CELLULAR LOCALIZATION OF MARINE-DERIVED EXCITATORY AMINO ACIDS IN THEIR PRODUCING ORGANISMS

用免疫组织化学方法研究了两种代表性的海洋兴奋性氨基酸：dysiherbaine（DH）和海人酸（KA）的细胞定位。通过将每种氨基酸偶联至载体蛋白如KLH来产生针对每种氨基酸的选择性多克隆抗体。使用成功制备的抗体，分别对产生DH和红藻氨酸的海绵Dysidea herbacea和红藻Digenea simplex的切片进行染色，然后用蛋白A-金显色。显微镜观察表明，DH仅定位于海绵中表皮的球形细胞中，并经DH结合亲和层析和SDS-PAGE电泳证实存在分子量为111 kD的DH结合蛋白。我们利用GluR靶向引物进行RT-PCR，初步观察到海绵中可能存在GluR样分子，KA主要定位于生长在藻主茎上的类继承人结构的细胞中。只有主茎的表面被染色。茎的表面染色较浓，可能是向耳状结构转化。耳状结构由三层细胞组成：外层小细胞、中层细胞和中央细胞。外小细胞中有大量叶绿体。所有这些细胞均可见KA染色。在周围细胞中观察到的小颗粒体（1 - 2 μm）比其他细胞结构染色更强。

Cellular localizations of two representative marine excitatory ammo acids : dysiherbaine (DH) and kainic acid (KA) were investigated using immunohistochemical methods. Selective polyclonal antibodies to each amino acid were raised by coupling each amino acid to carrier protein such as KLH. Using successfully prepared antibodies, section of Micronesian sponge Dysidea herbacea and red algae Digenea simplex, producing organism of DH and kainic acid, respectively, were separately stained then visualized with protein A-gold. Microscopic observations indicated that DH was localized exclusively in the spherical cell present hi the mesohyl of the sponge.We also showed a presence of putative DH-binding protein of 111kD using DH-conjugated affinity chromatography and SDS-PAGE. We preliminary observed by RT-PCR using GluR-targeted primers that there might be a GluR-like molecule in the sponge.KA was mainly located in the cells of heir-like structure of the algae which grows from the main stem of the algae. Only very surface of the main stem was stained. The surface of the stem which likely be transforming into the hear-like structure was stained densely. The hear-like structure composed of three cell layers : outer small cells, middle layer cells and the central cells. Numbers of chloroplasts were present in the outer small cells. KA-stain was observed all these cells. A small (1 to 2 μm) granular body observed in the surrounding cells was stained somewhat stronger then other cellular structures.