Björk P, Forsgren B, Gustafsson J A, Pousette A, Högberg B
Cancer Res. 1982 May;42(5):1935-42.
The [3H]estramustine-binding macromolecule in human prostate was partially characterized using a number of chromatographic procedures. Although human estramustine-binding protein (HEMBP) had a marked tendency to aggregate in several systems, a molecular weight of about 54,000 was determined by gel filtration on Sephacryl S-200 Superfine and high-performance liquid chromatography. A sedimentation-coefficient of about 3.6S was obtained for HEMBP when analyzed by sucrose density gradient centrifugation. Isoelectric focusing in polyacrylamide gels indicated an isoelectric point of 4.7 to 4.8, which was in agreement with the elution position of HEMBP following chromatofocusing on Polybuffer Exchanger 94. Furthermore, HEMBP was eluted from diethylaminoethyl-Sepharose with 0.23 M KCl, was retained by concanavalin A-Sepharose (indicating that HEMBP is a glycoprotein), but did not interact with Affi-Gel Blue. Special efforts were concentrated on establishing that HEMBP was a species distinct from human serum albumin. Separation between the [3H]estramustine-labeled HEMBP and the [3H]estramustine-human serum albumin complex was obtained both on sucrose density gradients by chromatography on Affi-Gel Blue, by chromatofocusing, by gel filtration, by isoelectric focusing, and on concanavalin A-Sepharose by affinity chromatography. Twenty-two of 27 human benign hyperplastic prostate cytosol samples were found to contain protein immunochemically similar to estramustine-binding protein (EMBP) purified from rat ventral prostate as determined by the EMBP radioimmunoassay method. Concentrations from 0.2 to 139.6 ng EMBP per mg of total cytosolic protein (mean, 19.3) were determined. Furthermore, four of seven prostatic cancer specimens as well as two of two normal prostatic specimens were also found to contain rat EMBP-immunoreactive material. The unequivocal demonstration of the presence of a HEMBP is of great potential interest in consideration of estramustine phosphate (Estracyt) therapy against prostatic carcinoma. It is not inconceivable that the concentration of HEMBP in the carcinomatous tissue will be of significance in determining the drug uptake in the malignant tissue.
利用多种色谱方法对人前列腺中的[3H]雌莫司汀结合大分子进行了部分特性鉴定。尽管人雌莫司汀结合蛋白（HEMBP）在多个系统中具有明显的聚集倾向，但通过在Sephacryl S - 200 Superfine上进行凝胶过滤和高效液相色谱法测定其分子量约为54,000。通过蔗糖密度梯度离心分析时，HEMBP的沉降系数约为3.6S。在聚丙烯酰胺凝胶中进行等电聚焦表明其等电点为4.7至4.8，这与在Polybuffer Exchanger 94上进行色谱聚焦后HEMBP的洗脱位置一致。此外，HEMBP用0.23 M KCl从二乙氨基乙基 - 琼脂糖上洗脱下来，被伴刀豆球蛋白A - 琼脂糖保留（表明HEMBP是一种糖蛋白），但不与Affi - Gel Blue相互作用。特别致力于确定HEMBP是一种不同于人血清白蛋白的物质。通过蔗糖密度梯度、Affi - Gel Blue柱色谱、色谱聚焦、凝胶过滤、等电聚焦以及伴刀豆球蛋白A - 琼脂糖亲和色谱法，均实现了[3H]雌莫司汀标记的HEMBP与[3H]雌莫司汀 - 人血清白蛋白复合物的分离。通过EMBP放射免疫测定法确定，27份人良性增生性前列腺细胞溶质样品中有22份含有与从大鼠腹侧前列腺纯化的雌莫司汀结合蛋白（EMBP）免疫化学相似的蛋白质。测定的浓度为每毫克总细胞溶质蛋白含0.2至139.6 ng EMBP（平均为19.3）。此外，在7份前列腺癌标本中有4份以及2份正常前列腺标本中的2份也发现含有大鼠EMBP免疫反应性物质。考虑到磷酸雌莫司汀（癌腺治）治疗前列腺癌，明确证明HEMBP的存在具有极大的潜在意义。不难想象，癌组织中HEMBP的浓度对于确定恶性组织中的药物摄取可能具有重要意义。
