Mechanisms on amylase secretion from parotid gland

1)蛋白激酶C在淀粉酶分泌中的作用--a）以合成肽合成酶-2（Pro-Leu-Ala-Arg-Thr-Leu-Ser-Val-Ala-Gly-Leu-Pro-Gly-Lys-Lys）为底物，测定大鼠唾液腺中钙/磷脂依赖性蛋白激酶（蛋白激酶C）和钙/钙调素依赖性蛋白激酶II（蛋白激酶II）的活性。腮腺和下颌下腺中蛋白激酶C和蛋白激酶II的水平低于0.05单位/g。蛋白激酶C抑制剂H-7可抑制1,2-O-十四酰佛波醇13-乙酸酯（PMA）或磷脂酰丝氨酸和1，2-二油酸甘油酯联合刺激的大鼠腮腺淀粉酶分泌。实验结果支持腮腺淀粉酶分泌机制为蛋白激酶C介导的假说。B）研究唾液腺中蛋白激酶C的磷酸化。当大鼠腮腺和下颌下腺的匀浆在加入额外的磷酸化剂的情况下， ...更多信息 SDS-PAGE显示细胞蛋白激酶C主要的Mr. 8 OK-Da条带。加入蛋白激酶C抑制剂H-7后，该条带消失。在腮腺的情况下，Mr. 80 k-Da底物主要定位于100，000 g上清液部分中。在下颌下腺的情况下，基质（80-KDa蛋白）似乎在100，000 g上清液级分之间大致均匀分布（胞浆部分）和100，000 g沉淀部分2）大鼠β_1-肾上腺素能受体基因的克隆--大鼠心脏cDNA文库（Clontech，USA）和大鼠基因组DNA文库（由大鼠基因组DNA的Eco RI文库和Lambda charon 35 Eco RI载体制备）。从cDNA中分离出三个克隆，从基因组文库中分离出一个克隆。对这些克隆的测序分析表明，大鼠β_1-AR基因是无内含子的。大鼠β_1-AR的氨基酸序列与人β_1-AR的氨基酸序列有90%相同。少

1)Role of protein kinase C on amylase secretion--a)The activities of Ca^2 /phospholipid- dependent protein kinase(protein kinase C) and Ca^2 /calmodulin-dependent protein kinase II(protein kinase II)in rat salivary gland were assayed using synthetic peptide syntide-2(Pro-Leu-Ala-Arg-Thr-Leu-Ser-Val-Ala-Gly-Leu-Pro-Gly-Lys-Lys) as substrate. Levels of the protein kinase C and protein kinase II were less than 0.05 units/g in the parotid and submandibular glands. The protein kinase C inhibitor, H-7, inhibited amylase secretion from rat parotid gland stimulated by 12-O-tetradecanoylphorbol 13- acetate(PMA) or the combination of phosphatidylserine and 1,2-diolein. The results supported the hypothesis of the secretory mechanism that protein kinase C mediates amylase secreiton in rat parotid gland. b) Phosphorylation by protein kinase C in salivary gland were investigated. When the homogenates of rat parotid and submandibular glands were subjected to phosphorylation with the addition of extra … More cellular protein kinase C, the major Mr. 8OK-Da band(SDS-PAGE) appeared. This band was disappeared by the addition of protein kinase C inhibitor, H-7. The Mr.80k-Da substrate was localized predominantly in the 100,000g supernatant fraction on the case of parotid gland. In the case of submandibular gland, the substrate(80-KDa protein) appeared to be approximately equally distributed between the 100,000g supernatant fraction(cytosolic fraction) and the 100,000g pellet fraction(granulate fraction).2)Cloning of rat beta_1-adrenergic receptor gene--- A rat heart cDNA library( Clontech, USA) and rat genomic DNA library(prepared from Eco RI digests of rat genomic DNA and Lambda charon 35 Eco RI vector) were screened with the synthetic oligonucleotide based on human beta_1-AR gene. Three clones from cDNA and one clone from genomic library were isolated. Sequencing analysis of these clones revealed that rat beta_1-AR gene is intronless. The amino acid sequence of rat beta_1-AR is 90% identical to that of the human beta_1-AR. Less