Bronk S F, Powers S P, Gores G J
Department of Internal Medicine, Mayo Medical School, Rochester, Minnesota 55905.
Anal Biochem. 1993 May 1;210(2):219-25. doi: 10.1006/abio.1993.1186.
Digitized video fluorescent microscopy (DVFM) is a powerful technique for quantitating multiple processes in living cells. However, techniques for measuring protease activity by DVFM are not available. Our aim was to develop an approach for measuring aminopeptidase activity using DVFM. We conjugated glycine-7-amino-4-methylcoumarin-3-acetic acid (glycine-AMC-3-acetic acid) to dextran using a PEG bridge. Glycine-AMC-3-acetic acid-PEG-dextran was microinjected into cultured rat hepatocytes along with rhodamine-dextran. Glycine-AMC-3-acetic acid-PEG-dextran is nonfluorescent, but aminopeptidase hydrolysis of the glycine-AMC bond liberates the fluorescent AMC-3-acetic acid-PEG-dextran within the cell. Following microinjection, rhodamine-dextran fluorescence remained constant while AMC-3-acetic acid-PEG-dextran fluorescence increased in a linear fashion over time reflecting proteolytic cleavage of the glycine-AMC bond. AMC-3-acetic acid-PEG-dextran and rhodamine-dextran fluorescence were cytosolic as evidenced by diffuse fluorescence and colocalized. Because rhodamine-dextran fluorescence remained constant and the probes colocalized, the fluorescent ratio of AMC-3-acetic acid-PEG-dextran/rhodamine-dextran could be used to measure proteolysis. Basal rates of proteolysis were 9 +/- 3 ratio units/10 min. Comicroinjection of the aminopeptidase inhibitor, bestatin, along with the dextran probes abolished proteolysis. Addition of the calcium ionophore, 4-Br-A23187, increased proteolysis 12-fold to 107 +/- 14/10 min (P < 0.01). We have developed a novel, dynamic technique for measuring pH-sensitive, Ca(2+)-dependent aminopeptidase activity in living cells using DVFM. This approach may be used for the measurement of other peptidase activities by synthesizing peptidase-specific peptidyl-AMC-3-acetic acid-PEG-dextran conjugates.
数字化视频荧光显微镜（DVFM）是一种用于定量活细胞中多种过程的强大技术。然而，通过DVFM测量蛋白酶活性的技术尚不存在。我们的目标是开发一种使用DVFM测量氨肽酶活性的方法。我们使用聚乙二醇（PEG）桥将甘氨酸-7-氨基-4-甲基香豆素-3-乙酸（甘氨酸-AMC-3-乙酸）与葡聚糖偶联。将甘氨酸-AMC-3-乙酸-PEG-葡聚糖与罗丹明-葡聚糖一起显微注射到培养的大鼠肝细胞中。甘氨酸-AMC-3-乙酸-PEG-葡聚糖无荧光，但甘氨酸-AMC键的氨肽酶水解会在细胞内释放出荧光性的AMC-3-乙酸-PEG-葡聚糖。显微注射后，罗丹明-葡聚糖荧光保持恒定，而AMC-3-乙酸-PEG-葡聚糖荧光随时间呈线性增加，反映了甘氨酸-AMC键的蛋白水解切割。AMC-3-乙酸-PEG-葡聚糖和罗丹明-葡聚糖荧光呈胞质分布，表现为弥漫性荧光且共定位。由于罗丹明-葡聚糖荧光保持恒定且探针共定位，AMC-3-乙酸-PEG-葡聚糖/罗丹明-葡聚糖的荧光比率可用于测量蛋白水解。基础蛋白水解速率为9±3比率单位/10分钟。将氨肽酶抑制剂苯丁抑制素与葡聚糖探针共同显微注射可消除蛋白水解。添加钙离子载体4-溴-A23187可使蛋白水解增加12倍，达到107±14/10分钟（P<0.01）。我们开发了一种新颖的动态技术，使用DVFM测量活细胞中对pH敏感、依赖Ca(2+)的氨肽酶活性。通过合成肽酶特异性的肽基-AMC-3-乙酸-PEG-葡聚糖偶联物，该方法可用于测量其他肽酶活性。
