Photoaffinity labelling of a receptor structure for macrophage cytotoxicity inducing factor (MCF).
Monocyte cytotoxicity inducing factor (MCF) is a cytokine derived from CD4+ lymphocytes which was isolated using its ability to cause human blood monocytes to become cytotoxic for tumour targets. An N-terminal derived biologically active peptide (GAAVLEDSQ) of the intact molecule was previously used to demonstrate a single class of high affinity binding sites on human blood monocytes and U937 cells. Photoaffinity labelling was carried out to identify the receptor. The N-terminal nonapeptide fragment (P2) could be cross-linked to a single molecular species on the surface of U937 cells when cross-linking was performed on intact cells. Under fully reducing conditions, this molecule had an average molecular weight of 58 kD. In contrast, the apparent molecular weight when determined under nonreducing conditions was 158-163 kD. Octylglucoside solubilized U937 membranes were then applied to a P2 ligand affinity column. The major protein peak when eluted had an apparent molecular weight of 73 kD when determined by a 12% SDS PAGE gel. Photoaffinity labelling of the ligand affinity product was carried out in solution. Gel electrophoresis under nonreducing conditions demonstrated cross-linking to a 163 kD and a 29 kD protein, while under reducing and denaturing conditions additional species were seen at 97 kD, 67 kD and 50 kD. Photoaffinity cross-linking performed both on whole cells and partially purified receptor in solution, and could be specifically abolished by the addition of unlabelled peptide. These data present evidence that the single high affinity binding site corresponds to a U937 membrane protein having an average molecular weight of 58 kD which exists as an apparent homodimer under native conditions, but may have additional low molecular weight components which were not available for binding and derivatization using intact cells.
Reyes
C J
CJ
Department of Medicine, University of Texas Health Science Center at Houston, USA.
Jones
C M
CM
D000345:Affinity Labels / Q000737:chemistry*; D000595:Amino Acid Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010777:Photochemistry; D018121:Receptors, Cytokine / Q000737:chemistry*
D000345:Affinity Labels; D018121:Receptors, Cytokine