VITAMIN D P450 FUNCTION--NATURAL & DIRECTED MUTAGENESIS

维生素 D P450 功能 - 天然

• 批准号: 6171147
• 负责人: JOHN L OMDAHL
• 金额: $ 24.2万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-22 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6171147
• 关键词: SDS polyacrylamide gel electrophoresis; affinity chromatography; animal genetic material tag; cytochrome P450; enzyme activity; enzyme substrate; gene mutation; high performance liquid chromatography; human tissue; isozymes; laboratory mouse; laboratory rabbit; point mutation; polymerase chain reaction; protein binding; protein structure function; site directed mutagenesis; swine; vitamin D; vitamin D resistant rickets

DESCRIPTION (Adapted from the Applicant's Abstract): This proposal is based upon the applicants' recent cloning of the rat 1-hydroxylase (cytochrome P450c1) enzyme and the human P450c1 gene. Using FISH analysis and human P450c1 DNA, they have determined the gene locus for P450c1 to be 12q13.1-13.3, the same site that was previously identified for pseudovitamin D-deficient rickets (PDDR). This application describes studies that are directed toward understanding the structure and function of P450c1, a regulatory enzyme that directs the bioactivation of vitamin D to the pleiotropic hormone 1,25-dihydroxy D3 [1,25(OH)2D3 or calcitriol], which is active in calcium homeostasis, cellular growth and differentiation, and the immune response. However, until recent advances, only a paucity of information existed on the molecular attributes of the 1-hydroxylase enzyme due to the lack of pure protein and enzyme coding information. Yet, a molecular understanding of the enzyme's binding and catalytic properties is fundamental to the biostructural design of 1-hydroxylase inhibitors for application in hyperparathyroidism and 1,25(OH)2D3-mediated hypercalcemic states. In addition, it is now possible to use the P450c1-gene information to investigate the molecular basis of PDDR, and in the design of gene therapeutic agents for the treatment of genetic disorders and cellular proliferation diseases (e.g., cancer). Therefore, the current investigation is designed to gain an understanding of the structural and functional attributes of cytochrome P450c1 in health and disease. It is hypothesized that a structural defect in P450c1 is the basis for PDDR, and that delineation of the genetic mutations in this disease will serve as a foundation for understanding the molecular function of cytochrome P450c1. Studies in the Specific Aims are designed to: 1) express and purify recombinant cytochrome P450c1, prepare antibodies and determine substrate-binding, spectral, kinetic and topological properties of the enzyme, and 2) to sequence analyze, and characterize the activity and substrate-binding properties of expressed natural mutants of the human and porcine.

描述（根据申请人的摘要改编）：此提案是基于的 申请人最近对大鼠1-羟化酶的克隆（细胞色素） P450C1）酶和人P450C1基因。 使用鱼类分析和人类 P450C1 DNA，他们确定了P450C1的基因基因座 12q13.1-13.3，以前鉴定为假硫胺的地点 D缺陷rick（PDDR）。 该应用描述的研究是 致力于理解P450C1的结构和功能 将维生素D的生物激活引导到 多效激素1,25-二羟基D3 [1,25（OH）2d3或Calcitriol]，即 活跃于钙稳态，细胞生长和分化以及 免疫反应。 但是，直到最近进步， 信息存在于1-羟化酶的分子属性上 由于缺乏纯蛋白质和酶编码信息。 但是， 分子对酶结合和催化特性的理解是 1-羟化酶抑制剂的生物结构设计的基础 在甲状旁腺功能亢进症和1,25（OH）2d3介导的高钙化中的应用 国家。 此外，现在可以使用P450C1基因信息 研究PDDR的分子基础和基因设计 治疗遗传疾病和细胞的治疗剂 增殖疾病（例如癌症）。 因此，当前的调查 旨在了解结构和功能 细胞色素P450C1在健康和疾病中的属性。 它是假设的 P450C1中的结构缺陷是PDDR的基础，并且 在该疾病中遗传突变的描述将作为一个 理解细胞色素P450C1的分子功能的基础。 特定目的的研究设计为：1）表达和净化 重组细胞色素P450C1，准备抗体并确定 底物结合，光谱，动力学和拓扑特性 酶，2）对分析进行测序，并表征活性和 人类表达的天然突变体的底物结合特性和 猪。