Human mast cell tryptase isoforms: separation and examination of substrate-specificity differences.
Human mast cell tryptase isoforms: separation and examination of substrate-specificity differences.
复制标题
人肥大细胞类胰蛋白酶亚型：底物特异性差异的分离和检查。
DOI：
10.1042/bj3070341
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Johnson,DA
中科院分区：
文献类型：
--
作者：
Little,SS;Johnson,DA
胰蛋白酶是在肥大细胞颗粒中发现的胰蛋白酶样酶，其似乎以四聚体存在。这些酶不受血浆蛋白酶抑制剂的控制，在体外仅切割少数生理底物，包括高分子量激肽原（HMMK）和血管活性肠肽（VIP）。纯化的人肺肥大细胞类胰蛋白酶（HLT）含有两条约为1000 bp的条带。SDS/PAGE上的分子量为29和33 kDa。这两种形式的HLT已通过磷酸纤维素柱色谱法分离，高分子量形式（高HLT）用10 μ M肝素洗脱，低分子量形式（低HLT）随后用1 M NaCl洗脱。去除天冬酰胺连接的碳水化合物导致这两种亚型在SDS/PAGE上以单一尖锐条带运行，分子量略有不同。这两种异构体的类胰蛋白酶的分离表明，四聚体由四个同源亚基，而不是两种异构体的混合物。使用HMMK和VIP作为底物，发现这两种形式的HLT在特异性和切割速率方面不同。高HLT最初在分子C末端阴离子结合区内的Arg-431处切割HMMK，而低HLT在分子C末端部分内的多个位点同时切割HMMK。HPLC肽图谱的基础上，每个亚型也切割VIP在不同的网站。基于滴定亚型活性位点浓度的裂解速率比较表明，低HLT裂解HMMK比高HLT更快。这两种异构体可能代表不同的基因产物，或者它们可能由翻译后修饰产生。
Tryptases are trypsin-like enzymes found in mast cell granules that appear to exist as tetramers. These enzymes are not controlled by blood plasma proteinase inhibitors and only cleave a few physiological substratesin vitro, including high-molecular-mass kininogen (HMMK) and vasoactive intestinal peptide (VIP). Purified human lung mast cell tryptase (HLT) contained two bands of approx. molecular mass 29 and 33 kDa on SDS/PAGE. These two forms of HLT have been separated by chromatography on a cellulose phosphate column, with the high-molecular-mass form (high-HLT) being eluted with 10 microM heparin and the low-molecular-mass form (low-HLT) subsequently eluted with 1 M NaCl. Removal of asparagine-linked carbohydrate caused both isoforms to run as single sharp bands on SDS/PAGE, differing slightly in molecular mass. Separation of these two isoforms of tryptase shows that tetramers consist of four homologous subunits rather than mixtures of the two isoforms. Using HMMK and VIP as substrates, these two forms of HLT were found to differ with regard to specificity and rate of cleavage. High-HLT initially cleaved HMMK at Arg-431 within the C-terminal anionic binding region of the molecule, whereas low-HLT cleaved HMMK simultaneously at multiple sites within the C-terminal portion of the molecule. On the basis of HPLC peptide mapping, each isoform also cleaved VIP at different sites. Comparison of cleavage rates based on the active-site concentrations of titrated isoforms showed that low-HLT cleaved HMMK more rapidly than did high-HLT. These two isoforms may represent different gene products or they may result from post-translational modification.