CHARACTERIZATION OF THE LEUKOCYTE INS (1,4,5)P3 RECEPTOR

白细胞 INS (1,4,5)P3 受体的表征

• 批准号: 3466863
• 负责人: PETER G BRADFORD
• 金额: $ 10.04万
• 依托单位: HAHNEMANN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3466863
• 关键词: G protein; calcium transporting ATPase; chemical models; chemoreceptors; complementary DNA; detergents; dimethylsulfoxide; endoplasmic reticulum; guanosine triphosphate; inositol phosphates; laboratory mouse; laboratory rabbit; lipids; membrane permeability; microsomes; neutrophil; p aminobenzoate; terpene saponin; thermodynamics

Studies will deal with a working model for mobilization of intracellular calcium by inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) in chemoattractant stimulated neutrophils. A specific receptor for Ins-1,4,5-P3 in saponin-permeabilized rabbit neutrophils and on microsomes has been identified. The characteristics of this receptor suggest that it is the physiological receptor in neutrophils which regulates the release of sequestered calcium from the endoplasmic reticulum. In an attempt to unify the data into a testable hypothesis, a model of an Ins-1,4,5-P3 dependent, GTP-regulated Ca2+ channel in the endoplasmic reticulum membrane is proposed. The major emphasis of the planned studies is placed on the purification and characterization of a GTP- regulated, Ins-1,4,5-P3 receptor from cell homogenates and microsomal fractions from DMSO-differentiated human promyelocytic HL-60 cells. Solubilized receptors will be purified by conventional ion exchange and molecular sieve chromatography using as an assay for receptor the specific binding of 32P-Ins- 1,4,5-P3 to isolated fractions following reconstitution into lipid vesicles and detergent removal. Attempts will be made to synthesize an affinity support using p-amino benzoiacid derivatized inositol 1,4,5-trisphosphate coupled to an activated CH-sepharose 4B matrix. Using the reconstituted, purified receptor, the mechanism of action of Ins-1,4,5-P3 in stimulating Ca2+ release and the modulatory role of GTP will be probed. The Ins-1,4,5-P3 binding data will be analyzed to obtain equilibrium (Kd and receptor number) and kinetic (thermodynamic) constants. These will be compared to those found for permeable cells. The purified receptor will be used to raise specific antibodies to be used in functional assays and purification protocols. Using oligonucleotide probes synthesized from N-terminus sequence data derived from trypsin fragments of the receptor, cDNA clones will be selected. Sequence data will then be derived. These studies may ultimately enable a better understanding of intracellular regulation of Ca2+ release in neutrophils.

研究将涉及动员妇女的工作模式， 1，4，5-三磷酸肌醇（Ins-1，4，5-P3）胞内钙 在化学引诱物刺激的中性粒细胞中。 特异性受体 对于皂苷透化兔中性粒细胞中的Ins-1，4，5-P3， 已被识别。 的特性进行 受体表明，它是生理受体， 调节螯合钙释放的中性粒细胞 来自内质网。 为了统一数据 转化为一个可检验的假设，一个Ins-1，4，5-P3依赖的模型， GTP调控的内质网Ca~（2+）通道 膜提出。 计划中的研究的主要重点是 是放置在纯化和表征的GTP- 调节的，来自细胞匀浆的Ins-1，4，5-P3受体， DMSO分化的人微粒体组分 早幼粒细胞HL-60细胞 将纯化溶解的受体 通过常规离子交换和分子筛色谱法 使用32P-Ins-1的特异性结合作为受体测定， 1，4，5-P3在复溶至脂质后分离至分离组分 囊泡和去污剂去除。 将试图 使用对氨基苯甲酸合成亲和载体 衍生的肌醇1，4，5-三磷酸偶联到活化的 CH-琼脂糖4B基质。 使用重组的，纯化的 Ins-1，4，5-P_3在促肾上腺皮质激素释放激素受体中的作用机制 Ca2+释放和GTP的调节作用将被探索。 的 将分析Ins-1，4，5-P3结合数据以获得平衡 (Kd和受体数）和动力学（热力学）常数。 这些将与渗透性细胞的结果进行比较。 的 纯化的受体将用于产生特异性抗体， 用于功能测定和纯化方案。 使用 从N-末端序列合成的寡核苷酸探针 数据来源于受体的胰蛋白酶片段，cDNA克隆 将被选中。 然后将导出序列数据。 这些 研究最终可能会使人们更好地了解 中性粒细胞中Ca 2+释放的细胞内调节。