Flow cytometric analysis of protease activities in vital cells.
The analysis of lysosomal proteases in cell lysates is complicated by pH-dependent and oxidative changes of their activity and complex formation with cytosolic inhibitors. Therefore, new flow cytometric methods were developed for the intracellular measurement of protease activities in viable cells. Intracellular cleavage of substrates such as Z-Arg-Arg-4-trifluoromethylcoumarinyl-7-amide to green fluorescent 7-amino-4-trifluoromethylcoumarin (AFC) in viable neutrophils and monocytes was only detected following phagocytosis of Escherichia coli. A measurement of the cysteine or serine proteinase activities in resting human leukocytes was, however, not possible with AFC derivatives as the overlapping blue fluorescence of the substrates reduces sensitivity. Nonfluorescent bis-substituted peptide derivatives of rhodamine 110 (R110), which are intracellularly cleaved to green fluorescent mono-substituted R110 and free R110 proved to be more sensitive substrates. The activity of the lysosomal cysteine proteinases of human monocytes or rat macrophages, i.e. cathepsin B and L, was specifically measured with (Z-Arg-Arg)2-R110, (Z-Phe-Arg)2-R110, or (Z-Ala-Arg-Arg)2-R110. Fluorescence generation was completely inhibited by Z-Phe-Ala-diazomethane or E-64. The serine proteinases of human neutrophils were analyzed with Elastase-substrates such as (Z-Ala-Ala)2-R110 or (Z-Ala-Ala-Ala)2-R110. Specificity was shown by inhibition with diisopropylfluorophosphate.
Rothe
G
G
Arbeitsgruppe Zellbiochemie, Max-Planck-Institut für Biochemie, Martinsried, FRG.
Klingel
S
S
Assfalg-Machleidt
I
I
Machleidt
W
W
Zirkelbach
C
C
Banati
R B
RB
Mangel
W F
WF
Valet
G
G
D000595:Amino Acid Sequence; D002401:Cathepsin B / Q000032:analysis; D056668:Cathepsin L; D002403:Cathepsins / Q000032:analysis; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000032:analysis*; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D008247:Lysosomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009000:Monocytes / Q000201:enzymology; D009504:Neutrophils / Q000201:enzymology; D010196:Pancreatic Elastase / Q000032:analysis / Q000378:metabolism; D010455:Peptides / Q000032:analysis / Q000138:chemical synthesis; D010587:Phagocytosis / Q000502:physiology; D012235:Rhodamines; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling
D010455:Peptides; D012235:Rhodamines; D002403:Cathepsins; D010450:Endopeptidases; D010196:Pancreatic Elastase; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; C534280:Ctsl protein, rat