Ash C, Atkinson H J
Exp Parasitol. 1984 Aug;58(1):27-40. doi: 10.1016/0014-4894(84)90018-3.
Eggshells of Nematodirus battus leaked trehalose 4 hr after being stimulated to hatch, and became permeable to trypan blue at their poles; 80% of eggs were stained blue 24 hr later. Exogenous application of ruthenium red significantly inhibited chill- and sodium fluoride-stimulated hatching, 50% hatch inhibition occurring in 44.67 +/- 2.2 and 8.5 +/- 1.5 microM, respectively. Lanthanum chloride, however, was not as inhibitory as ruthenium red on fluoride-stimulated hatching, 50% occurring at 31.60 +/- 1.25 microM. A Scatchard plot of the competitive binding of ruthenium red to eggshells demonstrated a high-affinity binding site for calcium, KCa' = 1.92 microM and a second, low-affinity site, KCa" = 1169.60 microM. Ruthenium red binding was significantly reduced by several enzymes, e.g., EGTA-buffered trypsin reduced binding by 73%. Radioiodinated concanavalin A also bound competitively to the eggshells in the presence of alpha-D-glucosyl-alpha-D-glucopyranoside and alpha-methyl-D-mannopyranoside. Eggshells incorporated phosphorus-32 from ATP after chilling or on exposure to sodium fluoride; gel filtration of solubilized homogenates of these samples showed that two proteins were radiolabelled with molecular weights of 38 X 10(3) and 8 X 10(3) Da, respectively. This phosphorylation was inhibited by N-ethylmaleimide, which also prevented hatching.
巴氏细颈线虫的卵在受到刺激孵化4小时后，卵壳会渗漏海藻糖，并且在两极对锥虫蓝具有通透性；24小时后，80%的卵被染成蓝色。外源施加钌红可显著抑制冷刺激和氟化钠刺激引起的孵化，50%孵化抑制的钌红浓度分别为44.67±2.2微摩尔和8.5±1.5微摩尔。然而，氯化镧对氟化钠刺激引起的孵化的抑制作用不如钌红，50%孵化抑制的氯化镧浓度为31.60±1.25微摩尔。钌红与卵壳竞争性结合的Scatchard图显示存在一个高亲和力的钙结合位点，KCa' = 1.92微摩尔，以及第二个低亲和力位点，KCa" = 1169.60微摩尔。几种酶可显著降低钌红的结合，例如，EGTA缓冲的胰蛋白酶可使结合减少73%。在α-D-葡萄糖基-α-D-吡喃葡萄糖苷和α-甲基-D-吡喃甘露糖苷存在的情况下，放射性碘化伴刀豆球蛋白A也与卵壳竞争性结合。冷藏或暴露于氟化钠后，卵壳会从ATP中摄取磷-32；对这些样品的溶解匀浆进行凝胶过滤显示，有两种蛋白质被放射性标记，分子量分别为38×10³和8×10³道尔顿。这种磷酸化作用受到N-乙基马来酰亚胺的抑制，N-乙基马来酰亚胺也会阻止孵化。
