HUMAN SKIN MAST CELL GRANULE ENZYMES--SKIN DISEASES

人体皮肤肥大细胞颗粒酶--皮肤病

• 批准号: 3156148
• 负责人: BRUCE U WINTROUB
• 金额: $ 24.46万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-07-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3156148
• 关键词: affinity chromatography; angiotensins; carboxypeptidase; clone cells; complementary DNA; enzyme complex; enzyme linked immunosorbent assay; enzyme mechanism; fibrinogen; genetic library; granule; high performance liquid chromatography; human subject; laminin; mast cell; monoclonal antibody; nucleic acid hybridization; pathologic process; pemphigus; peptidases; peptides; physical chemical interaction; protein biosynthesis; protein sequence; proteoglycan; proteolysis; radioimmunoassay; skin disorder; skin transplantation; substance P; urticaria; vesicular skin disorder

The goals of these studies are to isolate and characterize carboxypeptidase, a newly identified human mast cell secretory granule protease, and to localize and determine the pathobiologic role of mast cell proteolytic enzymes in mast cell mediated skin diseases. Proposed studies will address: 1) purification and physicochemical characterization of human skin mast cell carboxypeptidase; 2) characterization and in vitro reconstitution of the human skin mast cell secretory granule with respect to protease and proteoglycan components; 3) investigation of the biochemical action of purified carboxypeptidase, and enzyme- proteoglycan complexes on purified proteins, peptides, and potential cellular substrates in skin; and 4) examination of the pathobiologic role of human skin mast cell secretory granule proteases in mast cell-associated cutaneous diseases. These studies will add to understanding of the human skin mast cell, and the role of mast cell secretory granule proteases in mediating tissue damage in cutaneous disorders. To accomplish these goals, human skin mast cell carboxypeptidase, identified by an HPLC assay system, will be purified from whole skin extracts by affinity chromatography. A monospecific, polyclonal anticarboxypeptidase antibody will be developed. To understand regulation of carboxypeptidase expression, mast cells from various tissues will be assessed for carboxypeptidase activity by HPLC, and protein by ELISA or RIA, and a human mast cell cDNA library will be made to identify recombinant clones corresponding to carboxypeptidase. To understand their biochemical and biologic activity, enzyme-proteoglycan complexes from dispersed human mast cells or reconstituted from purified components, will be incubated with protein, peptide, and potential cellular substrates such as fibrinogen, laminin, substance P, angiotensin I, and cultured human umbilical vein endothelial cells, respectively. The proteolytic activity of these complexes will be examined in vitro using skin explants. The in situ release, localization and fate of carboxypeptidase will be examined in vitro using skin explants, and in vivo after intradermal injection of human C5a, and in a skin chamber model after challenge with allergens. Finally, the deposition of mast cell granule carboxypeptidase will be examined in vivo in diseases marked by increased vascular permeability, i.e. cold urticaria, and by tissue destruction, i.e. bullous pemphigoid.

这些研究的目的是分离和表征

项目成果

The granulocyte-angiotensin system. Angiotensin I-converting activity of cathepsin G.

粒细胞-血管紧张素系统。

• 发表时间: 1982
• 期刊: The Journal of biological chemistry
• 作者: Klickstein,LB;Kaempfer,CE;Wintroub,BU
• 通讯作者: Wintroub,BU

Separation of angiotensins and assay of angiotensin-generating enzymes by high-performance liquid chromatography.

通过高效液相色谱法分离血管紧张素并测定血管紧张素生成酶。

• DOI: 10.1016/0003-2697(82)90329-3
• 发表时间: 1982
• 期刊: Analytical biochemistry
• 影响因子: 2.9
• 作者: Klickstein,LB;Wintroub,BU
• 通讯作者: Wintroub,BU