MEMBRANE TRANSPORT AND FUSION

细胞必须合并细胞膜才能混合房水空间。我们有 今年在开发新的治疗方法方面取得了进展 融合孔的表征，鉴定和鉴定 真核融合蛋白的鉴定。使用双精度 全细胞记录技术。测量了结的电导 在杆状病毒介导的昆虫细胞合胞体形成过程中。 融合是由低pH引发的。每个电池中的对称电流 直接测量了熔融气孔电导。它 以突然(0.1-0.2毫秒上升时间)的气孔形成开始 以L北纬为中心的电导分布在#年缓慢加宽。 几十秒的时间尺度，然后陡然增加。我们的 以前的方法，时间分辨导纳测量，是 对两种方法进行比较：两种方法得出的结果相似。因此， 我们以前测量融合孔的方法得到了证实。这个 新的方法将使我们能够提高时间分辨率 熔融孔电导提高1-2个数量级。多个- 频率、数字锁定放大器在一台 微型计算机扩展我们对融合孔的测量，以便 中更精确地计算熔融孔电导。 存在噪音。确定参与融合的蛋白质 研究了孔道形成、硫醇基团的敏感性。氨基右旋糖苷 可逆硫醇试剂SPDP的衍生物抑制胞吐作用 与大小成反比。这意味着敏感的硫醇基团 不是埋得很深，而是在相对暴露的亲水性 环境。硫醇反应蛋白参与触发 聚变而不是下游事件，因为在地球上看不到聚变 DTT治疗去除SPDP阻滞剂。GTP触发 钠对大鼠肝微粒体融合的强烈抑制 偏碘酸根。DTT的恢复作用表明，在 这种抑制的最小可逆部分是由 蛋白质邻位硫醇基团的氧化与形成 二硫键。高碘酸根敏感硫醇的选择性标记 揭示了一个主要的标记多肽，其相对分子质量为15 kDa。这个 蛋白质氧化和还原的相关性以及 分别抑制和恢复微体融合， 提示该蛋白可能是依赖于GTP的 微粒融合机。

Cells must merge membranes in order to mix aqueous spaces. We have made progress this year in developing new methods for the characterization of fusion pores, and the identification and characterization of eukaryotic fusion proteins. Using double whole-cell recording technique. junctional conductance was measured during baculovirus-mediated syncytia formation of insect cells. Fusion was triggered by low pH. Symmetrical currents in each cell of the pair measured the fusion pore conductance directly. It started as abrupt (0.1-0.2 ms rise-time) pore formation with a distribution of conductances centered at l nS.- widened slowly in the time scale of tens of seconds and then increased abruptly. Our previous method, time-resolved admittance measurement, was performed to compare two methods: both gave similar results. Thus our previous methods of measuring fusion pores was confirmed. The new method will allow us to improve the time resolution of the fusion pore conductance by 1-2 orders of magnitude. A multiple- frequency, digital lock-in amplifier was implemented on a microcomputer to extend our measurements of fusion pores so as to calculate the fusion pore conductance more precisely in the presence of noise. To determine the proteins involved in fusion pore formation, thiol group sensitivity was studied. Amino-dextran derivatives of the reversible thiol reagent SPDP inhibit exocytosis inversely with size. This implies that the susceptible thiol groups are not buried deeply but are in a relatively exposed hydrophilic environment. Thiol-reactive proteins are involved in triggering fusion rather than downstream events, since no fusion is seen upon the removal of the SPDP block by DTT treatment. GTP-triggered fusion of rat liver microsomes was strongly inhibited by sodium metaperiodate. The restorative effect of DTT indicates that at least the reversible portion of the inhibition is caused by oxidation of protein vicinal thiol groups with the formation of disulfide bonds. Selective labeling of periodate-sensitive thiols revealed one major labeled polypeptide with M.W. of 15 kDa. The correlation between oxidation and reduction of the protein and inhibition and restoration of the microsomal fusion, respectively, suggests that this protein may be part of the GTP-dependent microsomal fusion machinery.