Tanaka S, Nemoto K, Yamamura E, Ichikawa A
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan.
J Biol Chem. 1998 Apr 3;273(14):8177-82. doi: 10.1074/jbc.273.14.8177.
To clarify the process of post-translational modification of L-histidine decarboxylase (HDC), we investigated the conversion of the 74-kDa form of HDC into the 53-kDa form in specialized organella of a rat basophilic/mast cell line (RBL-2H3). With treatment of streptolysin-O, RBL-2H3 cells released approximately 40% of HDC activity accompanied by over 90% of lactate dehydrogenase activity. Only the 74-kDa form of HDC was detected in the leaked fraction by SDS-polyacrylamide gel electrophoresis. The 74-kDa form in the homogenate of pulse-labeled cells was recovered in both the supernatant and particulate fractions, while the 53-kDa form was detected only in the particulate fraction containing marker proteins of microsomes, Golgi, and lysosomal granules. Confocal microscopic observation using double staining immunofluorescence with anti-GST fusion HDC antiserum showed that most of the HDC coexists with protein-disulfide isomerase, a typical marker of the luminal space of the ER. With treatment of digitonin, RBL-2H3 cells released only 74-kDa HDC. Trypsin digestion of digitonin-permeabilized cells resulted in the disappearance of the 74-kDa form but not the 53-kDa form. From these results, it is assumed that the 74-kDa form of HDC, synthesized in the cytosol, is translocated into the lumen of the ER, where it is converted to the 53-kDa form.
为阐明L-组氨酸脱羧酶（HDC）的翻译后修饰过程，我们研究了大鼠嗜碱性/肥大细胞系（RBL-2H3）的特殊细胞器中74 kDa形式的HDC向53 kDa形式的转化。用链球菌溶血素-O处理后，RBL-2H3细胞释放出约40%的HDC活性，同时伴有超过90%的乳酸脱氢酶活性。通过SDS-聚丙烯酰胺凝胶电泳在泄漏部分仅检测到74 kDa形式的HDC。脉冲标记细胞匀浆中的74 kDa形式在上清液和颗粒部分均有回收，而53 kDa形式仅在含有微粒体、高尔基体和溶酶体颗粒标记蛋白的颗粒部分检测到。使用抗GST融合HDC抗血清进行双重染色免疫荧光的共聚焦显微镜观察表明，大多数HDC与内质网腔的典型标记蛋白二硫键异构酶共存。用洋地黄皂苷处理后，RBL-2H3细胞仅释放74 kDa的HDC。对洋地黄皂苷通透的细胞进行胰蛋白酶消化导致74 kDa形式消失，但53 kDa形式未消失。从这些结果推测，在细胞质中合成的74 kDa形式的HDC被转运到内质网腔，在那里它被转化为53 kDa形式。
