Study on development of drugs for endocrine and metabolic disorders based on properties of new enzyme

乙酰乙酰辅酶a （AA-CoA）合成酶是一种新型的连接酶，它能特异性地激活乙酰乙酸酯与AA-CoA的连接。该酶主要参与肝脏脂质和胆固醇的生物合成，AA-CoA合成酶的生理作用尚不清楚。这使我们能够通过新酶的阐明来开发新药。首先，根据从大鼠肝脏纯化的酶制剂中确定的氨基酸序列进行酶cDNA的克隆。因此，获得的大鼠和人AA-CoA合成酶基因均由2016个核苷酸组成，编码672个氨基酸残基。大鼠与人氨基酸序列同源性为89.3%。该酶在脑内高表达，其表达谱与胆固醇生物合成限速酶HMG-CoA还原酶相似。在人脑中，该酶在大脑皮层，尤其是位于颞叶内侧的海马中表达量高，AA-CoA合成酶的表达谱与HMG-CoA还原酶的表达谱相似。注射链脲佐菌素（STZ）后，糖尿病大鼠肝脏AA-CoA合成酶活性开始逐渐下降，与HMG-CoA还原酶相似，降至初始活性的十分之一。在添加降胆固醇剂、普伐他汀和胆胺的饲粮中，stz诱导的AA-CoA合成酶活性降低程度减弱，血中酮体浓度降至正常水平。这些结果提示肝脏AA-CoA合成酶可能部分参与了血酮体的调节，该酶在人脑海马区高表达，可能在记忆功能中起重要作用。

Acetoacetyl-CoA (AA-CoA) synthetase is a novel ligase, which specifically activates acetoacetate to AA-CoA. This enzyme is mainly involved in lipid and cholesterol biosynthesis in the liver, and the physiological role of AA-CoA synthetase is not clear yet. This let us to develop new drugs by the elucidation of new enzyme. First, cloning of the enzyme cDNA was carried out being based on the amino acid sequence determined from the enzyme preparation purified from rat liver. Consequently, both the obtained rat and human AA-CoA synthetase genes consisted of 2016 nucleotides, and coded 672 amino acid residues. Homology of amino acid sequence between rat and human was 89.3%. Expression of this enzyme was high in brain, and its expression profile was similar to that of HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis. In human brain, expression of the enzyme was high in cerebral cortex, especially in hippocampus, which is located inner side part of temporal lobe, and expression profile of the AA-CoA synthetase was similar to that of HMG-CoA reductase. In diabetic rats injected with streptozotocin (STZ), hepatic AA-CoA synthetase activity started to gradually decrease similarly as HMG-CoA reductase and reached one tenth of the initial activity. When rats were fed with the diet supplemented with hypocholesterolemic agents, pravastatin and cholestyramine, the degree of STZ-induced decrease in AA-CoA synthetase activity was diminished, and ketone body concentration in blood was decreased to the normal level. These results indicate that hepatic AA-CoA synthetase may partly contribute to the regulation of blood ketone body, and that this enzyme expressed highly in hippocampus of human brain may have the important role in memory function.