Kanayama N, Himeda Y, Atomi H, Ueda M, Tanaka A
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University.
J Biochem. 1997 Sep;122(3):616-21. doi: 10.1093/oxfordjournals.jbchem.a021797.
In the n-alkane-assimilating yeast Candida tropicalis, there are two isozymes of acetoacetyl-CoA thiolase, peroxisomal acetoacetyl-CoA thiolase (peroxisomal Thiolase I), and cytosolic acetoacetyl-CoA thiolase (cytosolic Thiolase I). We have previously isolated two genes (CT-T1A and CT-T1B) which encode Thiolase I. In order to compare the expressed products of Thiolase I isozyme-encoding genes in C. tropicalis, cytosolic Thiolase I was first purified from glucose-grown C. tropicalis in which the proliferation of peroxisomes and the expression of peroxisomal Thiolase I were repressed. Cytosolic Thiolase I was virtually identical to peroxisomal Thiolase I in molecular mass, kinetic and immunochemical properties, and primary structure at the N-terminus. Amino acid sequence analysis revealed that cytosolic Thiolase I was the mixture of products of two genes (CT-T1A and CT-T1B), as in the case of the peroxisomal enzyme. CT-T1A and CT-T1B were expressed independently in the yeast Saccharomyces cerevisiae and the recombinant proteins were purified. Recombinant Thiolase IA and IB exhibited practically identical enzymatic properties to cytosolic and peroxisomal Thiolase Is from C. tropicalis. These results revealed that cytosolic Thiolase I and peroxisomal Thiolase I were encoded not by different genes, but by the same genes (CT-T1A and CT-T1B) and are present as a mixture of products expressed by both genes, although their subcellular localizations are different.
在同化正构烷烃的热带假丝酵母中，存在两种乙酰乙酰辅酶A硫解酶同工酶，即过氧化物酶体乙酰乙酰辅酶A硫解酶（过氧化物酶体硫解酶I）和胞质乙酰乙酰辅酶A硫解酶（胞质硫解酶I）。我们之前分离出了两个编码硫解酶I的基因（CT-T1A和CT-T1B）。为了比较热带假丝酵母中硫解酶I同工酶编码基因的表达产物，首先从葡萄糖培养的热带假丝酵母中纯化胞质硫解酶I，在这种培养条件下，过氧化物酶体的增殖和过氧化物酶体硫解酶I的表达受到抑制。胞质硫解酶I在分子量、动力学和免疫化学性质以及N端一级结构方面与过氧化物酶体硫解酶I几乎相同。氨基酸序列分析表明，胞质硫解酶I是两个基因（CT-T1A和CT-T1B）产物的混合物，就像过氧化物酶体酶的情况一样。CT-T1A和CT-T1B在酿酒酵母中独立表达，并对重组蛋白进行了纯化。重组硫解酶IA和IB表现出与热带假丝酵母的胞质和过氧化物酶体硫解酶I几乎相同的酶学性质。这些结果表明，胞质硫解酶I和过氧化物酶体硫解酶I不是由不同基因编码的，而是由相同基因（CT-T1A和CT-T1B）编码的，并且以两个基因表达产物的混合物形式存在，尽管它们的亚细胞定位不同。
