Research School of Biological Sciences, Australian National University, Canberra, A.C.T. 2601, Australia.
Plant Physiol. 1977 Apr;59(4):759-66. doi: 10.1104/pp.59.4.759.
An attempt has been made to use lactoperoxidase-catalyzed iodination of excised Cucurbita hypocotyl hooks to monitor the distribution of plasma membrane fragments relative to that of phytochrome in particulate fractions from this tissue. Upon fractionation, the iodinated tissue yields a 20,000g pellet which contains 58% of the trichloroacetic acid-precipitable (125)I at a specific radioactivity 12 times that of the proteins in the supernatant. On sucrose gradients, the labeled fraction has a mean isopycnic density of 1.15 g . cm(-3). The distribution profile is distinct from that of the total particulate protein and does not coincide with either mitochondrial or endoplasmic reticulum markers. These observations satisfy operational criteria commonly accepted in other systems as indices of selective labeling of the cell surface. The sucrose gradient profiles of the phytochrome and (125)I in the 20,000g pellets are noncoincident. In the absence of more direct evidence, this is readily interpreted to indicate a lack of association of the pigment with the plasma membrane.Autoradiographic analysis indicates, however, that the (125)I is almost exclusively associated with an amorphous film (possibly phloem-exudate protein) overlying the cut cells at the point of prelabeling excision and along the outer physical surface of the hypocotyl cuticle. No evidence of plasma membrane labeling is apparent. The observed membrane-like behavior of the iodinated material upon cell fractionation is attributed to the preferential posthomogenization association of this material with a particular membrane fraction(s). These data indicate that in addition to the well recognized potential for spurious labeling of the internal cytoplasmic proteins of leaky cells, a further source of ambiguity in surface-labeling experiments should be considered. That is, the potential for labeling extracellular proteins of nonplasma membrane origin but with a capacity to become associated with membranes upon homogenization.
已经尝试使用乳过氧化物酶催化的碘化作用来标记离体南瓜下胚轴钩，以监测质膜片段相对于组织中类囊体色素的分布。在分级分离后，碘化组织产生一个 20,000g 的沉淀，其中含有 58%的三氯乙酸沉淀（125）I，比上清液中的蛋白质具有 12 倍的比放射性。在蔗糖梯度上，标记的部分具有 1.15 g. cm(-3)的平均等密度。分布谱与总颗粒蛋白的分布谱明显不同，也与线粒体或内质网标记物不同。这些观察结果满足了在其他系统中普遍接受的作为细胞表面选择性标记的操作标准。20,000g 沉淀中类囊体色素和（125）I 的蔗糖梯度谱不重合。在没有更直接证据的情况下，这很容易解释为表明色素与质膜没有关联。然而，放射自显影分析表明，（125）I 几乎完全与覆盖在预标记切除点的细胞上的无定形膜（可能是韧皮部分泌物蛋白）以及下胚轴角质层的外物理表面相关联。没有明显的质膜标记的证据。在细胞分级分离时观察到碘化材料的类似膜的行为归因于该材料在特定膜部分（s）上的优先后匀化关联。这些数据表明，除了众所周知的渗漏细胞内部细胞质蛋白可能出现虚假标记的潜在可能性之外，还应该考虑表面标记实验中的另一个来源的模糊性。也就是说，有可能标记非质膜起源的细胞外蛋白质，但在匀化时具有与膜结合的能力。
