Figure 3

Adipocyte differentiation results in increased synthesis of a high molecular weight proteoglycan that consists of a mixture of heparan and chondroitin sulfate glycosaminoglycan moieties. Pre-adipocyte 3T3-L1 cells and differentiated adipocytes were incubated for 20 h with ³⁵SO₄ (125 μCi/ml). (A), cells were lysed with 8 M urea and proteins were fractionated by anion exchange chromatography as described in Methods. Radioactivity in each fraction was determined by scintillation counting. (B), fractions from (A) were separated by 7% SDS-PAGE and subjected to phosphorimager analysis (upper panel, pre-adipocytes; lower panel, adipocytes). (C), ³⁵SO₄-labeled proteoglycans, enriched by anion exchange chromatography, were incubated with or without heparinase I (5 Units/ml), chondroitinase ABC (5 Units/ml), or both enzymes for 20 h at 37°C. Labeled material was then fractionated by size exclusion chromatography and fractions were assessed by scintillation counting.