Gervasoni J E, Conrad D H, Hugli T E, Schwartz L B, Ruddy S
J Immunol. 1986 Jan;136(1):285-92.
Purified human C3a was iodinated (125I-C3a) and used to study the interaction of labeled peptide with rat peritoneal mast cells (RMC). Cellular binding of 125I-C3a occurred within 30 sec, followed by a rapid dissociation from the cell. Both the binding of 125I-C3a and the rate of dissociation from the cell were temperature dependent. At 0 degrees C, the binding of 125I-C3a was increased and the rate of dissociation reduced, as compared with 37 degrees C. Once 125I-C3a was exposed to RMC, it lost the ability to rebind to a second batch of RMC. Analysis of the supernatants by trichloroacetic acid (TCA) precipitation and electrophoresis in sodium dodecyl sulfate polyacrylamide gels (SDS PAGE) revealed a decrease in the fraction of 125I precipitable by TCA and the appearance of 125I-C3a cleavage fragments. Pretreatment of RMC with enzyme inhibitors specific for chymotrypsin, but not trypsin, abrogated the degradation of 125I-C3a. Treatment of RMC bearing 125I-C3a with bis (sulfosuccinimidyl) suberate (BS3) covalently cross-linked the 125I-C3a to chymase, the predominant enzyme found in the secretory granules. Antiserum directed against chymase precipitated 125I-C3a from extracts of RMC treated with BS3. Indirect immunofluorescence of RMC by using the IgG fraction of goat anti-rat chymase showed that chymase is present on the surface of unstimulated cells. Neither purified chymase nor heparin proteoglycan alone had any appreciable effect on 125I-C3a, but together they resulted in prompt degradation of the 125I-C3a. Immunoabsorption of RMC sonicates with specific antibody for chymase completely abrogated the ability of these sonicates to degrade 125I-C3a. The results indicate that 125I-C3a binds to RMC and is promptly degraded by chymase in the presence of heparin proteoglycan.
纯化的人C3a经碘化处理（125I-C3a），用于研究标记肽与大鼠腹膜肥大细胞（RMC）的相互作用。125I-C3a在30秒内即可与细胞结合，随后迅速从细胞上解离。125I-C3a的结合及其从细胞上的解离速率均与温度有关。与37℃相比，在0℃时，125I-C3a的结合增加，解离速率降低。一旦125I-C3a与RMC接触，它就失去了重新结合第二批RMC的能力。通过三氯乙酸（TCA）沉淀和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）对上清液进行分析，结果显示可被TCA沉淀的125I部分减少，并且出现了125I-C3a裂解片段。用针对胰凝乳蛋白酶而非胰蛋白酶的特异性酶抑制剂预处理RMC，可消除125I-C3a的降解。用双（磺基琥珀酰亚胺）辛二酸酯（BS3）处理带有125I-C3a的RMC，可使125I-C3a与分泌颗粒中主要的酶——糜酶共价交联。针对糜酶的抗血清可从用BS3处理的RMC提取物中沉淀出125I-C3a。用山羊抗大鼠糜酶的IgG组分对RMC进行间接免疫荧光检测，结果显示在未受刺激的细胞表面存在糜酶。单独的纯化糜酶或肝素蛋白聚糖对125I-C3a均无明显影响，但二者共同作用可导致125I-C3a迅速降解。用针对糜酶的特异性抗体对RMC超声裂解物进行免疫吸附，可完全消除这些超声裂解物降解125I-C3a的能力。结果表明，125I-C3a与RMC结合，并在肝素蛋白聚糖存在的情况下被糜酶迅速降解。
