Structural proteins of the mouse spermatozoan tail: an electrophoretic analysis.
Structural proteins of the mouse spermatozoan tail: an electrophoretic analysis.
复制标题
小鼠精子尾部的结构蛋白：电泳分析。
DOI：
10.1095/biolreprod24.3.691
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发表时间：
1981
影响因子：
3.6
通讯作者：
Bellvé,AR
中科院分区：
文献类型：
--
作者：
Bradley,FM;Meth,BM;Bellvé,AR
利用一、二维聚丙烯酰胺凝胶电泳技术，将小鼠精子尾部的结构成分分解为多个多肽种类。用十二烷基硫酸钠（SDS, 1%）或胰蛋白酶（0.1 mg/ml）短暂孵育精子。SDS和胰蛋白酶裂解后的精子分别采用蔗糖梯度离心和40-60%甲咪唑胺梯度超离心分离为纯头尾两部分。用单维sds -聚丙烯酰胺凝胶电泳分析胰蛋白酶裂解和sds裂解精子的sds不溶性结构尾部蛋白，发现约20条蛋白带。暴露于胰蛋白酶后，一些高分子量组分要么数量减少，要么完全丧失。此外，当sds不溶性尾部蛋白被羧甲基化时，多肽谱的复杂性大大降低，表明一些高分子量带代表了由分子间二硫键引起的蛋白质聚集。氨基乙基化和羧甲基化尾部蛋白的等电聚焦（IEF）凝胶也导致了显著不同的条带模式。最后，结构尾部蛋白的二维电泳，还原和羧甲基化，使29个主要蛋白点的明确分辨率。
Structural components of the mouse spermatozoan tail were resolved into multiple polypeptide species by using one- and two-dimensional polyacrylamide gel electrophoresis. Spermatozoa were decapitated by brief incubation with either sodium dodecyl sulfate (SDS, 1%) or trypsin (0.1 mg/ml). The SDS- and trypsin-cleaved spermatozoa were subsequently separated into pure head and tail fractions by using sucrose gradient centrifugation and ultracentrifugation in a 40–60% metrizamide gradient, respectively. Analysis of the SDS-insoluble structural tail proteins from trypsin-cleaved and SDS-cleaved spermatozoa by single dimension SDS-polyacrylamide gel electrophoresis revealed approximately 20 protein bands. Following exposure to trypsin several of the high molecular weight components were either reduced in quantity or lost completely. Furthermore, when SDS-insoluble tail proteins were carboxymethylated, the complexity of the polypeptide profile diminished substantially, indicating that some high molecular weight bands represented protein aggregation caused by intermolecular disulfide bonds. Isoelectric focusing (IEF) gels of aminoethylated and carboxymethylated tail proteins also resulted in remarkably different banding patterns. Finally, two-dimensional electrophoresis of the structural tail proteins, reduced and carboxymethylated, enabled definitive resolution of 29 major protein spots.