Abstract. The interaction of homologous and heterol-

Abstract. Isolated fat tissue microvessels and lung, whose capillary endothelia express in situ specific binding sites for albumin, were homogenized and subjected to SDS-gel electrophoresis and electroblotting. The nitrocellulose strips were incubated with either albumin-gold (Alb-Au) and directly visualized, or with [~25I]albumin (monomeric or polymeric) and autoradiographed. The extracts of both microvascular endothelium and the lung express albumin-binding proteins (ABPs) represented by two pairs of polypeptides with major components of molecular mass 31 and 18 kD. The ABP peptides have pls 8.05 to 8.75. Rabbit aortic endothelium, used as control, does not express detectable amounts of ABPs. The ABPs subjected to electrophoresis bind specifically and with high affinity (Kd = r~ 60 >( 10 -9 M) both monomeric and polymeric albumin: the binding is saturable at ~80 nM concentration and 50% inhibition is reached at 5.5 I.tg/ml albumin concentration. Sulfhydryl-reducing agents 13-mercaptoethanol and dithiothreitol do not markedly affect the ABPs electrophoretic mobility and binding properties. As indicated by cell surface iodination of isolated capillary endothelium followed by electroblotting, autoradiography, and incubation with Alb-Au, the bands specifically stained by this ligand are also labeled with radioiodine.

The topography of the charged residues on the endothelial cell surface of liver sinusoid capillaries was investigated by using electron microscopic tracers of different size and charge. The tracers used were native ferritin (pl 4 .2-4.7) and its cationized (pl 8.4) and anionized (pl 3 .7) derivatives, BSA coupled to colloidal gold (pl of the complex 5.1), hemeundecapeptide (pl 4.85), and alcian blue (pl >10) . The tracers were either injected in vivo or perfused in situ through the portal vein of the mouse liver. In some experiments, two tracers of opposite charge were sequentially perfused with extensive washing in between . The liver was processed for electron microscopy and the binding pattern of the injected markers was recorded . The electrostatic nature of the tracer binding was assessed by perfusion with high ionic strength solutions, by aldehyde quenching of the plasma membrane basic residues, and by substituting the cell surface acidic moieties with positively charged groups. Results indicate that the endothelial cells of the liver sinusoids expose on their surface both cationic and anionic residues. The density distribution of these charged groups on the cell surface is different . While the negative charge is randomly and patchily scattered all over the membrane, the cationic residues seem to be accumulated in coated pits. The charged groups co-exist in the same coated pit and bind the opposite charged macromolecule . It appears that the fixed positive and negative charges of the coated pit glycocalyx are mainly segregated in space . The layer of basic residues is located at 20-30-nm distance of the membrane, while most of the negative charges lie close to the external leaflet of the plasmalemma .

Plasma macromolecules can, in some tissues, cross the capillary wall and enter the interstitial space by a specific mechanism of transcytosis in which a macromolecule is first recognized by its receptor on the luminal surface of endothelial cells. A cluster of receptors bearing their cargo is confined to a vesicle that crosses the cells and discharges the macromolecules on the abluminal surface. The process may be especially important for transport of large molecules, such as albumin, transferrin, insulin, and low-density-lipoprotein, to the cells where they are needed.

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