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本文报道腺苷酸环化酶(AC)和环腺苷酸磷酸二酯酶(PDE)比活力的放射性同位素测定法。分别以~3H-ATP+ATP和~3H-cAMP+cAMP作为AC和PDE的底物,同肝组织制备液一起孵育,生成的或未水解的~3H-cAMP+cAMP,通过氧化铝微柱层析法进行分离,用液闪仪测读洗脱液中~3H-cAMP的cpm值。以cAMP生成量或水解量nmol/min·mg蛋白质分别表示AC或PD正比活力(u/mg蛋白质)。测得津白Ⅱ号小鼠肝组织上清液和沉淀中AC比活力分别为0.72和1.22u/mg蛋白质,PDE比活力分别为1.49和0.63u/mg蛋白质,AC/PDE比值分别为0.55和2.07。沉淀中AC比活力或AC/PDE比值大于上清液(P<0.05),而PDE比活力小于上清液(P<0.002)。将小鼠肝组织冻融对AC和PDE比活力无影响。
This article reports the radioisotope determination of the specific activities of adenylate cyclase (AC) and cyclic adenosine phosphodiesterase (PDE). The ~ 3H-cAMP + cAMP produced or not hydrolyzed with ~3H-ATP + ATP and ~ 3H-cAMP + cAMP as substrate for AC and PDE, respectively, Analytical method was used for separation, and the cpm value of ~ 3H-cAMP in the eluate was measured by liquid scintillation counting. The amount of cAMP produced or the amount of hydrolysis nmol / min · mg protein represent the AC or PD specific activity (u / mg protein) respectively. The specific activity of AC in the supernatant and sediment of Jinbai Ⅱ mice was 0.72 and 1.22 u / mg respectively, and the PDE specific activities were 1.49 and 0.63 u / mg protein, respectively. The AC / PDE ratios were 0.55 and 2.07. The specific activity of AC or AC / PDE in sediment was higher than that of supernatant (P <0.05), while the specific activity of PDE was lower than that of supernatant (P <0.002). Freezing and thawing of mouse liver tissue had no effect on the specific activities of AC and PDE.