Purification and distribution of a novel macrophage-specific calmodulin-binding glycoprotein.
The murine macrophage-like cell line J774.16 and peritoneal exudate cells elicited with thioglycollate or starch contain a major calmodulin (CaM)-binding protein (CaMBP) which is absent in trifluoperazine-resistant variants of J774, resident peritoneal macrophages and peritoneal macrophages elicited with concanavalin A, lipopolysaccharide, proteose peptone, or Bacillus Calmette-Guerin. Resident murine peritoneal cells maintained in tissue culture for 3 days begin to accumulate this protein, as do human peripheral blood monocytes after 7 days of culture. A specific competitive displacement radioimmunoassay was developed with the use of a rabbit antiserum raised to the partially purified CaM-binding protein and [125I]CaM covalently cross-linked to the principal CaM-binding protein in the preparation. The radioimmunoassay confirmed the unique cellular distribution of this protein, suggesting that it may be a marker for certain stages of macrophage differentiation. Monoclonal antibodies were prepared, and one of these was used to further purify the protein by immunoaffinity chromatography. A protein of Mr 50,000 to 60,000 was isolated. The protein could be selectively adsorbed to wheat germ agglutinin agarose and subsequently eluted with N-acetyl glucosamine. This property, plus the sensitivity of the protein to endoglycosidase F, led to the conclusion that it is a glycoprotein. The cellular distribution, subcellular localization, and evidence for glycosylation suggest that this protein may be a macrophage-specific receptor with a high affinity for Ca2+-CaM.
Orlow
S J
SJ
Rosenstreich
D L
DL
Pifko-Hirst
S
S
Rosen
O M
OM
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008264:Macrophages / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification*; D051379:Mice; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000276:immunology / Q000302:isolation & purification*; D011863:Radioimmunoassay; D051381:Rats; D011917:Rats, Inbred Lew
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D008565:Membrane Proteins; D010749:Phosphoprotein Phosphatases