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目的建立大鼠心肌多胺代谢限速酶鸟氨酸脱羧酶(ODC)及精脒/精胺乙酰转移酶(SSAT)活性分析方法。方法以Langendorff离体灌流心肌为实验材料,制备心肌组织匀浆;分别以dl[114C]Ornithine及[114C]acetylCoenzymeA为底物,以液体闪烁计数仪记录生成的14CO2及[14C]acetylspermidine的放射活度,并以其代表ODC,SSAT的活性;计算大鼠心肌ODC、SSAT的酶促反应动力学参数,筛选出适宜的底物浓度;同时观察一氧化氮(NO)供体硝普钠(SNP)对酶活性的影响。结果①大鼠心肌ODC、SSAT基础活性分别为:(9.67±3.09)nmol·mg-1Pro·h-1;(3.59±0.91)nmol·mg-1Pro·min-1。②ODC催化LOrnithine的酶促反应动力学参数Km=(54.95±8.14)μmol·L-1;Vmax=(2.364±0.37)nmol·mg-1·h-1;SSAT催化AcetylCoenzymeA的酶促反应动力学参数Km=(12.87±1.88)μmol·L-1;Vmax=(0.50±0.07)nmol·mg-1·min-1。③大鼠心肌ODC、SSAT活性检测的底物浓度分别为:90μmol·L-1(18.5kBq)DL[114C]Ornithine及36μmol·L-1(2.96kBq)[114C]acetylCoA。④SNP呈浓度依赖性地抑制ODC的活性、诱导SSAT的活性。结论建立了大鼠心肌多胺代谢限速酶鸟氨酸脱羧酶(ODC)及精脒/精胺乙酰转移酶(SSAT)活性的分析方法,该方法简便易行;根据Km值确定测定大鼠心肌ODC及SSAT?
OBJECTIVE: To establish a method for analyzing the activity of ornithine decarboxylase (ODC) and spermidine / spermine acetyltransferase (SSAT) in myocardial polyamines. Methods The perfused myocardium of Langendorff was used as experimental material to prepare cardiac homogenate. The radioactive activities of 14CO2 and [14C] acetylspermidine were recorded by liquid scintillation counter with dl [114C] Ornithine and [114C] acetylCoenzymeA as substrates respectively (ODC) and SSAT activity were calculated. The enzymatic kinetic parameters of ODC and SSAT in rat myocardium were calculated, and the suitable substrate concentration was screened. At the same time, the nitric oxide (NO) donor sodium nitroprusside ) On the enzyme activity. Results ① The basal activity of ODC and SSAT in rat myocardium were (9.67 ± 3.09) nmol · mg-1Pro · h-1 and (3.59 ± 0.91) nmol · mg-1Pro · min-1, respectively. (2) The kinetic parameters of enzymatic reaction of LOrnithine catalyzed by COD were Km = (54.95 ± 8.14) μmol·L-1 and Vmax = (2.364 ± 0.37) nmol · mg-1 · h-1, respectively. The enzymatic kinetic parameters of AcetylCoenzyme A Km = (12.87 ± 1.88) μmol·L-1; Vmax = (0.50 ± 0.07) nmol · mg-1 · min-1. ③ The concentrations of ODC and SSAT in rat myocardium were 90μmol·L-1 (18.5kBq) DL [114C] Ornithine and 36μmol·L-1 (2.96kBq) [114C] acetylCoA. ④ SNP inhibited the activity of ODC in a concentration-dependent manner and induced SSAT activity. Conclusions The method for determining the activity of ornithine decarboxylase (ODC) and spermidine / spermine acetyltransferase (SSAT) in rat myocardium with polyamine exchange rate is established. The method is simple and easy to determine. Myocardial ODC and SSAT?