ABSTRACT
A series of KRIBB3 analogs were synthesized by modifying substituents at aryl moieties of KRIBB3 for examining structure-activity relationships, and their inhibitory activities on microtubule polymerization were evaluated. The presence of free phenolic hydrogens in aryl moieties of KRIBB3 analogs plays an important role in inhibition of microtubule polymerization.
Subject(s)
Anisoles/chemistry , Isoxazoles/chemistry , Tubulin Modulators/chemistry , Tubulin/chemistry , Anisoles/chemical synthesis , Anisoles/pharmacology , Humans , Isoxazoles/chemical synthesis , Isoxazoles/pharmacology , Polymerization , Structure-Activity Relationship , Tubulin/metabolism , Tubulin Modulators/chemical synthesis , Tubulin Modulators/pharmacologyABSTRACT
A new simplified procedure for identifying human plasmin was developed using a DTT copolymerized agarose stacking gel (ASG) system. Agarose (1 %) was used for the stacking gel because DTT inhibits the polymerization of acrylamide. Human plasmin showed the lowest activity at pH 9.0. There was a similar catalytically active pattern observed under acidic conditions (pH 3.0) to that observed under alkaline conditions (pH 10.0 or 11.0). Using the ASG system, the primary structure of the heavy chain could be established at pH 3.0. This protein was found to consist of three fragments, 45 kDa, 23 kDa, and 13 kDa. These results showed that the heavy chain has a similar structure to the autolysed plasmin (Wu et al., 1987b) but there is a different start amino acid sequence of the N-termini.
Subject(s)
Biochemistry/methods , Dithiothreitol/pharmacology , Fibrinolysin/chemistry , Amino Acid Sequence , Disulfides/chemistry , Dithiothreitol/chemistry , Electrophoresis, Agar Gel/methods , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Fibrinogen/chemistry , Humans , Hydrogen-Ion Concentration , Plasminogen/chemistry , Protein Structure, Tertiary , Sepharose/chemistry , Thrombin/chemistryABSTRACT
In general, a SYPRO Ruby dye is well known as a sensitive fluorescence-based method for detecting proteins by one-or two-dimensional SDS-PAGE (1-DE or 2-DE). Based on the SYPRO Ruby dye system, the combined two-dimensional fibrin zymography (2-D FZ) with SYPRO Ruby staining was newly developed to identify the Bacillus sp. proteases. Namely, complex protein mixtures from Bacillus sp. DJ-4, which were screened from Doen-Jang (Korean traditional fermented food), showed activity on the zymogram gel. The gel spots on the SYPRO Ruby gel, which corresponded to the active spots showing on the 2-D FZ gel, were analyzed by a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis. Five intracellular fibrinolytic enzymes of Bacillus sp. DJ-4 were detected through 2-D FZ. The gel spots on the SYPRO Ruby dye stained 2-D gel corresponding to 2-D FZ were then analyzed by MALID-TOF MS. Three of the five gel spots proved to be quite similar to the ATP-dependent protease, extracellular neutral metalloprotease, and protease of Bacillus subtilis. Also, the extracellular proteases of Bacillus sp. DJ-4 employing this combined system were identified on three gels (e.g., casein, fibrin, and gelatin) and the proteolytic maps were established. This combined system of 2-D zymography and SYPRO Ruby dye should be useful for searching the specific protease from complex protein mixtures of many other sources (e.g., yeast and cancer cell lines).
Subject(s)
Biological Assay/methods , Europium/metabolism , Fibrin/metabolism , Fluorescent Dyes/metabolism , Nanotechnology , Proteomics/methods , Animals , Bacillus/chemistry , Bacillus/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cattle , Endopeptidases/chemistry , Endopeptidases/metabolism , Isoelectric Focusing , Molecular Sequence Data , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Based on the zymography analysis, Bacillus sp. DJ-4 (screened from Doen-Jang, a Korean traditional fermented food) secretes seven extracellular fibrinolytic enzymes (EFEs; 68, 64, 55, 45, 33, 27, and 13 kDa) in culture broth. These seven EFEs were analyzed by newly applied SDSfibrin zymography combined with gradient polyacrylamide (SDS-FZGP). This improved gel system was used with a 5-20% acrylamide gradient in a fibrin zymogram gel for the separation of proteins with molecular masses from below 10 kDa to over 100 kDa on one gel plate. Using this system, high molecular weight bands (HMWBs) were clearly and sharply resolved. We also examined the relationship between an acrylamide concentration and the enzymatic activity of EFE using densitometric analysis.
