Sensitive method to identify and characterize proteinases in situ after SDS-PAGE.
AuthorsWilliams J, McGrath WJ, Mangel WF
JournalBiotechniques
PubMed ID11084874
'Cells and body fluids contain numerous, different proteinases; to identify and characterize them are both important and difficult tasks. Especially difficult to identify and characterize are highly specific proteinases. Here, we present an extremely sensitive and quantitative method to characterize proteinases fractionated by SDS-PAGE that cleave specific rhodamine-based fluorogenic substrates. ... More
A forward genetic strategy reveals destabilizing mutations in the Ebolavirus glycoprotein that alter its protease dependence during cell entry.
AuthorsWong AC, Sandesara RG, Mulherkar N, Whelan SP, Chandran K,
JournalJ Virol
PubMed ID19846533
'Ebolavirus (EBOV) entry into cells requires proteolytic disassembly of the viral glycoprotein, GP. This proteolytic processing, unusually extensive for an enveloped virus entry protein, is mediated by cysteine cathepsins, a family of endosomal/lysosomal proteases. Previous work has shown that cleavage of GP by cathepsin B (CatB) is specifically required to ... More
Quantification of intracellular cathepsin activities in human lung tumor cell lines by flow cytometry.
AuthorsUlbricht B, Spiess E, Schwartz-Albiez R, Ebert W
JournalBiol Chem Hoppe Seyler
PubMed ID7576237
'The cysteine proteases cathepsin B and cathepsin L are very likely involved in invasive processes of normal and malignant cells, they become relevant for a number of diseases and are possibly prognostic markers for the outcome of human lung cancer. Therefore, we have determined activities of these related enzymes in ... More
Membrane permeable fluorogenic rhodamine substrates for selective determination of cathepsin L.
AuthorsAssfalg-Machleidt I, Rothe G, Klingel S, Banati R, Mangel WF, Valet G, Machleidt W
JournalBiol Chem Hoppe Seyler
PubMed ID1515071
'The dipeptidyl rhodamine diamide substrates (Z-Phe-Arg)2-R110 and (Z-Arg-Arg)2-R110 are 820- and 360-fold more selective for cathepsin L than for cathepsin B allowing a sensitive determination of cathepsin L activity in the presence of high activity of cathepsin B. The results obtained with cell lysates suggest that the cysteine proteinase activity ... More
Saposin C is required for lipid presentation by human CD1b.
AuthorsWinau F, Schwierzeck V, Hurwitz R, Remmel N, Sieling PA, Modlin RL, Porcelli SA, Brinkmann V, Sugita M, Sandhoff K, Kaufmann SH, Schaible UE
JournalNat Immunol
PubMed ID14716313
'Lipids from Mycobacterium tuberculosis are presented through CD1 proteins to T lymphocytes in humans, but the accessory molecules required for antigen loading and presentation remain unidentified. Here we show that fibroblasts deficient in sphingolipid activator proteins (SAPs) transfected with CD1b failed to activate lipid-specific T cells. However, the T cell ... More
Enzyme cytochemical techniques for metabolic mapping in living cells, with special reference to proteolysis.
AuthorsBoonacker E, Van Noorden CJ
JournalJ Histochem Cytochem
PubMed ID11724895
Specific enzymes play key roles in many pathophysiological processes and therefore are targets for therapeutic strategies. The activity of most enzymes is largely determined by many factors at the post-translational level. Therefore, it is essential to study the activity of target enzymes in living cells and tissues in a quantitative ... More
A homogeneous, nonradioactive high-throughput fluorogenic protein kinase assay.
AuthorsKupcho K, Somberg R, Bulleit B, Goueli SA
JournalAnal Biochem
PubMed ID12758259
Protein kinases play an important role in many cellular processes and mediate cellular responses to a variety of extracellular stimuli. They have been identified by many pharmaceuticals as valid targets for drug discovery. Because of the large number of protein kinases, and the large number of compounds to be screened, ... More
A homogeneous, nonradioactive high-throughput fluorogenic protein phosphatase assay.
AuthorsKupcho K, Hsiao K, Bulleit B, Goueli SA
JournalJ Biomol Screen
PubMed ID15140384
Protein phosphatases are critical components in cellular regulation; they do not only act as antioncogenes by antagonizing protein kinases, but they also play a positive regulatory role in a variety of cellular processes that require dephosphorylation. Thus, assessing the function of these enzymes necessitates the need for a robust, sensitive ... More
Flow cytometric analysis of protease activities in vital cells.
AuthorsRothe G, Klingel S, Assfalg-Machleidt I, Machleidt W, Zirkelbach C, Banati RB, Mangel WF, Valet G
JournalBiol Chem Hoppe Seyler
PubMed ID1381188
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 ... More
Squamous cell carcinoma antigen 2 is a novel serpin that inhibits the chymotrypsin-like proteinases cathepsin G and mast cell chymase.
AuthorsSchick C, Kamachi Y, Bartuski AJ, Cataltepe S, Schechter NM, Pemberton PA, Silverman GA
JournalJ Biol Chem
PubMed ID8999871
The squamous cell carcinoma antigen (SCCA) serves as a serological marker for more advanced squamous cell tumors. Molecular cloning of the SCCA genomic region revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2. Analysis of the primary amino acid sequences shows that both genes are members of the ... More
Detection of lysosomal cysteine proteinases in microglia: flow cytometric measurement and histochemical localization of cathepsin B and L.
AuthorsBanati RB, Rothe G, Valet G, Kreutzberg GW
JournalGlia
PubMed ID8432559
The activation and differentiation of microglia is a prominent pathophysiological process in numerous inflammatory and demyelinating diseases of the central nervous system, including Alzheimer's disease and the AIDS encephalopathy. The tissue damage during these diseases has partly been attributed to lipid peroxidating reactive oxygen intermediates for which activated microglia are ... More
The Caenorhabditis elegans muscle specific serpin, SRP-3, neutralizes chymotrypsin-like serine peptidases.
AuthorsPak SC, Tsu C, Luke CJ, Askew YS, Silverman GA
JournalBiochemistry
PubMed ID16584183
Members of the intracellular serpin family may help regulate apoptosis, tumor progression, and metastasis. However, their in vivo functions in the context of a whole organism have not been easily defined. To better understand the biology of these serpins, we initiated a comparative genomics study using Caenorhabditis elegans as a ... More