Expression And Activity Analysis Of Human Alcohol Dehydrogenase ADH1B2 Gene

Human alcohol dehydrogenases (ADHs) are a family of isoenzymes that catalyze oxidation and reduction of a wide variety of alcohols including ethanol. Among the six types of ADHs, ClassⅠADH is the most highly expressed type in human liver, and it plays a key role in ethanol metabolism. It is reported that Class I ADH not only oxidize ethanol without generating toxic radicals, but also further oxidize aldehydes into the corresponding carboxylic acids; it is an effective detoxifying enzyme in ethanol oxidation. According to kinetic studies,β2β2-ADH encoded by ADH1B2 oxidizes ethanol more efficiently, it has a low Km for ethanol and high Kmax. Thus, we transform liver ADH1B2 gene into E.coli. BL21(DE3), P.pastoris GS115 and liver cells to analyze enzyme activity and its relationship with ethanol-induced cytotoxicity.Liver ADH1B2 gene was amplified by PCR using pUCm-T-adh as template and sequenced to be right. Then according to restrict site on primers, we insert ADH1B2 gene into pET-32a vector to construct pET-32a-ADH1B2, which was transformed into E.coli.BL21 (DE3).The protein was efficient expressed in inclusion body, however, no activity was detected after renaturation. Thus, pPIC9K-ADH1B2 and pPICZB-ADH1B2 were constructed and electrotransformed into P.pastoris GS115. Positive clones with pPICZB-ADH1B2 expressed intracellular soluble ADH1B2 protein. Activity of ethanol oxidation was detected in the supernatant of crushed P.pastoris GS115 cells.After purification by DEAE-Sepharose FF, enzyme activity was illustrated. We also constructed plasmid pCDNA3.1-ADH1B2 which was transfected into human hepatocarcinoma cell HepG2 and normal liver cell LO2. As a result, HepG2 cells transfected with ADHIB2 gene showed ROS decrease and consequent Akt phosphorylation decrease, finally resulted in depressed cell viability. LO2 cells transfected with ADH1B2 gene showed a protective effect from cytotoxicity induced by ethanol.In this stuy, we obtained Class I ADH with activity by P.pastoris GS115 and analyzed the enzyme activity. Transfection research showed the effects of ADH1B2 on cell viability and helps to get more insights into ethanol-induced cytotoxicity, alcoholism prevetion and cure.