Cheng C Y, Bardin C W
Biochemistry. 1986 Sep 9;25(18):5276-88. doi: 10.1021/bi00366a044.
During a search for hormonally responsive products in media from Sertoli cell enriched cultures, a major follicle stimulating hormone responsive and testosterone-responsive protein was identified and designated CMB-1. The results of the present study indicate that this protein is related immunologically to rat albumin and rat alpha-fetoprotein (AFP) and is concentrated in the testis of the adult rat. CMB-1 was therefore termed testibumin. Testibumin was purified from Sertoli cell enriched cultures to apparent homogeneity by sequential high-performance liquid chromatography on anion-exchange, chromatofocusing, gel permeation, and hydroxylapatite columns. The purified protein consists of two concanavalin A (Con A) reactive forms: one which does not interact with Con A and the other which binds to this lectin and is eluted with methyl alpha-mannoside. Testibumin is a monomer with an apparent molecular weight of 69,000 and a pI ranging between 4.5 and 4.85. The heterogeneity of this protein was further demonstrated by crossed-immunoelectrophoresis and two-dimensional gel electrophoresis. A monospecific antiserum and highly purified testibumin were used to develop a specific radioimmunoassay which permitted studies of the hormonal responsiveness of Sertoli cell enriched culture and of the content of testibumin in the reproductive tract fluids in vivo. Even though testibumin was found in serum of both sexes, it was highly concentrated in the testicular and epididymal compartments in adult rats. This protein was compared to rat serum albumin and rat AFP immunologically. With the use of immunoblots, antiserum developed against testibumin showed partial cross-reactivity with albumin and AFP when these latter proteins were denatured and were present in amounts several orders of magnitude greater than testibumin. The extent of this cross-reactivity was then examined by comparing the ability of native and S-carboxymethylated albumin to compete with 125I-testibumin for binding to a monospecific testibumin antiserum. It was shown that the unfolded derivative of albumin showed partial cross-reactivity with testibumin. We conclude that testibumin is immunologically related to albumin and AFP as these latter proteins are related to one another and that testibumin is possibly the homologue of albumin in the seminiferous tubular compartment.
在从富含支持细胞的培养物的培养基中寻找激素反应性产物的过程中，鉴定出一种主要的促卵泡激素反应性和睾酮反应性蛋白，并将其命名为CMB-1。本研究结果表明，该蛋白在免疫学上与大鼠白蛋白和大鼠甲胎蛋白（AFP）相关，且在成年大鼠的睾丸中富集。因此，CMB-1被称为睾丸白蛋白。通过在阴离子交换柱、色谱聚焦柱、凝胶渗透柱和羟基磷灰石柱上依次进行高效液相色谱，从富含支持细胞的培养物中纯化出了纯度明显的睾丸白蛋白。纯化后的蛋白由两种伴刀豆球蛋白A（Con A）反应形式组成：一种不与Con A相互作用，另一种与该凝集素结合并能用α-甲基甘露糖苷洗脱。睾丸白蛋白是一种单体，表观分子量为69,000，pI在4.5至4.85之间。这种蛋白的异质性通过交叉免疫电泳和二维凝胶电泳进一步得到证实。使用单特异性抗血清和高度纯化的睾丸白蛋白建立了一种特异性放射免疫测定法，该方法可用于研究富含支持细胞的培养物的激素反应性以及体内生殖道液中睾丸白蛋白的含量。尽管在两性血清中都发现了睾丸白蛋白，但它在成年大鼠的睾丸和附睾中高度富集。将这种蛋白与大鼠血清白蛋白和大鼠AFP进行了免疫学比较。使用免疫印迹法，当后两种蛋白变性且其含量比睾丸白蛋白高几个数量级时，针对睾丸白蛋白产生的抗血清与白蛋白和AFP表现出部分交叉反应性。然后通过比较天然和S-羧甲基化白蛋白与125I-睾丸白蛋白竞争结合单特异性睾丸白蛋白抗血清的能力，来研究这种交叉反应性的程度。结果表明，白蛋白的未折叠衍生物与睾丸白蛋白表现出部分交叉反应性。我们得出结论，睾丸白蛋白在免疫学上与白蛋白和AFP相关，就如同后两者彼此相关一样，并且睾丸白蛋白可能是生精小管区室中白蛋白的同源物。
