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目的:研究GL62 酵母细胞中磷酸二酯酶4A(PDE4A)的诱导表达和茶碱、咯利普兰、N-对氟苄基-3-[(N-4-吡啶)-乙酰胺]-吲哚45(acetamide-45)对GL62酵母细胞诱导提取物中PDE4A活性的抑制作用.方法:克隆了人PDE4A基因的GL62酵母细胞在CuSO_4 150μmol/L条件下诱导表达PDE4A并提取,用高效液相色谱法测定PDE4A的活性.结果:GL62酵母细胞在CuSO_4诱导下的表达产物蛋白分子在62 kDa和83 kDa之间,表达产物的PDE4A的活性在3 h达到最大为(188±23)μmol·g~(-1)·min~(-1),其米氏常数 K_m为(17.7±2.6)μmol·L~(-1).茶碱,咯利普兰和acetamide-45对诱导提取物PDE4A活性的IC_(50)(95 ％可信限)分别为1642(989-2727),4.58(3.45-6.08)和275(170-444)μmol·L~(-1).结论:GL62酵母细胞经CuSO_4诱导可表达PDE4A,茶碱、咯利普兰和 acetamide-45能抑制其活性,GL62酵母细胞的诱导表达提取物可用来研究PDE4A及其抑制剂.
OBJECTIVE: To study the expression of phosphodiesterase 4A (PDE4A) in GL62 yeast cells and the effects of theophylline, rolipram, N-p-fluorobenzyl-3 - [(N- 45 (acetamide-45) on the activity of PDE4A induced by yeast extract from GL62.Methods: GL62 yeast cells cloned with human PDE4A gene were induced to express PDE4A under the condition of CuSO_4 150μmol / L and extracted by high performance liquid chromatography And the activity of PDE4A was measured.Results: The expression of PDE4A in GL62 yeast cells induced by CuSO_4 was between 62 kDa and 83 kDa, and reached the maximum of 188 ± 23 μmol · g ~ (-1) at 3 h -1) · min ~ (-1), and the K value of Km was (17.7 ± 2.6) μmol·L -1 .The inhibitory effect of theophylline, rolipram and acetamide-45 on the activity of PDE4A 50) (95% CI) were 1642 (989-2727), 4.58 (3.45-6.08) and 275 (170-444) μmol·L -1, respectively.Conclusion: The GL62 yeast cells can be induced by CuSO_4 PDE4A, theophylline, rolipram and acetamide-45 can inhibit their activity, and the induced expression of GL62 yeast cells extract can be used to study PDE4A and its inhibitors.