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syntide-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离子/磷脂依赖的蛋白激酶C和钙离子/钙调蛋白依赖性的蛋白激酶II的活性。腮腺和颌下腺的蛋白激酶C和蛋白激酶II水平均低于0.05单位/克。蛋白激酶C抑制剂H-7可抑制12-O-十四酰佛波醇13-乙酸酯(PMA)或磷脂酰丝氨酸和1，2-甘油三酯联合刺激的大鼠腮腺淀粉酶的分泌。结果支持了蛋白激酶C介导大鼠腮腺淀粉酶分泌机制的假说。B)研究唾液腺蛋白激酶C的磷酸化。大鼠腮腺和颌下腺匀浆在添加额外…的情况下发生磷酸化较多的细胞蛋白激酶C出现主要的Mr 8OK-Da条带(SDS-PAGE)。加入蛋白激酶C抑制剂H-7后，该条带消失。在腮腺的病例中，80k-Da底物主要定位在100000 g的上清液中。以颌下腺为例，底物(80-KDa蛋白)在100,000g上清液部分(胞浆部分)和100,000 g颗粒部分(颗粒部分)之间似乎大致均匀分布。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(从大鼠基因组DNA的Eco RI酶切和Lambda Charon 35 Eco RI载体制备)用基于人β_1-AR基因的合成寡核苷酸进行筛选。从基因文库中分离到3个克隆和1个克隆。对克隆的序列分析表明，大鼠β1-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%的同源性。较少

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