Alexson S E, Finlay T H, Hellman U, Svensson L T, Diczfalusy U, Eggertsen G
Department of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, Sweden.
J Biol Chem. 1994 Jun 24;269(25):17118-24.
We have cloned and sequenced a carboxylesterase from rat liver and purified the corresponding protein from rat blood. The cDNA encodes the entire mature serum esterase protein. It is apparently identical to cDNAs cloned from rat liver by several groups (Long, R. M., Satoh, H., Martin, B. M., Kimura, S., Gonzales, F. J., and Pohl, L. R. (1988) Biochem. Biophys. Res. Commun. 156, 866-873; Takagi, Y., Morohashi, K.-i., Kawabata, S.-i., Go, M., and Omura, T. (1988) J. Biochem. (Tokyo) 104, 801-806; and Robbi, M., and Beaufay, H. (1992) Biochem. Biophys. Res. Commun. 183, 836-841). However, the identification of the protein encoded by this cDNA has not been previously reported. The COOH-terminal -TEHT sequence found in the rat serum carboxylesterase does not possess retention properties and is therefore responsible for its secretion and presence in the circulation. The rat serum carboxylesterase was purified to apparent homogeneity by affinity chromatography on immobilized antibody to rat liver microsomal acyl-CoA thioesterase followed by ion exchange chromatography. The purified protein, with a M(r) of approximately 70,000, was cleaved in situ in a polyacrylamide gel with trypsin, and two peptides were isolated and sequenced. Sequence analysis showed that both peptides were identical only to the corresponding deduced amino acid sequence of the cloned cDNA. Antibodies raised to the COOH-terminal amino acid sequence deduced from the cDNA cross-reacted with the purified rat serum carboxylesterase. Changes in serum esterase activity levels followed changes in protein mass in rat serum and changes in liver mRNA levels in response to various nutritional conditions while total liver esterase activity was essentially unchanged. The above experiments confirm the identity of the protein isolated from rat sera with the cDNA cloned from rat liver and suggest a function for the serum esterase in lipid metabolism.
我们已从大鼠肝脏中克隆并测序了一种羧酸酯酶，并从大鼠血液中纯化了相应的蛋白质。该cDNA编码了整个成熟的血清酯酶蛋白。它显然与几个研究小组从大鼠肝脏中克隆的cDNA相同（Long, R. M., Satoh, H., Martin, B. M., Kimura, S., Gonzales, F. J., and Pohl, L. R. (1988) Biochem. Biophys. Res. Commun. 156, 866 - 873; Takagi, Y., Morohashi, K.-i., Kawabata, S.-i., Go, M., and Omura, T. (1988) J. Biochem. (Tokyo) 104, 801 - 806; 以及Robbi, M., and Beaufay, H. (1992) Biochem. Biophys. Res. Commun. 183, 836 - 841）。然而，此前尚未报道过该cDNA所编码蛋白质的鉴定情况。在大鼠血清羧酸酯酶中发现的COOH末端-TEHT序列不具备保留特性，因此负责其分泌及在循环中的存在。通过用针对大鼠肝脏微粒体酰基辅酶A硫酯酶的固定化抗体进行亲和层析，随后进行离子交换层析，将大鼠血清羧酸酯酶纯化至表观均一性。纯化后的蛋白质，其相对分子质量约为70,000，在聚丙烯酰胺凝胶中用胰蛋白酶进行原位切割，分离并测序了两个肽段。序列分析表明，这两个肽段仅与克隆cDNA相应的推导氨基酸序列相同。针对从cDNA推导的COOH末端氨基酸序列产生的抗体与纯化的大鼠血清羧酸酯酶发生交叉反应。在各种营养条件下，大鼠血清中酯酶活性水平的变化随蛋白质质量的变化以及肝脏mRNA水平的变化而变化，而肝脏总酯酶活性基本保持不变。上述实验证实了从大鼠血清中分离的蛋白质与从大鼠肝脏中克隆的cDNA的一致性，并提示了血清酯酶在脂质代谢中的功能。
