Coffe G, Le Caer J P, Lima O, Adoutte A
Laboratoire de Biologie Cellulaire 4, URA 1134, Université de Paris-Sud, Orsay, France.
Cell Motil Cytoskeleton. 1996;34(2):137-51. doi: 10.1002/(SICI)1097-0169(1996)34:2<137::AID-CM5>3.0.CO;2-8.
The epiplasmic layer, a continuous rigid granulo-fibrillar sheet directly subtending the surface membranes of Paramecium, is one of the outermost of the various cytoskeletal networks that compose it cortex. We have previously shown that the epiplasm consists of a set of 30 to 50 protein bands on SDS-PAGE in the range 50 to 33 kDa, the epiplasmins. We report a purification procedure for the set of epiplasmic proteins, a description of their physicochemical and reassembly properties, and a preliminary characterization of their sequence. The conditions for solubilization of the epiplasm and for in vitro reassembly of its purified constituents ar described. Reassembly of the entire set of proteins and of some (but not all) subsets are shown to yield filamentous aggregates. Microsequences of two purified bands of epiplasmins reveal a striking amino acid sequence consisting of heptad repeats of only three main amino acids, P, V, and Q. These repeats were confirmed by DNA sequencing of polymerase chain reaction products. The motif is QPVQ-h, in which h is a hydrophobic residue. This may constitute the core of the epiplasmin sequence and, in view of the tendency of such a sequence to form a coiled-coil, may account for the remarkable self-aggregation properties of epiplasmins.
表质层是直接位于草履虫表面膜下方的连续刚性颗粒纤维状薄片，是构成其皮层的各种细胞骨架网络中最外层的结构之一。我们之前已经表明，在SDS-PAGE上，表质由一组分子量在50至33 kDa范围内的30至50条蛋白带组成，即表质蛋白。我们报告了一套表质蛋白的纯化方法，描述了它们的物理化学性质和重新组装特性，并对其序列进行了初步表征。文中描述了表质溶解以及其纯化成分进行体外重新组装的条件。整套蛋白以及部分（但不是全部）子集的重新组装显示会产生丝状聚集体。对两条纯化的表质蛋白带的微序列分析揭示了一个惊人的氨基酸序列，该序列仅由三种主要氨基酸P、V和Q的七肽重复组成。这些重复序列通过聚合酶链反应产物的DNA测序得到了证实。该基序为QPVQ-h，其中h是一个疏水残基。这可能构成了表质蛋白序列的核心，鉴于这样的序列倾向于形成卷曲螺旋，这可能解释了表质蛋白显著的自我聚集特性。
