Adsorbed soluble [3H]elastin as substrate for proteinase activity. A new microassay technique.
Ontology highlight
ABSTRACT:
SUBMITTER: Jensen HS
PROVIDER: S-EPMC1153386 | biostudies-other | 1984 Mar
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Bovine ligamentum nuchae elastin that had been partially digested by porcine pancreatic elastase and then thoroughly washed with 0.2 M-NaC1/0.05 M-sodium borate solution continued to release peptide fragments in solution. Complete removal of elastase from the elastin surface required extraction with dilute alkali, a procedure that does not irreversibly inactivate the enzyme or hydrolyse elastin.
| S-EPMC1172896 | biostudies-other
Project description:The isolation of a salt-soluble homogeneous elastin from the aortas of lathyritic chicks by chromatography on DEAE-cellulose and salt precipitation is described. These new techniques, as well as some previously published by other workers, were evaluated with the help of antiserum raised in sheep against insoluble chick elastin. The purified elastin was very basic and behaved in a predictable manner in coacervation studies. The protein migrated in sodium dodecyl sulphate-polyacrylamide gels as a single band moving slightly faster than pyruvate kinase (mol.wt. 57000).
| S-EPMC1168111 | biostudies-other
Project description:Implantation S1 family serine proteinases (ISPs) are tryptases involved in embryo hatching and uterine implantation in the mouse. The two different ISP proteins (ISP1 and ISP2) have been detected in both pre- and post-implantation embryo tissue. To date, native ISP obtained from uterus and blastocyst tissues has been isolated only as an active hetero-dimer that exhibits trypsin-like substrate specificity. We hypothesised that in isolation, ISP1 might have a unique substrate specificity that could relate to its role when expressed alone in individual tissues. Thus, we isolated recombinant ISP1 expressed in Pichia pastoris and evaluated its substrate specificity. Using several chromogenic substrates and serine proteinase inhibitors, we demonstrate that ISP1 exhibits trypsin-like substrate specificity, having a preference for lysine over arginine at the P1 position. Phage display peptide mimetics revealed an expanded but mixed substrate specificity of ISP1, including chymotryptic and elastase activity. Based upon targets observed using phage display, we hypothesised that ISP1 might signal to cells by cleaving and activating proteinase-activated receptors (PARs) and therefore assessed PARs 1, 2 and 4 as potential ISP1 targets. We observed that ISP1 silenced enzyme-triggered PAR signaling by receptor-disarming. This PAR-disarming action of ISP1 may be important for embryo development and implantation.
| S-EPMC3223204 | biostudies-literature
Project description:The 3C-like proteinase of severe acute respiratory syndrome coronavirus (SARS) has been proposed to be a key target for structural based drug design against SARS. We have designed and synthesized 34 peptide substrates and determined their hydrolysis activities. The conserved core sequence of the native cleavage site is optimized for high hydrolysis activity. Residues at position P4, P3, and P3' are critical for substrate recognition and binding, and increment of beta-sheet conformation tendency is also helpful. A comparative molecular field analysis (CoMFA) model was constructed. Based on the mutation data and CoMFA model, a multiply mutated octapeptide S24 was designed for higher activity. The experimentally determined hydrolysis activity of S24 is the highest in all designed substrates and is close to that predicted by CoMFA. These results offer helpful information for the research on the mechanism of substrate recognition of coronavirus 3C-like proteinase.
| S-EPMC7092912 | biostudies-literature
Project description:Proteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site. On the basis of in silico site-saturation mutagenesis, we then generated KLK4S207A,L185D, a first-of-a-kind 'decoy' PAR1 inhibitor, by mutating the S207A and L185D residues in wild-type KLK4, which strongly binds to PAR1. KLK4S207A,L185D markedly inhibited PAR1 cleavage, and PAR1-mediated MAPK/ERK activation as well as the migration and invasiveness of melanoma cells. This 'substrate-capturing' KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 'proteinase substrate capture' approach to regulate proteinase-mediated PAR1 signaling.
| S-EPMC8352894 | biostudies-literature
Project description:Elastolysis is central to progression of emphysema and aortic aneurysms. Characterization of steady-state enzyme kinetics of elastolysis is fettered by the insolubility of mature elastin and the polydispersity of solubilized elastin. We prepared a fluor-tagged, 100-kDa fraction (fEln-100) from commercial ?-elastin. It is soluble, less heterogeneous in mass, cross-linked like mature elastin, and likely to retain the capacity of ?-elastin to self-assemble. fEln-100 has introduced the ability to compare quantitatively the apparent k(cat) and K(m) of elastases. For example, metalloelastase (MMP-12) displays higher apparent affinity for fEln-100, while MMP-2 displays faster catalytic turnover.
| S-EPMC2964420 | biostudies-literature
Project description:The interaction of hydrogen with platinum is enormously important in many areas of catalysis. The most significant of these are in polymer electrolyte membrane fuel cells (PEMFC), in which carbon-supported platinum is used to dissociate hydrogen gas at the anode. The nature of adsorbed hydrogen on platinum has been studied for many years on single-crystal surfaces, on high-surface area-platinum metal (Raney platinum and platinum black), and on supported catalysts. Many forms of vibrational spectroscopy have played a key role in these studies, however, there is still no clear consensus as to the assignment of the spectra. In this work, ab initio molecular dynamics (AIMD) and lattice dynamics were used to study a 1.1?nm nanoparticle, Pt44 H80 . The results were compared to new inelastic neutron scattering spectra of hydrogen on platinum black and of a carbon-supported platinum fuel cell catalyst and an assignment scheme that rationalises all previous data is proposed.
| S-EPMC6767033 | biostudies-literature
Project description:Moringa oleifera has been regarded as a food substance since ancient times and has also been used as a treatment for many diseases. Recently, various therapeutic effects of M. oleifera such as antimicrobial, anticancer, anti-inflammatory, antidiabetic, and antioxidant effects have been investigated; however, most of these studies described only simple biological phenomena and their chemical compositions. Due to the increasing attention on natural products, such as those from plants, and the advantages of oral administration of anticancer drugs, soluble extracts from M. oleifera leaves (MOL) have been prepared and their potential as new anticancer drug candidates has been assessed in this study. Here, the soluble cold Distilled Water extract (4°C; concentration, 300 µg/mL) from MOL greatly induced apoptosis, inhibited tumor cell growth, and lowered the level of internal reactive oxygen species (ROS) in human lung cancer cells as well as other several types of cancer cells, suggesting that the treatment of cancer cells with MOL significantly reduced cancer cell proliferation and invasion. Moreover, over 90% of the genes tested were unexpectedly downregulated more than 2-fold, while just below 1% of the genes were upregulated more than 2-fold in MOL extract-treated cells, when compared with nontreated cells. Since severe dose-dependent rRNA degradation was observed, the abnormal downregulation of numerous genes was considered to be attributable to abnormal RNA formation caused by treatment with MOL extracts. Additionally, the MOL extract showed greater cytotoxicity for tumor cells than for normal cells, strongly suggesting that it could potentially be an ideal anticancer therapeutic candidate specific to cancer cells. These results suggest the potential therapeutic implications of the soluble extract from MOL in the treatment of various types of cancers.
| S-EPMC3991666 | biostudies-literature
Project description:Sampling and extraction of chemical residues present on flat or curved surfaces as well as touch-sensitive objects are challenging. Hydrogels are characterized by high mechanical flexibility and water content. Thus, they are an ideal medium for transferring water-soluble analytes from a sampled surface to the next stage of an analytical workflow. Here, we demonstrate gel-phase microextraction (GPME), in which disks of blended hydrogels are utilized to lift traces of water-soluble substances adsorbed on surfaces. The protocol has been optimized in a series of tests involving fluorometric and mass spectrometric measurements. Compared with the pure agarose hydrogel, most of the tested blended hydrogels provide a higher efficiency for the sampling/extraction of a model analyte, fluorescein. The blended hydrogel disks are incorporated into three-dimensional (3D)-printed acrylonitrile-butadiene-styrene chips to create easy-to-use sampling probes. We exemplify the suitability of this improved GPME approach in sampling chemical residues present on the skin, glass, and daily use objects. In these tests, the extracts were analyzed on a triple quadrupole mass spectrometer fitted with an electrospray ion source operated in the positive- and negative-ion modes. The method enabled the detection of diclofenac on excised porcine skin fragments exposed to a topical nonsteroidal anti-inflammatory drug and sweat residues (lactic acid) left on surfaces touched by humans. The limits of detection for diclofenac and lactic acid in hydrogel extract were 6.4 × 10-6 and 2.1 × 10-5 M, respectively. In a model experiment, conducted using the presented approach, the amount of lactic acid on a glass slide with fingerprints was estimated to be ?1.4 × 10-7 mol cm-2.
| S-EPMC6868603 | biostudies-literature
Project description:BackgroundElastin-like polypeptides (ELPs) are a fascinating biomaterial that has undergone copious development for a variety of therapeutic applications including as a nanoscale drug delivery vehicle. A comprehensive understanding of ELP self-assembly is lacking and this knowledge gap impedes the advancement of ELP-based biomaterials into the clinical realm. The systematic examination of leucine-containing ELPs endeavors to expand existing knowledge about fundamental assembly-disassembly behaviours.ResultsIt was observed that these marginally soluble, short ELPs tend to behave consistently with previous observations related to assembly-related ELP phase transitions but deviated in their disassembly. It was found that chain length, concentration and overall sequence hydrophobicity may influence the irreversible formation of sub-micron particles as well as the formation of multi-micron scale, colloidally unstable aggregates. Amino acid composition affected surface charge and packing density of the particles. Particle stability upon dilution was found to vary depending upon chain length and hydrophobicity, with particles composed of longer and/or more hydrophobic ELPs being more resistant to disassembly upon isothermal dilution.ConclusionsTaken together, these results suggest marginally soluble ELPs may self-assemble but not disassemble as expected and that parameters including particle size, zeta potential and dilution resistance would benefit from widespread systematic evaluations. This information has the potential to reveal novel preparation methods capable of expanding the utility of all existing ELP-based biomaterials.
| S-EPMC5816514 | biostudies-literature