Watabe S, Kohno H, Kouyama H, Hiroi T, Yago N, Nakazawa T
Radioisotope Research Institute, St. Marianna University School of Medicine, Kawasaki.
J Biochem. 1994 Apr;115(4):648-54. doi: 10.1093/oxfordjournals.jbchem.a124390.
We have purified SP-22, a substrate protein for mitochondrial ATP-dependent protease in bovine adrenal cortex. Native SP-22 showed an M(r) of 350,000 +/- 20,000, and was composed of more than 10 molecules of an M(r) 21,600 subunit. Subcellular and submitochondrial fractionation of adrenocortical tissues revealed that SP-22 was localized in the mitochondrial matrix, suggesting that SP-22 is a natural substrate for ATP-dependent protease, a matrix enzyme. The concentration of SP-22 in adrenocortical mitochondrial fractions was 16 +/- 3 micrograms/mg proteins (mean +/- SD, n = 6) as determined by radioimmunoassay using specific anti-SP-22 antibody. Adrenal cortex showed the highest concentration among the 15 bovine tissues tested, followed by liver, renal cortex, adrenal medulla, heart, and renal medulla. We determined the amino acid sequence of SP-22, which is composed of 195 amino acids. Amino acid 47 was not identified by the sequencer. FAB-mass spectrometry of AA47-AA55 fragment revealed that AA47 was cysteine-sulfinic acid (Cys-SO2H). By a homology search in the NBRF-PIR data base, SP-22 was found to be 91% homologous to murine erythroleukemia cell MER-5 protein, which may have an important role in the induction of differentiation. SP-22 was also homologous to the C22 component of alkyl hydroperoxide reductase in Salmonella typhimurium, thiol-specific antioxidant in Saccharomyces cerevisiae, and some other proteins. Since a segment around AA47 was highly conserved, this residue may be important for the biochemical functions of SP-22.
我们已经纯化了SP - 22，它是牛肾上腺皮质中线粒体ATP依赖性蛋白酶的一种底物蛋白。天然的SP - 22的相对分子质量为350,000±20,000，由10多个相对分子质量为21,600的亚基分子组成。肾上腺皮质组织的亚细胞和亚线粒体分级分离显示，SP - 22定位于线粒体基质中，这表明SP - 22是ATP依赖性蛋白酶（一种基质酶）的天然底物。使用特异性抗SP - 22抗体通过放射免疫测定法测定，肾上腺皮质线粒体部分中SP - 22的浓度为16±3微克/毫克蛋白质（平均值±标准差，n = 6）。在所检测的15种牛组织中，肾上腺皮质的浓度最高，其次是肝脏、肾皮质、肾上腺髓质、心脏和肾髓质。我们测定了SP - 22的氨基酸序列，它由195个氨基酸组成。测序仪未鉴定出第47位氨基酸。对AA47 - AA55片段的快原子轰击质谱分析表明，AA47是半胱氨酸亚磺酸（Cys - SO2H）。通过在NBRF - PIR数据库中进行同源性搜索，发现SP - 22与小鼠红白血病细胞MER - 5蛋白有91%的同源性，后者可能在诱导分化中起重要作用。SP - 22还与鼠伤寒沙门氏菌中的烷基过氧化氢还原酶的C22成分、酿酒酵母中的硫醇特异性抗氧化剂以及其他一些蛋白质同源。由于第47位氨基酸周围的一段序列高度保守，该残基可能对SP - 22的生化功能很重要。
