PROTEOLIPIDS & MINERALIZATION OF BONES & TEETH

蛋白脂

• 批准号: 3219669
• 负责人: Barbara D. Boyan
• 金额: $ 14.08万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-03-01 至 1991-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3219669
• 关键词: 1,25 dihydroxycholecalciferol; 25 hydroxycholecalciferol; 5' nucleotidase; adenosinetriphosphatase; alkaline phosphatase; bone development; bone metabolism; calcification; cell membrane; chemical structure function; chondrocytes; dental development; enzyme induction /repression; high performance liquid chromatography; hormone regulation /control mechanism; hydrolysis; hydroxyapatites; laboratory rabbit; laboratory rat; membrane structure; messenger RNA; normal ossification; nutrition related tag; phospholipids; proteolipids; sodium potassium exchanging ATPase; tissue /cell culture

This proposal examines the role of proteolipids in biologic mineral formation and the mechanism of their action in situ. Matrix vesicle proteolipid (MVP) produced by rat chondrocytes in culture is used as a model system to test the hypothesis that MVP is a site of initial hydroxyapatite formation and that changes in the membrane environment, such as those produced by the action of vitamin D, will alter its functional properties. A model membrane system will be used to mimic the membrane environment in vitro. MVP will be purified by differential extraction in organic solvents of varying polarities, reverse phase and chromatofocusing HPLC. Heterogeneity will be assessed by ISF and SDS-PAGE. Polyclonal antibodies generated to MVP will be affinity purified and then used to affinity purify specific subsets of MVP, to characterize multimeric aggregates, to purify mRNA for individual proteolipids, and to chart MVP synthesis and incorporation into matrix vesicles. MVP will be incorporated into planar bilayers of known composition and ion transport compared to that of the intact matrix vesicle membrane. The ability of specific functional units to support hydroxyapatite formation will be tested in vitro. To examine the effect of regulation of the membrane microenvironment on MVP structure and function in culture and in vitro, reserve zone (RC) or growth region (GC) chondrocytes will be incubated with 1,25(OH)2D3 or 24R,25(OH)2D3. Changes in matrix vesicle and plasma membrane fluidity will be assessed using flourescence spectroscopy; membrane structure and function monitored by enzymatic activity (alkaline phosphatase, 5' nucleotidase, Na+/K+ ATPase) and phospholipid composition. Incorporation of vitamin D into the membrane will be measured using 3H-labeled metabolites. Phospholipid turnover will also be assessed as acylation and hydrolysis of radiolabeled fatty acids. Synthesis of MVP and its incorporation into matrix vesicles will be charted using specific antibodies. Membrane and MVP function will be measured using planar bilayer membranes or by in vitro hydroxyapatite formation.

该建议研究了蛋白脂质在生物矿物质中的作用 形成及其作用的机制。 矩阵 大鼠软骨细胞在培养物中产生的囊泡蛋白脂脂（MVP） 被用作模型系统，以测试MVP是一个站点的假设 初始羟基磷灰石的形成，并改变 膜环境，例如由 维生素D将改变其功能特性。 模型 膜系统将用于模仿膜 体外环境。 MVP将通过差分纯化 在不同极性的有机溶剂中提取，反相反向 和染色体HPLC。 异质性将由 ISF和SDS-PAGE。 与MVP产生的多克隆抗体将 亲和力纯化，然后用于纯化特定 MVP的子集，以表征多聚体聚集体，以净化 单个蛋白脂质的mRNA，并绘制MVP合成和 掺入基质囊泡中。 MVP将合并到 比较已知成分和离子运输的平面双层 完整的基质囊泡膜的膜。 能力 支持羟基磷灰石形成的特定功能单元将 在体外进行测试。 检查调节的影响 MVP结构和功能的膜微环境 培养和体外，储备区（RC）或生长区域（GC） 软骨细胞将与1,25（OH）2d3或 24R，25（OH）2d3。 基质囊泡和质膜的变化 流动性将使用粉状光谱进行评估； 通过酶促监测的膜结构和功能 活性（碱性磷酸酶，5'核苷酸酶，Na+/K+ ATPase） 和磷脂组成。 将维生素D掺入 膜将使用3H标记的代谢物测量。 磷脂周转率也将被评估为酰化和 放射标记脂肪酸的水解。 MVP及其合成 将合并到基质囊泡中，将使用特定 抗体。 膜和MVP功能将使用 平面双层膜或通过体外羟基磷灰石形成。