Paolicchi A, Pezzini A, Saviozzi M, Piaggi S, Andreuccetti M, Chieli E, Malvaldi G, Casini A F
Dipartimento di Biomedicina, Università di Pisa, Italy.
Arch Biochem Biophys. 1996 Sep 15;333(2):489-95. doi: 10.1006/abbi.1996.0419.
A novel GSH-dependent dehydroascorbate (DHA) reductase from rat liver cytosol has been recently purified and partially characterized in our laboratory. A further characterization study has been carried out in order to determine intracellular and tissue distribution of the enzyme. A modified purification method, yielding a threefold increase in enzyme activity recovery, has been used. Polyclonal antibodies were obtained in rabbits and specific anti-DHA reductase IgG were purified by affinity chromatography employing the homogeneous enzyme as ligand. Immunoblotting analysis of subcellular fractions showed the exclusively cytosolic location of the enzyme. Immunotitration experiments, performed in order to determine the percentage of cytosolic DHA reductase activity ascribable to our enzyme, revealed that purified enzyme activity was completely titrable, while only 70% of DHA reducing activity was titrable in liver cytosol preparation. When immunoblotting analysis was employed to determine tissue distribution of the enzyme, liver, intestinal mucosa, kidney, adrenals, submaxillary gland, testis, and pancreas appeared most endowed with the enzyme, and lower levels were observed in all the other tissues examined. Immunohistochemical studies showed clear zonal distributions in kidney and intestinal tract and overall homogeneous patterns in the other tissues.
最近，我们实验室从大鼠肝脏胞质溶胶中纯化出一种新型的谷胱甘肽依赖性脱氢抗坏血酸（DHA）还原酶，并对其进行了部分特性鉴定。为了确定该酶在细胞内和组织中的分布，我们开展了进一步的特性研究。我们采用了一种改良的纯化方法，使酶活性回收率提高了两倍。通过以纯化后的该酶作为配体，利用亲和层析法从兔体内获得了多克隆抗体，并纯化出了特异性抗DHA还原酶IgG。亚细胞组分的免疫印迹分析表明，该酶仅存在于胞质溶胶中。为了确定胞质溶胶中可归因于我们所研究的这种酶的DHA还原酶活性百分比而进行的免疫滴定实验显示，纯化后的酶活性可被完全滴定，而在肝脏胞质溶胶制剂中，只有70%的DHA还原活性可被滴定。当采用免疫印迹分析来确定该酶的组织分布时，发现肝脏、肠黏膜、肾脏、肾上腺、颌下腺、睾丸和胰腺中该酶的含量最高，而在所有其他检测的组织中含量较低。免疫组织化学研究表明，该酶在肾脏和肠道中呈现明显的区域分布，而在其他组织中呈现整体均匀的分布模式。
