大鼠心肌多胺代谢限速酶ODC、SSAT活性分析

Enzyme activity assay of ornithine decarboxylase(ODC) and spermidine/spermine N^1-acetyltransferase(SSAT) in rat myocardium

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?

Aim To establish the enzyme activity assay method for ornithine decarboxylase(ODC)and spermidine/spermine N 1-acetyltransferase(SSAT) in rat myocardium and observe the effects of nitric oxide(NO)donor sodium nitroprusside(SNP)on ODC and SSAT activity. Method Radioactivity of 14CO_2 and [ 14C] acetyl-spermidine released from DL-[1- 14C] Ornithine and [1- 14C]acetyl-CoA in rat myocardial homogenate was counted with LS6500 liquid scintillation counter, which represented ODC and SSAT activities. Enzyme activity parameter of ODC and SSAT was calculated, suitable substrate concentration was determined, and the effects of SNP on ODC and SSAT activity in rat myocardial were observed.Results ①ODC and SSATbasal activities in rat myocardium were(9.67±3.09)nmol·mg -1 Pro·h -1 and (3.59±0.91) nmol·mg -1 pro)·min -1 respectively.②The K_m value and V_m value of ODC catalyzing L-Ornithine were(54.95±8.14)μmol·L -1 and (2.364±0.37)nmol·mg -1·h -1 respectively; while the K_m value and V_m value of SSAT catalyzing [1- 14C]Acetyl-COA were(54.95±8.14)μmol·L -1 and (2.364±0.37)nmol·mg -1·h -1 respectively. ③The suitable substrate concentration for ODC and SSAT assay were 18.5 kBq DL-[1- 14C]Ornithine and 2.96 kBq[1- 14C]acetyl-Coenzyme respectively.④ODC activity was inhibited and SSAT activity was induced by SNP dose-dependently.Conclusion A kind of simple and practicable enzyme assay method for the key enzyme of polyamine biosynthesis (ODC) and terminal degradation(SSAT)was established and the substrate concentration for ODC and SSAT action was determined in rat myocardium. The test also showed that NO not only induced polyamine synthesis but also accelerated polyamine breakdown.