Department of Biochemistry, V.P. Chest Institute, University of Delhi, Delhi 1100 07, India.
Biochimie. 2010 Sep;92(9):1173-9. doi: 10.1016/j.biochi.2010.01.016. Epub 2010 Jan 28.
Our earlier investigations have identified a unique enzyme in the endoplasmic reticulum (ER) termed Acetoxy Drug: Protein Transacetylase (TAase) catalyzing the transfer of acetyl group from polyphenolic acetates (PA) to certain receptor proteins (RP). An elegant assay procedure for TAase was developed based on the inhibition of glutathione S-transferase (GST) due to acetylation by a model acetoxycoumarin, 7, 8-Diacetoxy-4-methylcoumarin (DAMC). TAase purified from various mammalian tissue microsomes to homogeneity exhibited a molecular weight (M.wt) of 55kDa. Further, by N-terminal sequencing TAase was identified as Calreticulin (CR), a multifunctional Ca2+-binding protein in ER lumen. The identity of TAase with CR was evidenced by proteomics studies such as immunoreactivity with anti-CR antibody and mass spectrometry. This function of CR was termed Calreticulin transacetylase (CRTAase). CRTAase was also found to mediate the transfer of acetyl group from DAMC to RP such as NADPH Cytochrome c Reductase (CYPR) and Nitric Oxide Synthase (NOS). The autoacetylation of purified human placental CRTAase concomitant with the acetylation of RP by DAMC was observed. CRTAase activity was found to be inhibited by Ca2+. Our investigations on the individual domains (N, P and C) of CR from a nematode Haemonchus contortus revealed that the P-domain alone was found to possess CRTAase activity. Based on the observation that the autoacetylated CR was a stable intermediate in the CRTAase catalyzed protein acetylation by PA, a putative mechanism was proposed. Further, CRTAase was also found capable of transferring propionyl group from a propoxy derivative of polyphenol, 7,8-Dipropoxy-4-methylcoumarin (DPMC) to RP and concomitant autopropionylation of CR was encountered. Hence, CRTAase was assigned the general term Calreticulin Transacylase. Also, CRTAase was found to act upon the biological acyl group donors, acetyl CoA and propionyl CoA. CRTAase mediated modulation of specific functional proteins by way of acylation was exploited to elicit the biological applications of PA.
我们早期的研究在内质网（ER）中鉴定出一种独特的酶，称为乙酰氧基药物：蛋白质转乙酰酶（TAase），它催化乙酰基从多酚乙酸酯（PA）转移到某些受体蛋白（RP）。基于模型乙酰氧基香豆素7,8 - 二乙酰氧基 - 4 - 甲基香豆素（DAMC）的乙酰化作用对谷胱甘肽S - 转移酶（GST）的抑制作用，开发了一种用于TAase的精密检测方法。从各种哺乳动物组织微粒体中纯化至同质的TAase表现出55kDa的分子量（M.wt）。此外，通过N端测序，TAase被鉴定为钙网蛋白（CR），一种内质网腔中的多功能Ca2 +结合蛋白。蛋白质组学研究，如与抗CR抗体的免疫反应性和质谱分析，证明了TAase与CR的一致性。CR的这种功能被称为钙网蛋白转乙酰酶（CRTAase）。还发现CRTAase介导乙酰基从DAMC转移到RP，如NADPH细胞色素c还原酶（CYPR）和一氧化氮合酶（NOS）。观察到纯化的人胎盘CRTAase的自乙酰化与DAMC对RP的乙酰化同时发生。发现CRTAase活性受到Ca2 +的抑制。我们对线虫捻转血矛线虫的CR的各个结构域（N、P和C）的研究表明，仅P结构域具有CRTAase活性。基于自乙酰化的CR是PA催化的CRTAase蛋白乙酰化中的稳定中间体这一观察结果，提出了一种推测机制。此外，还发现CRTAase能够将多酚的丙氧基衍生物7,8 - 二丙氧基 - 4 - 甲基香豆素（DPMC）中的丙酰基转移到RP，并伴随CR的自丙酰化。因此，CRTAase被赋予了通用术语钙网蛋白转酰酶。此外，发现CRTAase作用于生物酰基供体乙酰辅酶A和丙酰辅酶A。利用CRTAase通过酰化作用对特定功能蛋白进行的调节来引发PA的生物学应用。
