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Mono(ADP-ribosyl)ation reaction and its functions in primary cultured hepatocytes

原代培养肝细胞中的单(ADP-核糖基)化反应及其功能

基本信息

批准号：
60570112
负责人：
TANIGAWA Yoshinori
金额：
$ 0.9万
依托单位：
Depertment of biochemistry, Shimane medical university
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1986
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-60570112/
关键词：
Mono(ADP-ribose)Protein phosphorylation Membran proteins 肝初代培養

项目摘要

(1)Phosphorylation of Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) by cyclic AMP- or <Ca^(2+)> , phospholipid-dependent protein kinase occured the serine residue,and also phosphorylation of Reverse-kemptide (Gly-Leu-Ser-Ala-Arg-Arg-Leu) by <Ca^(2+)> ,phospholipid-dependent protein kinase occured at the serine residue, but Kemptide dose not served as substrate for cyclic AMP-dependent protein kinase.(2)ADP-ribosylation of Kemptide or Reverse-kemptide by hen liver nuclear ADP-ribosyltransferase occured at the two arginine residues sequenced at the <NH_2> -terminal(Kemptide)or COOH-terminal(Reverse-kemptide) side of the phosphate-accepting serine residue.(3)Phosphorylations of Kemptide and Reverse-kemptide by cyclic AMP-dependent and <Ca^(2+)> , phospholipid-de-pendent protein kinase, respectively,were markedly reduced when both synthetic heptapeptides were ADP-ribosylated. Kinetic studies of phosphorylation revealed that ADP-ribosylated both synthetic peptides were a linear competitive inhibitor of each synthetic peptide and a linear noncompetitive inhibitor of ATP.(4)Primary cultured hepatocite were labeled with [ <^(32)P> ]orthophosphate and [ <^3H> ]adenosine, respectively.Polyacrylamide gel analyses of membrane preparation from these cells revealed that over ten proteins are ADP-ribosylated and at least eight proteins are phosphorylated. Four proteins has molecular weight of 85,000, 60,000, 42,000, and 20,000 proved to be effectve acceptor for both modifications.(5)When 20 mM nicotinamide was added into the praimary culture medium, 30 % decrease of total protein phosphorylation were observed. The phosphorylation reduced was predominantly observed to two proteins which has molecular weight of 60,000 and 42,000.

（1）Kemptide（Leu-Arg-Arg-Ala-Ser-Leu-Gly）被cAMP-或<Ca^（2+）>磷酸化，磷脂依赖性蛋白激酶发生在丝氨酸残基; Kemptide（Gly-Leu-Ser-Ala-Arg-Arg-Leu）被<Ca^（2+）>磷酸化，磷脂依赖性蛋白激酶发生在丝氨酸残基，但Kemptide不作为cAMP依赖性蛋白激酶的底物。（2）Kemptide和Reverse-kemptide的ADP核糖基化作用发生在<NH_2>磷酸受体丝氨酸残基的-末端（Kemptide）或COOH-末端（Reverse-kemptide）的两个精氨酸残基上。（3）当两种合成的七肽被ADP-核糖基化时，分别由环AMP依赖性和磷脂依赖性蛋白激酶<Ca^（2+）>引起的Kemptide和Reverse-kemptide的磷酸化作用明显减少。磷酸化的动力学研究表明，ADP-核糖基化的合成肽是每个合成肽的线性竞争性抑制剂和ATP的线性非竞争性抑制剂。（4）原代培养的肝细胞分别用[ <^（32）P> ]正磷酸和[ <^3H> ]腺苷标记，经聚丙烯酰胺凝胶电泳分析，发现10多个蛋白质被ADP核糖基化，至少8个蛋白质被磷酸化。分子量分别为85，000、60，000、42，000和20，000的四种蛋白质被证明是两种修饰的有效受体。（5）当在原代培养液中加入20 mM烟酰胺时，总蛋白磷酸化水平降低30%。主要观察到两种分子量为60，000和42，000的蛋白质的磷酸化减少。