PROPERTIES OF CHEMICALLY MODIFIED PHOSPHOLIPASE A2

化学修饰磷脂酶 A2 的特性

• 批准号: 3395615
• 负责人: PHILIP ROSENBERG
• 金额: $ 17.57万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-07-01 至 1987-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3395615
• 关键词: acid base balance; amino group; aminoacid analyzer; arginine; carboxyl group; chemical structure function; cyclic aminoacid; drug administration routes; drug metabolism; electron microscopy; electrophysiology; enzyme induction /repression; gel electrophoresis; gel filtration chromatography; high voltage electrophoresis; hydrolysis; ion exchange chromatography; membrane potentials; methionine; nerve endings; neuromuscular junction; neurophysiology; neurotoxins; paper chromatography; phospholipase A2; phospholipids; reptile poison; synapses

Our long term objective is to determine why proteins with phospholipase A2 (PLA2) activity differ so markedly in their toxicological and pharmacological properties even though they have similar enzymatic activities. For example, we shall use in our studies: 1. presynaptically acting PLA2 neurotoxins (Beta bungarotoxin, notexin, caudoxin) which are very toxic and show specific effects on motor nerve terminals. 2. PLA2 from N. nigricollis snake venom which has strong direct hemolytic, anticoagulant, cardiotoxic and central effects. 3. PLA2 from N.n. atra snake venom which has relatively low toxicity with no organ specific action even though it has high enzymatic activity. To determine whether there exists (and if so its chemical nature) a region of the molecule responsible for pharmacological properties and independent from the enzymatic active site, we shall prepare known specific chemical modifications of the PLA2 enzymes and presynaptic PLA2 neurotoxins. Modifications to be performed include: 1. alkylation and methylation of histidine at the enzymatic active site, 2. carbamylation, acylation and guanidination of lysines, 3. semicarbazide addition to free carboxyls (aspartic and glutamic acids), 4. alkylation of tryptophans, 5. sulfonylation of tyrosines, 6. oxidation of methionines, 7. Cleavage of the n-terminal octapeptide (micellar recognition site). Biochemical and pharmacological properties of the native enzymes and their modified derivatives which will be tested include: enzymatic activity on substrates in various states of organization (monomers, micelles, lipoproteins, natural membranes), phospholipid substrate preferences, enzyme-phospholipid complex formation, hemolytic, and anticoagulant action, intravenous and intraventricular lethal potencies, electro-physiological effects on isolated heart tissue and the phrenic nerve-diaphragm preparation, hydrolysis of phospholipids and binding of PLA2 to heart sarcolemma and brain synaptic plasma membranes, microscope alterations of tissues. Information obtained in these studies using model PLA2 enzymes and presynaptic PLA2 neurotoxins may be relevant to the functioning of mammalian PLA2 enzymes whose activities are associated with prostaglandin formation and membrane turnover, in addition to possible roles in modulating transmitter action and membrane fluidity. Clinical disorders could be associated with abnormal enzymatic activity or toxic effects unrelated to enzymatic activity.

我们的长期目标是确定为什么磷脂酶A2的蛋白质 （PLA2）活性在毒理学和 药理特性即使它们具有相似的酶促 活动。 例如，我们将在研究中使用：1。 作用PLA2神经毒素（Beta Bungarotoxin，Notexin，caudoxin） 非常有毒，并对运动神经末端显示特定的影响。 2。PLA2 来自N. nigricollis蛇毒液，具有强烈的直接溶血， 抗凝剂，心脏毒性和中心作用。 3. n.n.的PLA2 Atra 蛇毒的毒性相对较低，没有器官特定作用 即使它具有较高的酶活性。 为了确定是否存在（如果是这样的化学性质） 负责药理特性和独立的分子的 从酶促活性位点，我们将准备已知的特定化学化学物质 PLA2酶和突触前PLA2神经毒素的修饰。 要进行的修改包括：1。烷基化和甲基化 酶促活性位点的组氨酸，2。氨基甲基化，酰化和 赖氨酸的鸟苷，3。自由羧基添加半棕榈 （天冬氨酸和谷氨酸），4。色氨酸的烷基化，5。 酪氨酸的磺酰基，6。甲基蛋白的氧化，7。裂解 N末端八肽（胶束识别位点）。 生化和 天然酶的药理特性及其修饰 将要测试的衍生物包括：基材上的酶促活性 在各种组织状态（单体，胶束，脂蛋白， 天然膜），磷脂底物偏好，酶 - 磷脂 复合形成，溶血和抗凝作用，静脉注射和 脑室内致命效力，电生理学对 孤立的心脏组织和神经抑制肌的制剂， 磷脂的水解以及PLA2与心脏肌膜和心脏的结合 脑突触质膜，组织的显微镜改变。 在这些研究中使用模型PLA2酶和 突触前PLA2神经毒素可能与 活动与前列腺素相关的哺乳动物PLA2酶 形成和膜转换，除了可能的作用 调节发射器作用和膜流动性。 临床疾病 可能与异常酶活性或有毒作用有关 与酶活性无关。