrane fractions, and affinity chromatography of solubilized membranes employing a phenyl-Sepharose adsorbent with subsequent specific elution of receptor using fluorescein ligand. The final product was a protein with a molecular mass of approximately 180 kDa. Characterization of the purified receptor involved absorption and fluorescence spectroscopy, circular dichroism, fluorescence quenching analyses, various ligand binding assays and an immunological analysis. Spectroscopic analyses revealed that the receptor possessed aromatic amino acids while circular dichroism suggested significant alpha-helical secondary structure. Binding specificity of the purified receptor was confirmed in a spectrofluorometric assay where the fluorescence of fluorescein ligand was quenched approximately 97%. Finally, specific binding activity of the receptor with FITC-BSA was demonstrated in Western blot analysis under native conditions. Receptor purity was confirmed in amino acid sequencing analysis when the amino-terminal residue was found to be totally blocked. Results are discussed in terms of the possible identity of the isolated macrophage receptor and its biological-immunological role.
Department of Microbiology, University of Illinois, Urbana-Champaign, B103 Chemical and Life Sciences Laboratory, 601 South Goodwin Ave, Urbana, IL 61801, USA.
A
A
Cherukuri
J
J
Nelson
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EW
Voss
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D002942:Circular Dichroism; D005260:Female; D005434:Flow Cytometry; D019793:Fluorescein / Q000378:metabolism*; D005470:Fluorometry; D006241:Haptens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology; D008024:Ligands; D008264:Macrophages / Q000737:chemistry*; D051379:Mice; D010081:Oxazolone / Q000031:analogs & derivatives* / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011817:Rabbits; D013347:Subcellular Fractions / Q000737:chemistry
D006241:Haptens; D007074:Immunoglobulin G; D008024:Ligands; C018361:2-phenyloxazolone; D010081:Oxazolone; D019793:Fluorescein