Using flow cytometry and fluorescence polarization analysis, specific muramyl peptide‐binding sites were shown to be located inside T‐lymphocytes, macrophages and neuroblastoma cells, but not inside B‐cells. No binding sites were found on the cell surface. The number of binding sites for each cell type was determined. Two types of binding sites were observed for myelomonocytic WEH1‐3 cells with K fd values of 21 and 540 nM. Inhibition analysis demonstrated that for effective binding, an intact glycopeptide molecule and D‐configuration of isoglutamine residue are important.

By using radioligand analysis, murine peritoneal macrophages were shown to express several hundred high-affinity cell surface GMDPbiding sites (& 350 PM). PhotoaBinity labeling followed by SDS-PAGE enabled us to identify 32-34 and 38 kDa proteins inside these cells that bound GMDP specifically.

Intracellular N-acetylglucosaminylmuramyl peptidebinding proteins of murine macrophages and myelomonocytic WEHI-3 cells were characterized. SDS-PAGE and Western blotting revealed proteins with molecular masses of 18, 32 and 34 kDa retaining the ability to specifically bind glucosaminylmuramyl dipeptide. The inhibition analysis demonstrated that only biologically active muramyl peptides but not inactive analogs or fragments of glucosaminylmuramyl dipeptide could inhibit glucosaminylmuramyl dipeptide-binding to these proteins. Purification of these proteins and sequencing of peptides obtained after in-gel trypsin digestion enabled us to identify the above mentioned proteins as histones H1 and H3. These findings suggest that nuclear histones might be target molecules for muramyl peptides.z 1999 Federation of European Biochemical Societies.

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