The role of phospholipase D associated with apical plasma membrabes in post-docking steps of exocytosis

与顶端质膜相关的磷脂酶 D 在胞吐作用的对接后步骤中的作用

• 批准号: 12671820
• 负责人: FUJITA Atsushi
• 金额: $ 1.73万
• 依托单位: Asahi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671820/
• 关键词: apical plasma membranes; phospholipase D; phospholipase A_2; inositol phospholipids; free fatty acid; membrane fusion; 開口分泌; 耳下腺; 形質膜

To clarify the involvement of phospholipase D (PLD) in producing fusogenic mediators during exocytosis, we investigated the regulatory mechanism of PLD activity associated with apical plasma membranes from rat parotid glands. Then, using a cell-free fusion system, coupling between conformational changes in the membranes induced by PLD and the fusion reaction was also investigatedPhosphatidylinositol 4, 5 ・ bisphosphate (PIP_2), which is an authentic activator of PLDs 1 and 2, markedly activated the apical PLD. GTP-y-S, another activator of PLD1, affected neither the basal activity of apical PLD nor its PIP_2, dependent activation. Oleic acid activated the apical PLD and the activation was amplified in the presence of PIP_2. Neomysin, an amino glycoside antibiotic with high affinity for PIP_2, partially inhibited this PLD activity. Pathways producing lipid activators such as PIP_2 and oleic acid were investigated in apical plasma membranes. Then we detected the activities of phosphatidy … More linositol 4-kinase and phosphatidylinositol-4-phosphate kinase in apical plasma membrabes, suggesting that PIP_2 was produced via the sequential pathway of the two enzymes. The Ca^<2+> independent phospholipase A_2 that selectively released unsaturated fatty acids from neutral phospholipids and the Ca^<2+> dependent deacylation from phosphatidylinositol were also found in apical plasma membranes. These enzymes may contribute to a full activation of the apical PLD. In addition, to clarify the post-docking role of the apical PLD, the fusion of exgenous PLD-treated membranes with secretory granules was examined by fluorescence dequenching assay. This study demonstrated the facilitation of fusion by PLD-trearment. To date, our data are insufficient to allow the conclusion that the apical PLD enhances membrane fusion in vivo. However, Dohke et al. [BBRC, 299, 699 (2002)] reported that neomysin partially suppressed Camp-dependent exocytosis in saponin- permeabilized parotid acinar cells. This finding seems to support our conclusion, since apical PLD was also inhibited by neomycin. Further investigation is needed to clarify the physiological role of apical PLD Less

为了阐明磷脂酶D（PLD）在胞吐过程中产生融合介质的参与，我们研究了与大鼠腮腺顶端质膜相关的PLD活性的调节机制。磷脂酰肌醇4，5 ·二磷酸（PIP_2）是磷脂酰肌醇1和2的真正激活剂，它能显著激活顶端的磷脂酰肌醇。PLD 1的另一种激活剂GTP-γ-S既不影响顶端PLD的基础活性，也不影响其PIP_2依赖的激活。油酸激活顶端PLD，并且在PIP_2存在下激活被放大。新霉素（Neomysin）是一种与PIP_2具有高度亲和性的氨基糖苷类抗生素，它能部分抑制PLD的活性。研究了顶端质膜产生脂质激活剂如PIP_2和油酸的途径。然后检测磷脂酶的活性 ...更多信息 PIP_2的产生可能是由肌醇4-激酶和磷脂酰肌醇-4-磷酸激酶的顺序途径引起的。在顶端质膜中还发现了选择性地从中性磷脂中释放不饱和脂肪酸的不依赖Ca^<2 +>的磷脂酶A_2和依赖于Ca^<2+>的磷脂酰肌醇脱酰作用。这些酶可能有助于顶端PLD的完全激活。此外，为了阐明顶端PLD的后对接作用，通过荧光去猝灭法检测外源PLD处理的膜与分泌颗粒的融合。本研究证明PLD处理促进融合。迄今为止，我们的数据不足以得出结论，顶端PLD增强膜融合在体内。然而，Dohke等人[BBRC，299，699（2002）]报道了新霉素部分抑制皂苷透化的腮腺腺泡细胞中的cAMP依赖性胞吐作用。这一发现似乎支持我们的结论，因为顶端PLD也被新霉素抑制。需要进一步的研究来阐明顶端PLD的生理作用

项目成果

Inokuchi, Hiroshi: "Comparison of Ca^<2+>-independent phospholipase A_2 activity in apical plasma membranes and secretory granules from the rat parotid gland"Japanese Journal of Oral Biology. 43・6. 666-675 (2001)

Inokuchi，Hiroshi：“大鼠腮腺顶端质膜和分泌颗粒中Ca ^ 2+ 独立的磷脂酶A_2活性的比较”日本口腔生物​​学杂志43·6（2001）。

Inokuchi, H., Mizuno-Kamiya, M. and Fujita, A: "Comparison of Ca^<2+> independent phospholipase A_2 activity in apical plasma membranes and secretory granules from the rat paroid gland"Japanese Journal of Oral Biology. 43(6). 666-675 (2001)

Inokuchi, H.、Mizuno-Kamiya, M. 和 Fujita, A：“大鼠腮腺顶端质膜和分泌颗粒中 Ca^2 独立磷脂酶 A_2 活性的比较”日本口腔生物​​学杂志。

Mizuno-Kamiya, Masako: "Ca^<2+>-independent phospholipase A_2 activity in apical plasma membranes from t e rat parotid gland"Archives of Oral Biology. 46・9. 789-799 (2001)

Mizuno-Kamiya，Masako：“来自大鼠腮腺的顶端质膜中Ca ^ 2+ -独立的磷脂酶A_2活性”口腔生物学档案46·9（2001）。

Yashiro, Koji: "Microsomal thiol S-metyltransferase activity in rat salivary glands"Japanese Journal of Oral Biology. 43・2. 133-139 (2001)

Yashiro，Koji：“大鼠唾液腺中的微粒体硫醇S-甲基转移酶活性”日本口腔生物​​学杂志43·2（2001）。

Kameyama, Y. and Shinkai, A: "Characterization of 1-alkenyl-sn-glycero-3-phosphorylcholime acyltransferase activity in the microsomes from rat parotid gland"apanese Journal of Oral Biology. 42(2). 319-325 (2000)

Kameyama, Y. 和 Shinkai, A：“大鼠腮腺微粒体中 1-烯基-sn-甘油-3-磷酰胆碱酰基转移酶活性的表征”日本口腔生物​​学杂志。