Endothelin Receptor-Effector Coupling and Signaling in Cardiac Cells
Malcolm J. Lewis, Ajay M. Shah in Endothelial Modulation of Cardiac Function, 2020
As a peptide, endothelin is also subject to proteolysis; indeed, proteolytic degradation may terminate ET’s actions. Using isolated organs, Rimar and Gillis (1992) demonstrated that clearance of ET by the heart is minimal (5% ± 2%) but that a single pass through the pulmonary circulation can remove 49 ± 4%. In some tissues, there may be relatively specific proteases that degrade endothelin: Deng and Jeng (1992) report partial purification from rat kidney of a cytosolic acid protease that cleaves the C-terminal tryptophan from endothelin, producing a foreshortened peptide that exhibits only one thousandth the vasoconstrictor activity of native ET-1. Kidney, spleen and liver have this enzyme; brain, heart and lung do not. Thiol protease inhibitors and phenylmethylsulfonylfluoride (PMSF) inhibit this activity. The absence of this enzyme from lung indicates that the avid extraction of ET by lung (Rimar and Gillis, 1992) is due to a different and as yet undefined mechanism. Neither the means by which ET gains access to this intracellular protease nor the mechanism of extraction of ET from extracellular fluids has been defined.
Using a Recombinant Metagenomic Lipase for Enantiomeric Separation of Pharmaceutically Important Drug Intermediates
Peter Grunwald in Pharmaceutical Biocatalysis, 2019
Since the active site has been proposed to contain a conserved serine, PMSF was used as an inhibitor. To our surprise, PMSF did not inhibit the LipR1 lipase activity (Table 3.3). We observed temperature dependent inhibition with PMSF. At 55°C, LipR1 protein loses its 40–50% activity even with 1 mM of PMSF. Lipases have been shown to remain active in presence of PMSF in some cases (Li and Zhang, 2005; Soliman et al., 2007). It is very likely that active site serine is somehow inaccessible to PMSF yet at higher temperature some conformational changes in the protein makes the catalytic serine accessible to the inhibitor. Contrary to this DEPC, a histidine modifier inhibited the enzyme activity at very low concentration suggesting the easy accessibility of the catalytic histidine residue to DEPC. Eserine, an esterase’s inhibitor did not affect the enzyme activity, which suggested that it is a true lipase.
Eukaryotic Dna-Dependent Rna Polymerases: An Evaluation of Their Role in the Regulation of Gene Expression
Gerald M. Kolodny in Eukaryotic Gene Regulation, 2018
6. In attempting to ascribe mechanisms to polymerase-mediated alterations of transcription, several groups have detected changes in subunit sizes or composition of the eukaryotic enzymes. It is necessary to sound a note of caution here; in addition to the general difficulty of ascertaining functional requirements of individual subunits,29 the problems of proteolytic conversion during extraction (perhaps arising through changes in protease activities during regulatory sequences) warrant continued attention. Coupar et al.41 have shown that the low-salt extraction method for isolating rat liver polymerase I results in the proteolytic degradation of the heavy subunit (from 195K to 175K daltons). A similar situation may be responsible for the microheterogeneity of form II RNA polymerase. Careful use of the serine protease inhibitor PMSF results in the isolation of a single enzyme II from yeast; a protease can be extracted which selectively reduces the size of the heavy subunit to create the diversity usually observed.46 Of course, if changes in the subunit size do have a regulatory function, there would clearly have to be an enzyme to bring about the change, and the demonstration of proteolytic effects may be interpreted as a fruitful area of research rather than just an in vitro artifact.
Exploring the mechanisms of action of Zengye decoction (ZYD) against Sjogren’s syndrome (SS) using network pharmacology and animal experiment
Published in Pharmaceutical Biology, 2023
Jiake Yu, Shuying Wang, Jie Yang, Wuxinrui Huang, Beikang Tang, Weijun Peng, Jing Tian
TNF-α, IL-1β, IL-6, and IL-17 enzyme-linked immunosorbent assay (ELISA) kits was purchased from Elabscience, China. Phenylmethylsulfonyl fluoride (PMSF) was purchased from Aladdin, China. A phosphatase inhibitor, radioimmunoprecipitation assay (RIPA) lysis buffer, and bicinchoninic acid (BCA) protein assay kit were ordered from Beyotime Biotechnology, China. Antibodies, including GAPDH, Bax, Bcl-2, Caspase-3 (CASP3), HRP-labeled rabbit anti-sheep IgG, and HRP-labeled rabbit anti-rats IgG, were purchased from Wuhan Sanyin Biotechnology Co., Ltd., China, and Wuhan Doctor De Biological Engineering Co., Ltd., China. An ECL substrate fluid was obtained from Beijing Plilai Gene Technology Co., Ltd., China. An X-ray photograph was purchased from Ruike Medical Equipment Co., Ltd., China. A developing and fixing kit was purchased from Tianjin Hanzhong Photography Materials Factory, China.
m-Calpain is released from striatal synaptosomes
Published in International Journal of Neuroscience, 2023
Nina Pestereva, Irina Ivleva, Alexander Zubov, Maria Tikhomirova, Marina Karpenko
Rat striatal synaptosomes were prepared on discontinuous Percoll gradients [9]. In brief, striatum from five rats was carefully dissected, homogenized in ice-cold 320 mM sucrose, 0.5 mM EDTA and 20 mM Tris–HCl (pH 7.4), and centrifuged for 5 min at 1,000 g, and the resulting supernatants were spun for 30 min at 15,000 g. The resulting pellet was gently resuspended in the same solution, layered onto a three-step gradient composed of 3, 10 and 23% Percoll, and centrifuged at 25,000 g for 20 min. The interface between the 10 and 23% layers containing the synaptosomal-enriched fraction was removed, added to 10 volumes of calcium-free Krebs–Ringer buffer containing 124 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 1.2 mM NaH2PO4, 26 mM NaHCO3, 10 mM D(+)-glucose and 20 mM Hepes-NaOH at a pH of 7.4, and saturated with O2/CO2 mixture (95:5). The final synaptosomal pellet was resuspended in a Krebs–Ringer buffer at a total protein concentration of 1 mg/ml. Aliquots of synaptosomes were incubated at 37 °C for the indicated time in the presence of different concentrations of CaCl2 or EDTA. After incubation, the reaction tubes were centrifuged at 4 °C for 1 min at 15,000 g. PMSF (final concentration, 1 mM) was added to the supernatant, which was additionally centrifuged for 5 min at 15,000 g to remove tissue remains.
Misrouting of glucagon and stathmin-2 towards lysosomal system of α-cells in glucagon hypersecretion of diabetes
Published in Islets, 2022
Farzad Asadi, Savita Dhanvantari
αTC1-6 cells were incubated for 24 h in serum-free medium containing 16.7 mM glucose and 0.1% BSA. Cells were then washed twice with HBSS containing 16.7 mM glucose, HEPES, and 0.5% BSA and pre-incubated for 30 min in this medium. For lysosomal inhibition, cells were treated with 10 nM Bafilomycin A1 (Cat# B1793, Sigma) for 2 h. Secretion studies were done by addition of 55 mM KCl for 20 min as we have done previously18 in the presence of Bafilomycin A1. Media were then collected into microfuge tubes containing PMSF (45 mM) and Aprotinin (5 µg/mL), centrifuged at 14000 × g for 15 minutes at 4°C, and then stored at -80°C until analysis. Cells were washed in ice-cold HBSS, lysed in ice-cold lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100 plus 45 mM PMSF, and 5 μg/mL Aprotinin), and centrifuged at 14000 × g for 15 minutes at 4°C. The supernatant was collected and stored at -80°C until analysis. Cell protein level was measured by BCA, as mentioned above, and used for normalization of glucagon secretion.
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