Hubbard A L, Cohn Z A
J Cell Biol. 1975 Feb;64(2):461-79. doi: 10.1083/jcb.64.2.461.
The fate of the L-cell plasma membrane proteins labeled by enzymatic iodination was studied. The disappearance of label from growing cells exhibits a biphasic behavior, with 5-20% lost rapidly (t1/2 similar to 2 h) and 80-90% lost relatively slowly (t1/2 similar to 25-33 h). The loss is temperature dependent and serum independent, and is accompanied by the appearance of 51% (125-I)monoiodotyrosine (MIT) in the medium by 47 h. A variable amount (1-14%) of acid-insoluble label can be recovered in the medium over 47 h. Sodium dodecyl sulfate (SDS)-polyacrylamide gel labeling patterns from cells cultured up to 48 h after iodination reveal no change in the relative distribution of radioactivity, indicating similar rates of degradation for most of the labeled membrane proteins. The fate of the labeled membrane proteins was studied at various times after phagocytosis of nondigestible polystyrene particles. Iodinated L cells phagocytose sufficient 1.1 mum latex beads in 60 min to interiorize 15-30% of the total cell surface area. Electron microscope autoradiography confirmed that labeled membrane is internalized during phagocytosis. The latex-containing phagocytic vacuoles are isolated by flotation in a discontinuous sucrose gradient. 15-30% of the total incorporated label and a comparable percentage of alkaline phosphodiesterase I activity (PDase, a plasma membrane enzyme marker) are recovered in the phagocytic vacuole fraction. Lysosomal enzyme activities are found in the latex vacuole fraction, indicating formation of phagolysosomes. SDS gel analyses reveal that all of the radioactive proteins initially present on the intact cell's surface are interiorized to the same relative extent. Incorporated label and PDase activity disappear much more rapidly from the phagolysosomes than from the whole cell. In the phagolysosomal compartment, greater than 70% of the TCA-precipitable labeled proteins and all of the PDase activity are lost rapidly (t1/2 equals 1-2 h) but similar 30% of the labeled proteins in this compartment are degraded with a 17-20 h half-life. The slowly degraded label is due to specific long-lived polypeptides, of 85,000 and 8,000-15,000 daltons, which remain in the phagolysosomal membrane up to 40 h after phagocytosis.
研究了经酶促碘化标记的L细胞质膜蛋白的命运。生长细胞中标记物的消失呈现双相行为，5 - 20%迅速丢失（半衰期约为2小时），80 - 90%丢失相对缓慢（半衰期约为25 - 33小时）。这种丢失与温度有关，与血清无关，到47小时时，培养基中出现51%的（125 - I）单碘酪氨酸（MIT）。在47小时内，培养基中可回收可变数量（1 - 14%）的酸不溶性标记物。碘化后培养至48小时的细胞的十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶标记模式显示放射性的相对分布没有变化，表明大多数标记的膜蛋白的降解速率相似。在吞噬不可消化的聚苯乙烯颗粒后的不同时间研究了标记的膜蛋白的命运。碘化的L细胞在60分钟内吞噬足够的1.1μm乳胶珠，使细胞总表面积的15 - 30%内化。电子显微镜放射自显影证实标记的膜在吞噬过程中被内化。含乳胶的吞噬泡通过在不连续蔗糖梯度中浮选分离。在吞噬泡部分回收了15 - 30%的总掺入标记物和相当比例的碱性磷酸二酯酶I活性（PDase，一种质膜酶标记物）。在乳胶泡部分发现了溶酶体酶活性，表明形成了吞噬溶酶体。SDS凝胶分析表明，最初存在于完整细胞表面的所有放射性蛋白都以相同的相对程度内化。掺入的标记物和PDase活性从吞噬溶酶体中消失的速度比从整个细胞中快得多。在吞噬溶酶体区室中，超过70%的三氯乙酸沉淀的标记蛋白和所有的PDase活性迅速丢失（半衰期等于1 - 2小时），但该区室中类似30%的标记蛋白以17 - 20小时的半衰期降解。缓慢降解的标记物归因于85,000和8,000 - 15,000道尔顿的特定长寿多肽，它们在吞噬后长达40小时仍保留在吞噬溶酶体膜中。
