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Two proteoglycan-hyaluronate complexes were extracted from bovine aorta with 4.0 M guanidinium chloride in the presence of protease inhibitors and purified by cesium chloride density gradient centrifugation under associative conditions. Complex I with higher buoyant density had 22% protein and 18% uronate, whereas Complex II had 11% protein and 23.6% uronate. Glycosaminoglycan (GAG) analysis of the complexes indicated that both complexes contained large amounts of hyaluronic acid, 13.6% and 24.0% of the total GAG in Complexes I and II, respectively. Chondroitin sulfates and dermatan sulfate constituted the remainder of the GAG. Chromatography on Sepharose CL 6B suggested that Complex I had an estimated molecular weight of 800,000 and Complex II, 250,000. The complexes were dissociated into proteoglycan and hyaluronate by cesium chloride density gradient centrifugation under dissociative conditions. The proteoglycans free of hyaluronic obtained from the complexes had similar protein and GAG composition. The molecular weight of the proteoglycan was estimated to be 80,000 and that of the hyaluronate of the complex, 90,000.

Proteoglycans were extracted from bovine lung gas exchange tissue, pleura, and bronchioles with 4.0 M guanidinium chloride at 5 degrees C in the presence of protease inhibitors. Preliminary purification of the proteoglycans was achieved by an initial CsCl isopycnic centrifugation (rho 0 = 1.33) and through precipitation with cetylpyridinium chloride in 0.5 M KCl. Further purification and fractionation of proteoglycans was achieved by a second CsCl isopycnic centrifugation (rho 0 = 1.45) in 4.0 M guanidinium chloride. Based on the ultracentrifuge profiles and electrophoretic behavior, the major fractions were pooled. They were purified further by gel filtration on Sepharose CL-2B and characterized by extensive analyses. A heparan sulfate proteoglycan was the major proteoglycan identified in the gas exchange tissue and in the pleura. The major proteoglycan component from the bronchioles was a chondroitin sulfate proteoglycan. Approximate molecular weight of 2 x 10(6) for the heparan sulfate proteoglycan from the pleura and chondroitin sulfate proteoglycan from the bronchioles and 1 x 10(6) for the heparan sulfate proteoglycan from the gas exchange tissue were estimated from gel filtration analyses. After incubation with hyaluronic acid, the chondroitin sulfate proteoglycans from the bronchioles showed an increase in specific viscosity and a higher molecular weight compound eluting near the void volume in Sepharose CL-2B column chromatography. The proteoglycans exhibited varied anticoagulant activities in Stypven, partial thromboplastin and thrombin clotting times and inhibited thrombin-induced platelet aggregation.

The interactions of a chondroitin sulfate-dermatan sulfate proteoglycan with serum very low (VLDL), low (LDL), and high density (HDL3) lipoproteins were studied with special reference to the nature of the interaction of LDL and the proteoglycan. The proteoglycan formed insoluble complexes with VLDL and LDL, but no complex was formed with HDL3. The proteoglycan (40 micrograms/ml) converted 98% of added LDL (150 micrograms of cholesterol/ml) into insoluble complex at a Ca2+ concentration of 30 mM. Physiologic concentrations of albumin inhibited insoluble complex formation with VLDL and LDL by 61.5 and 40.7%, respectively. The proteoglycan formed soluble complexes with LDL even in the absence of Ca2+. Optimum soluble complex formation occurred when the medium contained 66.6 micrograms of proteoglycan and 166.6 micrograms of LDL cholesterol/ml. Specific modifications of the lysine and/or arginine residues of LDL prevented complex formation with the proteoglycan, thus indicating the requirement of the positive charges of the protein moiety of LDL in the interaction. Removal of the protein core or the glycosaminoglycan chains of the proteoglycan prevented interaction with LDL. Desulfation of the proteoglycan molecule also inhibited complex formation with LDL. Thus, the native state of the arterial proteoglycan molecule confers optimum charge density for specific interaction with serum apoprotein B-containing lipoproteins.