Wu H, He C L, Jehn B, Black S J, Fissore R A
Molecular and Cellular Biology Program, University of Massachusetts, Amherst, Massachusetts, 01003, USA.
Dev Biol. 1998 Nov 15;203(2):369-81. doi: 10.1006/dbio.1998.9070.
Injection of sperm cytosolic extracts into mammalian eggs has been shown to elicit intracellular calcium ([Ca2+]i) oscillations that are similar in amplitude, duration, and frequency to those observed following fertilization. Thus, to characterize the Ca2+-release component(s) in porcine sperm cytosolic extracts, a combination of fractionation techniques was used. The fraction with Ca2+ releasing activity was precipitated by 50% saturating solutions of ammonium sulfate and Western blot analysis showed that the pellets contained glucosamine-6-phosphate deaminase (gpd)/oscillin, a protein which has been suggested to be the sperm's active component. Single and double isoelectrofocusing (IEF) of porcine sperm extracts generated fractions with different Ca2+-releasing activities. Fractions with maximal Ca2+-releasing activity did not contain material that was immunoreactive with antibodies against gpd/oscillin; adjacent fractions containing gpd/oscillin had no Ca2+-releasing activity. These findings were confirmed by IEF coupled with size exclusion chromatography on Superose 12 and with hydroxyapatite chromatography. These procedures predict an isoelectric point of our active component of 6.5-7.0 and a relative molecular weight ranging from 29 to 68 kDa. In summary, the data show that the Ca2+ release-inducing component(s) of porcine sperm extracts can be fractionated and that gpd/oscillin is not the pig sperm Ca2+ oscillogen.
已证明将精子胞质提取物注入哺乳动物卵子可引发细胞内钙（[Ca2+]i）振荡，其振幅、持续时间和频率与受精后观察到的振荡相似。因此，为了表征猪精子胞质提取物中的钙释放成分，使用了多种分级分离技术。具有钙释放活性的级分可被50%饱和度的硫酸铵溶液沉淀，蛋白质印迹分析表明沉淀中含有6-磷酸葡糖胺脱氨酶（gpd）/振荡蛋白，一种被认为是精子活性成分的蛋白质。猪精子提取物的单次和双次等电聚焦（IEF）产生了具有不同钙释放活性的级分。具有最大钙释放活性的级分不含与抗gpd/振荡蛋白抗体发生免疫反应的物质；含有gpd/振荡蛋白的相邻级分没有钙释放活性。这些发现通过IEF与Superose 12上的尺寸排阻色谱以及羟基磷灰石色谱联用得到证实。这些方法预测我们的活性成分的等电点为6.5-7.0，相对分子质量范围为29至68 kDa。总之，数据表明猪精子提取物中诱导钙释放的成分可以被分级分离，并且gpd/振荡蛋白不是猪精子钙振荡原。
