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

研究将涉及动员的工作模式 肌醇1，4，5-三磷酸(INS-1，4，5-P3)诱导的细胞内钙离子 在化学诱导剂刺激的中性粒细胞中。一种特殊的受体 皂苷通透性兔中性粒细胞INS-1，4，5-P3和 在微粒子上的基因已经被发现。它的特点是 受体提示它是体内的生理受体。 调节隔离钙释放的中性粒细胞 来自内质网。试图将数据统一起来 变成一个可检验的假设，一个依赖INS-1，4，5-P3的模型， GTP对内质网钙通道的调节 提出了膜的概念。计划研究的主要重点 致力于一种GTP的纯化和表征- 受调节的INS-1，4，5-P3受体来自细胞匀浆和 二甲基亚砜分化人的微粒体组分 早幼粒细胞HL-60细胞。溶解的受体将被提纯 通过常规的离子交换和分子筛层析 用~(32)P-INS-R的特异性结合作为受体的测定 1，4，5-P3重组为脂类后的分离部分 去除水泡和洗涤剂。我们将尝试 用对氨基苯甲酸合成亲和载体 衍生的1，4，5-三磷酸肌醇偶联到活化的 CH-Sepharose4B基质。使用重组的、提纯的 受体--INS-1，4，5-P3的兴奋作用机制 探讨钙离子释放和GTP的调节作用。这个 将分析INS-1，4，5-P3结合数据以获得平衡 (Kd和受体数目)和动力学(热力学)常数。 这些数据将与可渗透细胞的数据进行比较。这个 纯化的受体将用于将特异性抗体提高到 用于功能分析和纯化方案。vbl.使用 由N-末端序列合成的寡核苷酸探针 来自受体的胰酶片段的数据，cDNA克隆 将被选中。然后将导出序列数据。这些 研究可能最终会使我们更好地理解 中性粒细胞内钙离子释放的调节。

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.