Department of Horticulture, Ohio State University, 43210, Columbus, OH, USA.
Planta. 1989 Sep;179(2):137-47. doi: 10.1007/BF00393683.
The usefulness of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) for in-situ studies of the chloroplast phosphate translocator was evaluated by fluorescence microscopy and radiolabeling of spinach (Spinacia oleracea L.) (C3 plant) and maize (Zea mays L.) (C4 plant) chloroplasts. In maize mesophyll and bundle-sheath chloroplasts and in spinach chloroplasts that were either intact, broken or swollen, DIDS fluorescence was only associated with the chloroplast envelope. Intact chloroplasts often had fluorescent patches corresponding to concave regions of the chloroplast which we assume to be regions enriched in DIDS-binding sites.Incubation of intact or broken spinach chloroplasts or maize mesophyll chloroplasts with [(3)H2]DIDS resulted in the labeling of a single polypeptide (relative molecular mass, Mr, ∼30 kDa) in the envelope fraction, in each case. Label in the stromal fraction was not detected when intact chloroplasts were incubated with [(3)H2]DIDS. However, when broken chloroplasts were incubated with [(3)H2]DIDS, several polypeptides of various molecular masses were labeled, but not the 30×31-kDa polypeptide. In thylakoid fractions from both broken and intact chloroplasts, a single 30×31-kDa polypeptide was labeled inconsistently. When a mixture of intact maize mesophyll and bundle-sheath chloroplasts was labeled with [(3)H2]DIDS, extracts of whole chloroplasts displayed radioactivity only in the 30×31-kDa band.We conclude that DIDS is a valuable probe for the in-situ identification and characterization of the ∼30-kDa protein - the presumptive phosphate translocator - in C3 and C4 chloroplasts since DIDS (1) does not penetrate the inner membrane of the envelope of intact chloroplasts and, therefore, (2) does not bind internal sites in intact chloroplasts, and (3) only binds the ∼30-kDa protein in the inner membrane of the envelope.
4,4'-二异硫氰酸根合二苯乙烯-2,2'-二磺酸(DIDS)在原位研究叶绿体磷酸盐转运体中的有用性，通过荧光显微镜和放射性标记菠菜（Spinacia oleracea L.）（C3 植物）和玉米（Zea mays L.）（C4 植物）叶绿体来评估。在玉米叶肉和束鞘叶绿体以及完整、破裂或肿胀的菠菜叶绿体中，DIDS 荧光仅与叶绿体被膜相关。完整的叶绿体通常具有荧光斑块，对应于叶绿体的凹面区域，我们假设这些区域富含 DIDS 结合位点。用 [(3)H2]DIDS 孵育完整或破裂的菠菜叶绿体或玉米叶肉叶绿体，在每种情况下，都会导致被膜部分标记出一个单一的多肽（相对分子质量，Mr，约 30 kDa）。当完整的叶绿体用 [(3)H2]DIDS 孵育时，在基质部分未检测到标记。然而，当破裂的叶绿体用 [(3)H2]DIDS 孵育时，各种大小的多个多肽被标记，但不是 30×31-kDa 多肽。在破裂和完整叶绿体的类囊体部分，一个单一的 30×31-kDa 多肽被不一致地标记。当用 [(3)H2]DIDS 标记完整的玉米叶肉和束鞘叶绿体的混合物时，整个叶绿体提取物仅在 30×31-kDa 带中显示放射性。我们得出结论，DIDS 是一种有价值的探针，可用于原位鉴定和表征 C3 和 C4 叶绿体中约 30-kDa 蛋白 - 假定的磷酸盐转运体 -，因为 DIDS (1) 不会穿透完整叶绿体被膜的内膜，因此，(2) 不会与完整叶绿体中的内部位点结合，并且 (3) 仅与被膜的内膜中的约 30-kDa 蛋白结合。
