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

(1)环状AMP-或&lt；Ca^(2+)&gt；磷脂依赖的蛋白激酶发生丝氨酸残基的磷脂依赖性蛋白激酶Kemptide(Leu-Arg-Arg-Ala-Ser-Leu-Leu-Gly)的磷酸化；(2)鸡肝细胞核ADP-核糖基转移酶的ADP-核糖基化发生在接受磷酸盐的丝氨酸残基的氨基末端(Kemptide)或COOH末端(Reverse-kemptide)一侧的两个精氨酸残基上。和磷脂依赖的蛋白激酶，当两个合成的七肽都被ADP-核糖化时，它们的活性都显著降低。磷酸化动力学研究表明，ADP-核糖化的两种合成肽分别是合成肽的线性竞争性抑制物和ATP的线性非竞争性抑制物。(4)原代培养的肝细胞分别用正磷酸和腺苷标记。这些细胞的膜制备的聚丙烯酰胺凝胶分析表明，超过10种蛋白质被ADP-核糖化，至少8种蛋白质被磷酸化。分子量分别为85,000，60,000，42,000和20,000的四种蛋白质是这两种修饰的有效受体。(5)在初级培养基中加入20 mM烟酰胺后，总蛋白磷酸化水平下降了30%。降低的磷酸化主要观察到两个相对分子质量为60,000和42,000的蛋白质。

(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.