Furuta S, Hayashi H
Department of Biochemistry, Shinshu University School of Medicine, Nagano.
J Biochem. 1990 May;107(5):708-13. doi: 10.1093/oxfordjournals.jbchem.a123113.
Catalase is a characteristic enzyme of peroxisomes. To study the molecular mechanisms of the biogenesis of peroxisomes and catalase in a less complex system than rat liver cells, we expressed recombinant rat catalase in Escherichia coli, which has no peroxisomes. The concentration of recombinant catalase produced in E. coli transformed with the expression vector carrying the complete coding region of rat catalase cDNA was about 0.1% of the total soluble protein. The recombinant catalase was purified by DEAE-cellulose column chromatography followed by acidic ethanol precipitations. The properties of rat liver catalase and those of the recombinant were similar with respect to molecular mass, catalytic properties, profiles of absorption spectra, and iron contents. The NH2-terminal amino acid sequence of the purified recombinant catalase, as determined by Edman degradation, was in complete agreement with the amino acid sequence predicted from the nucleotide sequence of rat catalase cDNA, except that the first initiator methionine was not detected. The COOH-terminal amino acid sequence was determined by carboxypeptidase A digestion and the sequence, -Ala-Asn-Leu-OH, matched the predicted COOH-terminal amino acid sequence of rat catalase. Recombinant rat catalase gave almost the same multiple protein bands on native polyacrylamide gel isoelectric focusing as observed with authentic rat liver catalase.
过氧化氢酶是过氧化物酶体的一种特征性酶。为了在比大鼠肝细胞更简单的系统中研究过氧化物酶体和过氧化氢酶生物发生的分子机制，我们在没有过氧化物酶体的大肠杆菌中表达了重组大鼠过氧化氢酶。用携带大鼠过氧化氢酶cDNA完整编码区的表达载体转化的大肠杆菌中产生的重组过氧化氢酶浓度约为总可溶性蛋白的0.1%。重组过氧化氢酶通过DEAE-纤维素柱色谱法，随后进行酸性乙醇沉淀进行纯化。大鼠肝脏过氧化氢酶和重组过氧化氢酶在分子量、催化特性、吸收光谱图谱和铁含量方面的性质相似。通过埃德曼降解法测定的纯化重组过氧化氢酶的NH2末端氨基酸序列，与从大鼠过氧化氢酶cDNA核苷酸序列预测的氨基酸序列完全一致，只是未检测到第一个起始甲硫氨酸。通过羧肽酶A消化确定COOH末端氨基酸序列，序列-Ala-Asn-Leu-OH与大鼠过氧化氢酶预测的COOH末端氨基酸序列匹配。重组大鼠过氧化氢酶在天然聚丙烯酰胺凝胶等电聚焦上产生的多条蛋白带与天然大鼠肝脏过氧化氢酶几乎相同。
