Isolation of heparan sulfate proteoglycan from beneath the monolayers of rat hepatocytes and its binding to type IV collagen.
Primary cultures of rat hepatocytes maintained as monolayer in a serum-free medium synthesise and secrete sulphated proteoglycans. Nearly 5% of the total 35(S)-sulphated material was obtained in a soluble form from beneath the cell layer. A shift in gel filtration pattern on beta-elimination with alkali suggested that it is a sulphated proteoglycan. On ion exchange chromatography over Dowex AG 1 x 2, the major fraction was eluted with 1.25 M NaCl. Further, nearly 80% of the 35(S)-labeled material was susceptible to nitrous acid degradation and more than 90% of the material was resistant to chondroitinase ABC digestion suggesting that it is predominantly a heparan sulphate proteoglycan (HSPG). Since HSPG is a major component of basement membrane, its binding with collagen was studied by a solid phase binding assay. About 75% of the 35(S) HSPG bound to wells coated with type IV collagen whereas only about 20% bound to type I collagen at physiological pH. Binding to collagen IV was reduced by about 50% when free GAG chains were used indicating that the protein core is also involved in interaction with the collagen. These results indicate the possible role of this basal extracellular heparan sulphate proteoglycan in the basal lamina formation.
Babu
P B
PB
Department of Biochemistry, University of Kerala, Kariavattom, Trivandrum, India.
Sudhakaran
P R
PR
D000818:Animals; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003094:Collagen / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000302:isolation & purification* / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D011508:Chondroitin Sulfate Proteoglycans; D003094:Collagen; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate