Quantitation of specific binding of orosomucoid to cultured microvascular endothelium: role in capillary permeability.
Orosomucoid (also known as alpha 1-acid glycoprotein) is a highly sialylated, polyanionic serum glycoprotein. Its presence in vascular perfusates alters microvascular permeability, presumably by binding to the endothelial glycocalyx, but the rest of its functions remains unknown. In this study, we assess the binding of bovine serum orosomucoid (BSO) to the surface of bovine pulmonary microvascular endothelial cells (BLMVEC) in culture at 4 degrees C. The binding of radioiodinated BSO (125I-BSO) reached equilibrium after 10 min of incubation, was not calcium dependent, and was reversible by greater than 80% in 30 min. The binding of 125I-BSO was competed with unlabeled BSO but not by transferrin, immunoglobulin, gelatin, ovalbumin, mannan, fucoidan, mucin, or asialomucin. In addition, binding was not affected by the presence of galactose, glucose, fucose, mannose, N-acetyl-D-galactosamine, or N-acetyl-D-glucosamine. This binding behavior is not consistent with carbohydrate recognition by lectin-like molecules such as the asialoglycoprotein receptor. Scatchard analysis of the BSO binding to BLMVEC produced a concave-upward shaped curve, which revealed a higher affinity binding component with an apparent equilibrium affinity constant (Kd) of 8.6 nM and a maximum receptor number of 40,000/cell and revealed a moderate affinity component with a Kd of 9.1 microM with a maximum binding of 10(7) binding sites per cell. Other blood vessel-associated cells clearly bound less BSO with a lower binding affinity than the BLMVEC monolayers. From the binding data, we estimated the total increase in the negative charge of the glycocalyx with orosomucoid binding to be approximately 17 meq/l.(ABSTRACT TRUNCATED AT 250 WORDS)
Schnitzer
J E
JE
Department of Pathology and Medicine, University of California San Diego School of Medicine, La Jolla 92093-0651.
Pinney
E
E
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D002199:Capillary Permeability; D002417:Cattle / Q000097:blood; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D007700:Kinetics; D008833:Microcirculation; D009961:Orosomucoid / Q000378:metabolism*; D011975:Receptors, Mitogen / Q000502:physiology
D009961:Orosomucoid; D011975:Receptors, Mitogen; D002118:Calcium