Haldosén L A, Gustafsson J A
Department of Medical Nutrition F60, NOVUM, Huddinge University Hospital, Sweden.
Mol Cell Endocrinol. 1990 May 28;71(1):41-8. doi: 10.1016/0303-7207(90)90073-h.
Basal parameters for binding and cross-linking of 125I-rat prolactin (rPRL) to lactogenic (PRL) binding species present in crude membrane fraction (CMF) or detergent-solubilized preparations of rat liver have been investigated. (1) The highest specific binding to CMF was obtained with an incubation time of 50 h at 20 degrees C and with a 50 mM potassium phosphate buffer adjusted to pH 8.5. (2) Cross-linking of 125I-rPRL to binding sites in CMF with disuccinimidyl suberate (DSS) showed the autoradiographic appearance of an Mr 40,000 binding species. (3) No specific binding or cross-linking of rPRL was seen in Triton X-100-solubilized CMF. This is probably due to Triton X-100-induced changes in the physical properties of rPRL. (4) Specific binding of 125I-rPRL was detected in CHAPS-solubilized CMF. Following cross-linking the autoradiographic appearance of a binding species with an Mr value of 40,000 was shown. 125I-hGH was cross-linked to three PRL binding species with Mr 82,000, 40,000 and 35,000 in CHAPS-solubilized preparations. (5) In Golgi-enriched low-density membrane preparation 125I-rPRL was cross-linked to Mr 82,000, 40,000 and 35,000 species. It is proposed that the inability of rPRL to be cross-linked to Mr 82,000 and 35,000 species present in CHAPS-solubilized preparation is the result of CHAPS-induced changes of rPRL binding properties and low solubilizing capacity of CHAPS. (6) In conclusion, this study shows that also the iodinated endogenous hormone, rat prolactin, and not only hGH identifies high and low molecular forms of the rat liver prolactin receptor.
已对125I-大鼠催乳素（rPRL）与大鼠肝脏粗膜部分（CMF）或去污剂增溶制剂中存在的催乳素（PRL）结合物种的结合和交联的基础参数进行了研究。（1）在20℃下孵育50小时，并用pH值调至8.5的50 mM磷酸钾缓冲液，可获得与CMF的最高特异性结合。（2）用辛二酸二琥珀酰亚胺酯（DSS）将125I-rPRL与CMF中的结合位点交联，显示出Mr为40,000的结合物种的放射自显影外观。（3）在Triton X-100增溶的CMF中未观察到rPRL的特异性结合或交联。这可能是由于Triton X-100诱导的rPRL物理性质变化。（4）在CHAPS增溶的CMF中检测到125I-rPRL的特异性结合。交联后显示出Mr值为40,000的结合物种的放射自显影外观。在CHAPS增溶制剂中，125I-hGH与Mr为82,000、40,000和35,000的三种PRL结合物种交联。（5）在富含高尔基体的低密度膜制剂中，125I-rPRL与Mr为82,000、40,000和35,000的物种交联。有人提出，rPRL无法与CHAPS增溶制剂中存在的Mr为82,000和35,000的物种交联是CHAPS诱导的rPRL结合特性变化和CHAPS低增溶能力的结果。（6）总之，本研究表明，不仅hGH，碘化的内源性激素大鼠催乳素也能识别大鼠肝脏催乳素受体的高分子和低分子形式。
