Culp L A, Terry A H, Buniel J F
Biochemistry. 1975 Jan 28;14(2):406-12. doi: 10.1021/bi00673a030.
Balb/c 3T3, SV40-transformed 3T3 (SVT2), and Con A revertant variants of transformed cells leave a layer of glycoprotein on the culture substrate upon EGTA mediated removal of cells. The metabolic properties of this substrate-attached material (glycoprotein) have been examined. Pulse and cumulative radiolabeling experiments with glucosamine and leucine precursors established that this substrate-attached material accumulates on the substrate in growing cultures until cells have completely covered the substrate. The synthesis and/or deposition of the material diminished dramatically in cultures whose substrates had been completely covered with cells as observed microscopically, even though the contact-inhibited cell lines continued to make cell-associated and medium-secreted glycoproteins and transformed cells continued to divide and form multilayered cultures. Pulse-chase analysis using long periods of pulsing with radioactive leucine demonstrated that these glycoproteins are deposited directly on the substrate by cells and not subsequent to secretion into the medium. The substrate-attached material accumulated during long pulses was stably adherent to the substrate and displayed little appreciable turnover during 3 days of chasing of either sparse or dense cultures. Short-term pulse-chase analysis with leucine revealed two metabolically different pools of material-one which turns over very rapidly with a half-life of 2-3 hr (observed in both low-density and high-density cultures) and a second pool which is stably deposited on the substrate and whose proportion increased with the length of the radiolabeling period. No appreciable differences in the metabolic properties of substrate-attached material were observed in the three cell types studied during growth on a plastic substrate. These results are discussed with regard to the implicated roles of these glycoproteins in in mediating adhesion of normal and virus-transformed cells to the substrate.
Balb/c 3T3细胞、SV40转化的3T3细胞（SVT2）以及转化细胞的刀豆球蛋白A回复变体，在经乙二醇双四乙酸（EGTA）介导去除细胞后，会在培养底物上留下一层糖蛋白。已对这种附着于底物的物质（糖蛋白）的代谢特性进行了研究。用葡糖胺和亮氨酸前体进行的脉冲和累积放射性标记实验表明，在生长的培养物中，这种附着于底物的物质会在底物上积累，直至细胞完全覆盖底物。显微镜观察发现，即使接触抑制的细胞系继续产生细胞相关和培养基分泌的糖蛋白，且转化细胞继续分裂并形成多层培养物，但在底物已被细胞完全覆盖的培养物中，该物质的合成和/或沉积显著减少。使用放射性亮氨酸进行长时间脉冲的脉冲追踪分析表明，这些糖蛋白是由细胞直接沉积在底物上，而非分泌到培养基后再沉积。在长时间脉冲期间积累的附着于底物的物质稳定地附着于底物，在稀疏或密集培养物的3天追踪期内，其周转量很小。用亮氨酸进行的短期脉冲追踪分析揭示了两个代谢不同的物质池——一个周转非常迅速，半衰期为2 - 3小时（在低密度和高密度培养物中均观察到），另一个池则稳定地沉积在底物上，其比例随放射性标记期的延长而增加。在塑料底物上生长的过程中，在所研究的三种细胞类型中，未观察到附着于底物的物质的代谢特性有明显差异。讨论了这些糖蛋白在介导正常细胞和病毒转化细胞与底物粘附方面的潜在作用。
