Lin X, Tang J
Laboratory of Protein Studies, Oklahoma Medical Research Foundation, Oklahoma City.
J Biol Chem. 1990 Jan 25;265(3):1490-5.
A thermostable, acid proteolytic activity has been found to be associated with the cells and in the culture medium of Sulfolobus acidocaldarius, an archaebacterium. This acid protease, which has been named thermopsin, was purified to homogeneity from the culture medium by a five-step procedure including column chromatographies on DEAE-Sepharose CL-6B, phenyl-Sepharose CL-4B, Sephadex G-100, monoQ (fast protein liquid chromatography), and gel filtration (high pressure liquid chromatography). The purified thermopsin produced a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the proteolytic activity was associated with the band. Thermopsin is a single-chain protein as indicated by gel electrophoresis and by a single NH2-terminal sequence. It has maximal proteolytic activity at pH 2 and 90 degrees C. A genomic library of S. acidocaldarius was prepared and screened by an oligonucleotide probe designed from the NH2-terminal sequence of thermopsin. Five positive clones were isolated. From these clones the thermopsin gene was mapped and sequenced. The nucleotide sequence showed that the thermopsin structure is encoded in 1020 bases. In the deduced protein sequence, there are 41 amino acid residues (including the initiation Met) preceding the NH2-terminal position of thermopsin. Most of these residues appear to be characteristic of a leader sequence. However, the presence in this region of a short pro sequence cannot be ruled out. Thermopsin contains a single cysteine at residue 237 that is not essential for activity (Fusek, M., Lin, X.-L., Tang, J. (1990) J. Biol. Chem. 265, 1496-1501. Thermopsin has no apparent sequence similarity to aspartic proteases of the pepsin family nor to pepstatin-insensitive acid protease (Maita, T., Nagata, S., Matsuda, G., Murata, S., Oda, K., Murao, S., and Tsura, D. (1984) J. Biochem. 95, 465-475) and thus may represent a new class of acid proteases. Also absent is the characteristic active site aspartyl sequence of aspartic proteases. There are 11 potential N-glycosylation sites on each thermopsin molecule. The molecular weight estimated from gel filtration (45,000) is larger than that calculated from the sequence (32,651), suggesting that thermopsin is the sequence (32,651), suggesting that thermopsin is glycosylated at at least some of these 11 sites.
已发现嗜热栖热菌（一种古细菌）的细胞及培养基中存在一种热稳定的酸性蛋白水解活性。这种酸性蛋白酶已被命名为嗜热栖热菌素，通过五步程序从培养基中纯化至同质，这五步程序包括在DEAE-琼脂糖CL-6B、苯基-琼脂糖CL-4B、葡聚糖G-100、单Q（快速蛋白质液相色谱）上进行柱色谱以及凝胶过滤（高压液相色谱）。纯化后的嗜热栖热菌素在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上产生一条带，且蛋白水解活性与该带相关。如凝胶电泳和单一的NH₂末端序列所示，嗜热栖热菌素是一种单链蛋白。它在pH 2和90℃时具有最大蛋白水解活性。制备了嗜热栖热菌的基因组文库，并通过根据嗜热栖热菌素的NH₂末端序列设计的寡核苷酸探针进行筛选。分离出五个阳性克隆。从这些克隆中对嗜热栖热菌素基因进行了定位和测序。核苷酸序列表明嗜热栖热菌素结构由1020个碱基编码。在推导的蛋白质序列中，在嗜热栖热菌素的NH₂末端位置之前有41个氨基酸残基（包括起始甲硫氨酸）。这些残基中的大多数似乎是前导序列的特征。然而，不能排除该区域存在短前肽序列的可能性。嗜热栖热菌素在第237位残基处含有一个半胱氨酸，该半胱氨酸对活性并非必需（富塞克，M.，林，X.-L.，唐，J.（1990）《生物化学杂志》265，1496 - 1501）。嗜热栖热菌素与胃蛋白酶家族的天冬氨酸蛋白酶以及对胃蛋白酶抑制剂不敏感的酸性蛋白酶没有明显的序列相似性（今田，T.，永田，S.，松田，G.，村田，S.，小田，K.，村尾，S.，津良，D.（1984）《生物化学杂志》95，465 - 475），因此可能代表一类新的酸性蛋白酶。天冬氨酸蛋白酶特有的活性位点天冬氨酰序列也不存在。每个嗜热栖热菌素分子上有11个潜在的N-糖基化位点。从凝胶过滤估计的分子量（45,000）大于根据序列计算的分子量（32,651），这表明嗜热栖热菌素至少在这11个位点中的一些位点上进行了糖基化。
