Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, FL 33431, USA.
Anal Biochem. 2011 Feb 1;409(1):37-45. doi: 10.1016/j.ab.2010.09.040. Epub 2010 Oct 23.
Proteolytic enzymes play fundamental roles in many biological processes. Members of the matrix metalloproteinase (MMP) family have been shown to take part in processes crucial in disease progression. The current study used the ExcelArray Human MMP/TIMP Array to quantify MMP and tissue inhibitor of metalloproteinase (TIMP) production in the lysates and media of 14 cancer cell lines and 1 normal cell line. The overall patterns were very similar in terms of which MMPs and TIMPs were secreted in the media versus associated with the cells in the individual samples. However, more MMP was found in the media (in both amount and variety). TIMP-1 was produced in all cell lines. MMP activity assays with three different fluorescence resonance energy transfer (FRET) substrates were then used to determine whether protein production correlated with function for the WM-266-4 and BJ cell lines. Metalloproteinase activity was observed for both cell lines with a general MMP substrate (Knight SSP), consistent with protein production data. However, although both cell lines promoted the hydrolysis of a more selective MMP substrate (NFF-3), metalloproteinase activity was confirmed only in the BJ cell line. The use of inhibitors to confirm metalloproteinase activities pointed to the strengths and weaknesses of in situ FRET substrate assays.
蛋白水解酶在许多生物过程中发挥着基本作用。基质金属蛋白酶（MMP）家族的成员已被证明参与了疾病进展过程中至关重要的过程。本研究使用 ExcelArray 人类 MMP/TIMP 阵列定量分析了 14 种癌细胞系和 1 种正常细胞系的裂解物和培养基中 MMP 和金属蛋白酶组织抑制剂（TIMP）的产生。就单个样本中细胞相关的 MMP 和 TIMP 分泌而言，整体模式非常相似。然而，培养基中分泌的 MMP 更多（无论是数量还是种类）。所有细胞系均产生 TIMP-1。然后使用三种不同的荧光共振能量转移（FRET）底物的 MMP 活性测定来确定 WM-266-4 和 BJ 细胞系的蛋白产生是否与功能相关。两种细胞系均具有一般 MMP 底物（Knight SSP）的金属蛋白酶活性，与蛋白产生数据一致。然而，尽管两种细胞系均促进了更具选择性的 MMP 底物（NFF-3）的水解，但仅在 BJ 细胞系中证实了金属蛋白酶活性。抑制剂的使用证实了金属蛋白酶活性，指出了原位 FRET 底物测定的优缺点。
