Wypych J, Bennett L G, Schwartz M G, Clogston C L, Lu H S, Broudy V C, Bartley T D, Parker V P, Langley K E
Amgen, Inc, Amgen Center, Thousand Oaks, CA 91320-1789.
Blood. 1995 Jan 1;85(1):66-73.
c-kit encodes the transmembrane receptor tyrosine kinase (Kit) for the recently described ligand stem cell factor (SCF). We have developed an enzyme-linked immunosorbent assay for measuring soluble human Kit and we have used the assay to show high levels of soluble Kit in human serum. The distribution of soluble Kit levels was investigated among 112 normal human serum donors. The mean serum level (+/- SD) was found to be 324 +/- 105 ng/mL with the values falling between 163 ng/mL and 788 ng/mL. No correlation between soluble Kit levels and the sexes or ages of the donors was found. Partial purification using immunoaffinity chromatography allowed us to characterize the soluble Kit from pooled human serum. Antibodies generated to a 497-amino acid recombinant human soluble Kit corresponding to the N-terminal extracellular domain of the receptor recognized the serum-derived soluble Kit by immunoblotting. We found that the serum-derived soluble Kit is glycosylated, with mostly N-linked but also O-linked carbohydrate, and with terminal sialic acid residues. When compared with the recombinant human soluble Kit, the serum-derived material was similar both in size and glycosylation pattern. CNBr cleavage of the isolated serum-derived material followed by amino terminal sequencing confirmed the presence of five peptides expected for the extracellular portion of the Kit molecule. The immunoaffinity purified serum-derived soluble Kit inhibited binding of [125I]SCF to membrane-bound receptor in an in vitro assay. These results indicate that soluble Kit could modulate the activity and functions of SCF in vivo.
c-kit编码一种跨膜受体酪氨酸激酶（Kit），该激酶作用于最近发现的配体干细胞因子（SCF）。我们开发了一种酶联免疫吸附测定法来检测可溶性人Kit，并使用该测定法显示人血清中存在高水平的可溶性Kit。我们研究了112名正常人血清供体中可溶性Kit水平的分布情况。发现血清平均水平（±标准差）为324±105 ng/mL，数值在163 ng/mL至788 ng/mL之间。未发现可溶性Kit水平与供体的性别或年龄之间存在相关性。使用免疫亲和色谱法进行部分纯化，使我们能够对来自混合人血清的可溶性Kit进行表征。针对与受体N端细胞外结构域对应的497个氨基酸的重组人可溶性Kit产生的抗体，通过免疫印迹法识别血清来源的可溶性Kit。我们发现血清来源的可溶性Kit是糖基化的，主要是N-连接糖基化，但也有O-连接糖基化，并且带有末端唾液酸残基。与重组人可溶性Kit相比，血清来源的物质在大小和糖基化模式上均相似。对分离的血清来源物质进行溴化氰裂解，然后进行氨基末端测序，证实了Kit分子细胞外部分预期的五种肽的存在。免疫亲和纯化的血清来源可溶性Kit在体外测定中抑制了[125I]SCF与膜结合受体的结合。这些结果表明可溶性Kit可能在体内调节SCF的活性和功能。
