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Mechanisms on amylase secretion from parotid gland

腮腺分泌淀粉酶的机制

基本信息

批准号：
62570844
负责人：
SHIMOMURA Hiromi
金额：
$ 1.28万
依托单位：
The Nippon Dental 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：
1987
资助国家：
日本
起止时间：
1987 至 1989
项目状态：
已结题

项目摘要

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

1)蛋白激酶C对淀粉酶分泌的作用--a)利用合成肽测定大鼠唾液腺Ca^2/磷脂依赖性蛋白激酶(蛋白激酶C)和Ca^2/钙调蛋白依赖性蛋白激酶II(蛋白激酶II)的活性 syntide-2（Pro-Leu-Ala-Arg-Thr-Leu-Ser-Val-Ala-Gly-Leu-Pro-Gly-Lys-Lys）作为底物。腮腺和颌下腺中蛋白激酶 C 和蛋白激酶 II 的水平低于 0.05 单位/克。蛋白激酶C抑制剂H-7抑制12-O-十四烷酰佛波醇13-乙酸酯(PMA)或磷脂酰丝氨酸和1,2-二油酸联合刺激的大鼠腮腺淀粉酶分泌。该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设。 b) 研究了唾液腺中蛋白激酶 C 的磷酸化。当大鼠腮腺和颌下腺匀浆加入额外的细胞蛋白激酶C进行磷酸化时，出现主要的Mr. 8OK-Da条带（SDS-PAGE）。添加蛋白激酶 C 抑制剂 H-7 后该条带消失。 Mr.80k-Da 底物主要位于腮腺病例的 100,000g 上清液级分中。在下颌下腺的情况下，底物（80-KDa蛋白）似乎大致均匀地分布在100,000g上清液级分（胞质级分）和100,000g颗粒级分（颗粒级分）之间。2)大鼠β_1-肾上腺素受体基因的克隆---大鼠心脏cDNA文库（Clontech，美国）和大鼠基因组DNA文库（从使用基于人β_1-AR基因的合成寡核苷酸筛选大鼠基因组DNA的Eco RI消化物和Lambda charon 35 Eco RI载体。分离出来自 cDNA 的 3 个克隆和来自基因组文库的 1 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。较少的