Bienkowski M J, Conrad H E
J Biol Chem. 1984 Nov 10;259(21):12989-96.
The metabolism of heparan sulfate proteoglycan was studied in monolayer cultures of a rat hepatocyte cell line. Late log cells were labeled with 35SO4(2-) or [3H] glucosamine, and labeled heparan sulfate, measured as nitrous acid-susceptible product, was assayed in the culture medium, the pericellular matrix, and the intracellular pools. Heparan sulfate in the culture medium and the intracellular pools increased linearly with time, while that in the matrix reached a steady-state level after a 10-h labeling period. When pulse-labeled cells were incubated in unlabeled medium, a small fraction of the intracellular pool was released rapidly into the culture medium while the matrix heparan sulfate was taken up by the cells, and the resulting intracellular pool was rapidly catabolized. The structures of the heparan sulfate chains in the three pools were very similar. Both the culture medium pool and the cell-associated fraction of heparan sulfate contained proteoheparan sulfate plus a polydisperse mixture of heparan chains which were attached to little, if any, protein. Pulse-chase data suggested that the free heparan sulfate chains were formed as a result of catabolism of the proteoglycan. When NH4Cl, added to inhibit lysosomal function, was present during either a labeling period or a chase period, the total catabolism of the heparan sulfate chains to monosaccharides plus free SO2-4 was blocked, but the conversion of the proteoglycan to free heparan sulfate chains continued at a reduced rate.
在大鼠肝细胞系的单层培养物中研究了硫酸乙酰肝素蛋白聚糖的代谢。对数生长期后期的细胞用35SO4(2-)或[3H]葡糖胺进行标记，作为对亚硝酸敏感产物测定的标记硫酸乙酰肝素，在培养基、细胞周围基质和细胞内池进行检测。培养基和细胞内池中的硫酸乙酰肝素随时间呈线性增加，而基质中的硫酸乙酰肝素在标记10小时后达到稳态水平。当脉冲标记的细胞在未标记的培养基中孵育时，细胞内池的一小部分迅速释放到培养基中，而基质硫酸乙酰肝素被细胞摄取，产生的细胞内池迅速被分解代谢。三个池中的硫酸乙酰肝素链结构非常相似。培养基池和硫酸乙酰肝素的细胞相关部分都含有蛋白硫酸乙酰肝素以及硫酸乙酰肝素链的多分散混合物，这些链与很少或几乎没有蛋白质相连。脉冲追踪数据表明，游离硫酸乙酰肝素链是蛋白聚糖分解代谢的结果。当在标记期或追踪期加入NH4Cl以抑制溶酶体功能时，硫酸乙酰肝素链完全分解代谢为单糖加游离SO2-4被阻断，但蛋白聚糖向游离硫酸乙酰肝素链的转化以降低的速率继续进行。
