Faiers A A, Loh A Y, Osmond D H
Can J Biochem. 1977 Aug;55(8):869-75. doi: 10.1139/o77-128.
Pooled plasmas from normal or binephrectomized rats and perfusates of isolated livers were used as sources of renin substrate for isoelectric focusing. After desalting, preliminary fractionation (plasma only), and concentration, the preparations were focused in a pH 3--10 gradient on 20-cm glass plates layered with Sephadex slurry. The pH 4--6 region, containing all the substrate, was scraped from this plate and refocused in a pH 4--6 gradient. Substrate content of 1-cm strips of slurry from half of the plate was determined by both radioimmunoassay and bioassay of angiotensin resulting from incubation with added renin. Corresponding strips from the other half of the plate were incubated without renin as a control for any preformed angiotensin. The asymmetry and broad distribution (pH 4--5) of substrate from different sources suggested the existence of more than one form. Higher resolution achieved by using the high substrate concentration of postnephrectomy plasma and 0.5-cm strips of slurry on 20-cm or 40-cm plates revealed peaks and shoulders of substrate activity. Our data suggest that multiple forms of substrate are synthesized by the liver and circulate in plasma. Postnephrectomy rat plasma appears to contain relatively more substrate(s) with higher isoelectric points than in normal plasma, possibly an accumulation of forms ordinarily degraded by endogenous renal renin.
来自正常或双侧肾切除大鼠的混合血浆以及离体肝脏的灌流液被用作等电聚焦的肾素底物来源。脱盐、初步分级分离（仅针对血浆）并浓缩后，将制剂在铺有葡聚糖凝胶浆液的20厘米玻璃板上进行pH 3 - 10梯度聚焦。从该板上刮下含有所有底物的pH 4 - 6区域，并在pH 4 - 6梯度中重新聚焦。通过与添加的肾素孵育产生的血管紧张素的放射免疫测定和生物测定来确定来自板一半的1厘米凝胶条带的底物含量。作为对照，将来自板另一半的相应条带在不添加肾素的情况下孵育，以检测任何预先形成的血管紧张素。来自不同来源的底物的不对称性和广泛分布（pH 4 - 5）表明存在不止一种形式。通过使用肾切除术后血浆的高底物浓度以及在20厘米或40厘米板上的0.5厘米凝胶条带实现了更高的分辨率，揭示了底物活性的峰和肩。我们的数据表明，多种形式的底物由肝脏合成并在血浆中循环。肾切除术后大鼠血浆似乎比正常血浆含有相对更多等电点较高的底物，可能是通常被内源性肾素降解的形式的积累。
