Suc-Gly-Pro-Leu-Gly-Pro-AMC, a highly sensitive, fluorogenic substrate for thimet oligopeptidase as well as for post-proline cleaving enzyme (prolyl endopeptidase) [1].
The FAP activity in human plasma was measured using succinyl-pentapeptide composed of SUch-Gly-pro-Leu-Gly-pro-AMC, which contains two FAP cleavage sites.Only the cleavage between the last proline residue and the AMC group releases the fluorescent AMC group, resulting in a fluorescence reading, and the FAP activity of the measured samples ranges from 1.3 to 7 nM/min/ μL[2].
When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA, a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells[5].
In Male BALB/c mice ，using Suc-Gly-Pro-Leu-Gly-Pro-AMC test，no significant accumulations of other proteinases, such as matrix metalloproteinases, cathepsin D, and serine proteinases, were determined[3].Suc-GPLGP-MCA is hydro- lyzed at the Leu-Gly bond by CL-peptidase. The CL-peptidase activity in synovial fluid was significantly higher in patients with rheumatoid arthritis (RA) than in patients with osteoarthritis (OA) and in arthropathy-free controls[4].
References:
[1]: Kojima K, Kinoshita H, et,al. A new and highly sensitive fluorescence assay for collagenase-like peptidase activity. Anal Biochem. 1979 Nov 15;100(1):43-50. doi: 10.1016/0003-2697(79)90106-4. PMID: 232383.
[2]: Zhen EY, Jin Z, et,al. Circulating FGF21 proteolytic processing mediated by fibroblast activation protein. Biochem J. 2016 Mar 1;473(5):605-14. doi: 10.1042/BJ20151085. Epub 2015 Dec 3. PMID: 26635356; PMCID: PMC4764976.
[3]: Kakegawa H, Matano Y, et,al. Significant accumulations of cathepsin B and prolylendopeptidase in inflammatory focus of delayed-type hypersensitivity induced by Mycobacterium tuberculosis in mice. Biochem Biophys Res Commun. 2004 Mar 26;316(1):78-84. doi: 10.1016/j.bbrc.2004.01.176. PMID: 15003514.
[4]:Ito A, Hagihara M, et,al. Collagenase-like (CL) peptidase activity in synovial fluid from patients with rheumatoid arthritis. Clin Chim Acta. 1987 Dec;170(2-3):291-6. doi: 10.1016/0009-8981(87)90139-2. PMID: 2830060.
[5]:Okochi N, Kato-Murai M, et,al. Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface. Appl Microbiol Biotechnol. 2007 Dec;77(3):597-603. doi: 10.1007/s00253-007-1197-0. Epub 2007 Sep 27. PMID: 17899065.
Suc-Gly-Pro-Leu-Gly-Pro-AMC，一种高度敏感的荧光底物，可用于硫氨肽寡肽酶和脯氨酸后裂解酶（脯氨酰内肽酶）[1]。
使用由 SUch-Gly-pro-Leu-Gly-pro-AMC 组成的琥珀酰五肽测定人血浆中的 FAP 活性，其中包含两个 FAP 切割位点。仅最后一个脯氨酸残基和 AMC 基团之间的切割释放荧光AMC基团，产生荧光读数，被测样品的FAP活性范围为1.3-7 nM/min/μL[2]。
当每种细胞悬浮液分别与 BODIPY FL 酪蛋白和七种肽-MCA 底物反应时，在构建体之间观察到对 Suc-GPLGP-MCA（胶原酶样肽酶的底物）的水解活性存在显着差异: Lc-Triad展示细胞比Lc-WT展示细胞表现出更高的催化活性[5].
在雄性 BALB/c 小鼠中，使用 Suc-Gly-Pro-Leu-Gly-Pro-AMC 试验，未检测到其他蛋白酶如基质金属蛋白酶、组织蛋白酶 D 和丝氨酸蛋白酶的显着积累 [3].Suc-GPLGP-MCA 在 Leu-Gly 键处被 CL-肽酶水解。类风湿关节炎（RA）患者滑液中的CL-肽酶活性显着高于骨关节炎（OA）患者和无关节病的对照组[4]。