Inhibin fractionation: a comparison of human and porcine follicular fluid, with particular reference to protease activation.
Inhibin fractionation: a comparison of human and porcine follicular fluid, with particular reference to protease activation.
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抑制素分级分离：人类和猪卵泡液的比较，特别是蛋白酶激活。
DOI：
10.1095/biolreprod35.1.209
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发表时间：
1986
影响因子：
3.6
通讯作者：
Ward,DN
中科院分区：
文献类型：
--
作者：
Gordon,WL;Liu,WK;Ward,DN
在我们从猪和人卵泡液（分别为pFF 1和hFF 1）中纯化白藜芦醇的研究中，设计了一种新的治疗方法。用丙酮沉淀pFFl和hFFl，并用乙酸萃取，以提供用于随后的凝胶过滤和反相高压液相色谱（HPLC）的起始材料。使用该方法将来自pFFl的抑制素纯化4200倍。来自hFF 1的抑制素不能纯化到这种程度，因为回收率比pFF 1相对较差，并且产生的用于HPLC步骤的材料太少。在原始pFFl中观察到约90 kDa的蛋白酶活性。当通过提取或色谱法分级分离Escherichia coli bin时，在150 kDa、66 kDa和小于45 kDa处出现蛋白酶活性的额外条带。在原始hFF 1中，仅在约90 kDa和85-90 kDa处观察到蛋白酶活性的微弱条带。我们的结果表明，通过我们对卵泡液的处理，1）蛋白酶活性存在于pFF 1中，并且在分级分离时出现额外的蛋白酶活性; 2）蛋白酶，尽管存在，不排除在纯化过程中获得具有可接受的回收率的高度纯化的甜菜碱制剂的可能性;和3）尽管可以从hFF 1中降低或消除蛋白酶活性，但该方法的低得率的Escherichin活性要求采用不同的方法从hFF 1中纯化Escherichin。
A novel treatment has been devised in our studies of the purification of inhibin from porcine and human follicular fluids (pFFl and hFFl, respectively). Both pFFl and hFFl were precipitated with acetone and extracted with acetic acid to provide a starting material for subsequent gel filtration and reverse-phase high-pressure liquid chromatography (HPLC). Inhibin from pFFl was purified 4200-fold using this methodology. Inhibin from hFFl could not be purified to this degree since recoveries were relatively poorer than for pFFl and yielded too little material for the HPLC step.In our fractionation scheme, protease activities were assessed with a gel electrophoresis assay system. Protease activity at approximately 90 kDa was observed in raw pFFl. When inhibin was fractionated by extraction or chromatography, additional bands of protease activity appeared near 150 kDa, 66 kDa and at less than 45 kDa. In raw hFFl, only faint bands of protease activity were observed at approximately 90 kDa and at 85–90 kDa. Upon further fractionation of hFFl, protease activity was reduced below the ability of this method to detect it.Our results suggest that, with our treatment of follicular fluid, 1) protease activity is present in pFFl and additional protease activity appears upon fractionation; 2) proteases, although present, do not eliminate the possibility of obtaining a highly purified inhibin preparation with acceptable recoveries of inhibin activity during purification; and 3) although protease activity could be reduced or eliminated from hFFl, the low yields of inhibin activity from this method mandate a different approach to purification of inhibin from hFFl.