S-adenosylhomocysteine (AdoHcy) hydrolase is an enzyme that regulates biomethylation and some other physiological processes. Recombinant AdoHcy hydrolase was overexpressed in E. coli JM109 and purified with ion exchan...S-adenosylhomocysteine (AdoHcy) hydrolase is an enzyme that regulates biomethylation and some other physiological processes. Recombinant AdoHcy hydrolase was overexpressed in E. coli JM109 and purified with ion exchange and gel filtration chromatographies. The effects of copper ions (Cu2+) on the activity of AdoHcy hydrolase were investigated and the results showed that Cu2+ inhibited the enzyme’s activity by a concentration and time-dependent process. The inhibition constant (Ki) and the apparent rate constant (Kapp) were calculated to be (14±4) nmol·L-1 and (1.08±0.15) min-1, respectively. The existence of the natural substrate Ado could to some extent prevent Cu2+ from inactivating the enzyme, suggesting that copper ions possibly could compete with the natural substrate on enzyme’s substrate binding site. Further studies on the mechanism of inhibition are being carried out.展开更多
目的探讨S-腺苷同型半胱氨酸(S-adenosyl hom ocysteine;SAH)水解酶(SAH H)在同型半胱氨酸(H om ocysteine;H cy)致平滑肌细胞增殖中的作用机制。方法原代培养平滑肌细胞(VSM C s),用0、50、100、200、500μM浓度H cy干预VSM C s,高效...目的探讨S-腺苷同型半胱氨酸(S-adenosyl hom ocysteine;SAH)水解酶(SAH H)在同型半胱氨酸(H om ocysteine;H cy)致平滑肌细胞增殖中的作用机制。方法原代培养平滑肌细胞(VSM C s),用0、50、100、200、500μM浓度H cy干预VSM C s,高效液相色谱法(H PLC)检测S-腺苷甲硫氨酸(S-adenosylm ethionine,SAM)和SAH含量的变化;实时R T-PC R和W estern blot检测SAH H表达的变化;M TT法检测H cy对VSM C s增殖的影响。结果 H PLC结果表明,VSM C s与0、50、100、200、500μM H cy培养72 h后,细胞中SAH的含量明显增加,而细胞中SAM的含量和SAM/SAH的比值下降,与对照组比较,差异均有显著性(P<0.05),实时R T-PC R和W estern blot结果显示,SAH H m R N A和蛋白水平下降,与对照组比较差异有显著性(P<0.05),M TT显示,在50μM H cy组,VSM C s数量明显增加,而100、200、500μM H cy组VSM C s数量反较对照组减少。结论 SAH H表达下调致使SAM/SAH的比值下降可能是H cy引起VSM C s增殖的重要机制之一。展开更多
根据草菇(Volvariella volvacea)S-腺苷-L-高半胱氨酸水解酶基因(sahh)全长序列设计两个嵌套引物,与Takara LA PCR in vitro cloning试剂盒的接头引物结合进行巢式PCR扩增,得到sahh上游调控序列,通过序列分析发现,该序列含有1个TATA盒和...根据草菇(Volvariella volvacea)S-腺苷-L-高半胱氨酸水解酶基因(sahh)全长序列设计两个嵌套引物,与Takara LA PCR in vitro cloning试剂盒的接头引物结合进行巢式PCR扩增,得到sahh上游调控序列,通过序列分析发现,该序列含有1个TATA盒和4个CAAT盒。应用MatInspector程序分析sahh上游调控序列,发现该区域存在1个耐盐因子,1个热激因子,2个DRE元件和2个ABA应答元件。展开更多
Nonalcoholic steatohepatitis(NASH)is an important indication for liver transplantation in many Western countries due to the epidemic of obesity and insulin resistance.Unfortunately,no medication is approved for NASH a...Nonalcoholic steatohepatitis(NASH)is an important indication for liver transplantation in many Western countries due to the epidemic of obesity and insulin resistance.Unfortunately,no medication is approved for NASH and risk factor modification is often advised.Over the last decade,several clinical trials on NASH have been conducted with several ongoing and the future looks promising.Although betaine(trimethyl glycine)was evaluated for NASH,results were mixed in the clinical trials in large part due to the quality of the studies.It seems reasonable to re-evaluate betaine in clinical trials for NASH and alcoholic liver disease due to its low cost,tolerability and mechanism of action.展开更多
基金This work was supported by CMB, SRF for ROCS/SEM similar foundation from Peking University Health Science Center.
文摘S-adenosylhomocysteine (AdoHcy) hydrolase is an enzyme that regulates biomethylation and some other physiological processes. Recombinant AdoHcy hydrolase was overexpressed in E. coli JM109 and purified with ion exchange and gel filtration chromatographies. The effects of copper ions (Cu2+) on the activity of AdoHcy hydrolase were investigated and the results showed that Cu2+ inhibited the enzyme’s activity by a concentration and time-dependent process. The inhibition constant (Ki) and the apparent rate constant (Kapp) were calculated to be (14±4) nmol·L-1 and (1.08±0.15) min-1, respectively. The existence of the natural substrate Ado could to some extent prevent Cu2+ from inactivating the enzyme, suggesting that copper ions possibly could compete with the natural substrate on enzyme’s substrate binding site. Further studies on the mechanism of inhibition are being carried out.
文摘目的探讨S-腺苷同型半胱氨酸(S-adenosyl hom ocysteine;SAH)水解酶(SAH H)在同型半胱氨酸(H om ocysteine;H cy)致平滑肌细胞增殖中的作用机制。方法原代培养平滑肌细胞(VSM C s),用0、50、100、200、500μM浓度H cy干预VSM C s,高效液相色谱法(H PLC)检测S-腺苷甲硫氨酸(S-adenosylm ethionine,SAM)和SAH含量的变化;实时R T-PC R和W estern blot检测SAH H表达的变化;M TT法检测H cy对VSM C s增殖的影响。结果 H PLC结果表明,VSM C s与0、50、100、200、500μM H cy培养72 h后,细胞中SAH的含量明显增加,而细胞中SAM的含量和SAM/SAH的比值下降,与对照组比较,差异均有显著性(P<0.05),实时R T-PC R和W estern blot结果显示,SAH H m R N A和蛋白水平下降,与对照组比较差异有显著性(P<0.05),M TT显示,在50μM H cy组,VSM C s数量明显增加,而100、200、500μM H cy组VSM C s数量反较对照组减少。结论 SAH H表达下调致使SAM/SAH的比值下降可能是H cy引起VSM C s增殖的重要机制之一。
文摘根据草菇(Volvariella volvacea)S-腺苷-L-高半胱氨酸水解酶基因(sahh)全长序列设计两个嵌套引物,与Takara LA PCR in vitro cloning试剂盒的接头引物结合进行巢式PCR扩增,得到sahh上游调控序列,通过序列分析发现,该序列含有1个TATA盒和4个CAAT盒。应用MatInspector程序分析sahh上游调控序列,发现该区域存在1个耐盐因子,1个热激因子,2个DRE元件和2个ABA应答元件。
文摘Nonalcoholic steatohepatitis(NASH)is an important indication for liver transplantation in many Western countries due to the epidemic of obesity and insulin resistance.Unfortunately,no medication is approved for NASH and risk factor modification is often advised.Over the last decade,several clinical trials on NASH have been conducted with several ongoing and the future looks promising.Although betaine(trimethyl glycine)was evaluated for NASH,results were mixed in the clinical trials in large part due to the quality of the studies.It seems reasonable to re-evaluate betaine in clinical trials for NASH and alcoholic liver disease due to its low cost,tolerability and mechanism of action.
