Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A study of equilibrium binding of link protein to hyaluronate.

ABSTRACT: Link protein was extracted from bovine femoral-head cartilage, radiolabelled while in the proteoglycan-aggregate stage, and then purified by density-gradient centrifugation and gel chromatography. The purity of the preparation was assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and two species with approx. mol.wts. 45000 and 48000 were observed. Sedimentation-velocity experiments were performed in 0.5 M-guanidinium chloride/5 mM-phosphate, pH 7.4, and yielded an SO20, w of 4.75S. The proportion of link protein unable to interact with hyaluronate was determined by chromatography on Sepharose CL-4B. The binding of link protein to high-molecular-weight hyaluronate was studied by frontal-gel chromatography on Sepharose CL-4B in 0.5 M-guanidinium chloride/5 mM-phosphate/0.1% bovine serum albumin, pH 7.4. Experiments were performed at 10, 17 and 25 degrees C and the results were treated as described by Scatchard [(1949) Ann. N.Y. Acad. Sci. 51, 660-672]. Dissociation constants of approx. (1-4) X 10(-8) M were obtained. The length of hyaluronate occupied per link-protein molecule was determined to be six to seven disaccharides.

SUBMITTER: Lyon M

PROVIDER: S-EPMC1152146 | biostudies-other | 1983 Aug

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

A comparative study of the binding of cartilage link protein and the hyaluronate-binding region of the cartilage proteoglycan to hyaluronate-substituted Sepharose gel.

Project description:The hyaluronate-binding proteins from bovine nasal cartilage, i.e. the hyaluronate-binding region of the proteoglycan and the link protein, were labelled with 125I and separated from each other by gel chromatography. The proteins were characterized by molecular-weight determinations and their purity was established by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and immunodiffusion. The binding properties of the two proteins by hyaluronate-substituted Sepharose gel were compared. It was found that both proteins behaved similarly. They bound with the same efficiency to the gel, they showed the same time course of binding, had slightly different pH optima for binding and both proteins had a decreasing affinity for the gel with increasing ionic strength. The binding to the gel could be inhibited by soluble hyaluronate, and the minimum size of a hyaluronate oligosaccharide required for inhibition was in both cases a decasaccharide (only even-numbered oligosaccharides were tested). The proteins did not show any co-operative binding in the system tested, which could be explained by the large number of binding sites in the hyaluronate-substituted gel. Binding constants for the protein-hyaluronate interaction were estimated. A value of 1.3 x 10(7) M-1 was obtained for the hyaluronate-binding region of the proteoglycan, in agreement with literature data. The corresponding value for the link protein was 0.7 x 10(7) M-1.

| S-EPMC1163372 | biostudies-other

Equilibrium-binding studies of pig laryngeal cartilage proteoglycans with hyaluronate oligosaccharide fractions.

Project description:The binding of hyaluronate oligosaccharide fractions to proteoglycans from pig laryngeal cartilage has been studied by equilibrium dialysis in dilute solution. It has been shown that: (1) each proteoglycan monomer binds only one hyaluronate oligosaccharide molecule [containing about eighteen saccharide residues (HA approximately 18) and of number-average molecule weight (Mn) 37501]; (2) the dissociation constant, Kd, for interaction between proteoglycan monomer and oligosaccharide HA approximately 18 is 3 x 10(-8) M at 6 degrees C at I 0.15-0.5, pH 7.4; (3) the dissociation constant has little dependence on temperature, so that Kd at 54 degrees C is 3 x 10(-7) M under the same conditions; (4) the aggregatability is high at 6 degrees C, falls significantly at 54 degrees C, but much of it can be recovered on cooling to 6 degrees C again, demonstrating reversible denaturation; (5) a method for determining the proportion of the proteoglycan molecules capable of binding to hyaluronate by equilibrium dialysis was compared with gel-chromatographic and ultracentrifugal methods; (6) a hyaluronate oligosaccharide, HA approximately 56 (Mn 11 000), could bind more than one proteoglycan molecule; (7) consideration of ultracentrifugal data shows that when proteoglycans bind to a hyaluronate of larger size (mol..wt. 670 000), an average Kd of 12 x 10(7) M fits the data in 0.5 M-guanidine hydrochloride at 20 degrees C.

| S-EPMC1161275 | biostudies-other

Articular cartilage cultured with interleukin 1. Increased release of link protein, hyaluronate-binding region and other proteoglycan fragments.

Project description:Pig articular cartilage was maintained in culture for 3 days with and without porcine interleukin 1. The proteoglycans remaining in the cartilage and those released into the medium were analysed by using radioimmunoassays for the hyaluronate-binding region, link protein and keratan sulphate. In interleukin 1-treated cultures after 3 days there was 38% release of total glycosaminoglycans into the medium, 18% release of binding region, 14% release of link protein and 20% release of keratan sulphate epitope, whereas in control cultures the proportions released were much less (16, 9, 10 and 7% respectively). Characterization of the proteoglycans in the media after 1.5 days and 3 days of culture showed that interleukin 1 promoted the release of proteoglycan of large average size and also the release of link protein and of low-Mr binding region which was unattached to proteoglycan. Both the link protein and binding region released were able to bind to exogenously added hyaluronate, whereas the proteoglycan in the medium was not. The proteoglycans extracted from cultured cartilage were similar to those from fresh cartilage: they contained a high proportion of aggregating proteoglycans and some low-Mr binding region. The proportion of this binding region extracted from the interleukin 1-treated cartilage was increased. The presence of interleukin 1 in the cultures therefore appeared to increase the rate of proteolytic degradation of proteoglycan in the matrix and to lead to a more rapid loss of intact binding region, of link protein and of large proteoglycan fragments into the medium.

| S-EPMC1147171 | biostudies-other

Cartilage proteoglycans. Assembly with hyaluronate and link protein as studied by electron microscopy.

Project description:Aggregates formed by the interaction of cartilage proteoglycan monomers and fragments thereof with hyaluronate were studied by electron microscopy by use of rotary shadowing [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333]. The differences in shape and packing of the proteins bound along the hyaluronate strand in aggregates formed in the presence and in the absence of link protein were examined in detail. The high resolution of the method allowed examination of the involvement in hyaluronate binding of the globular core-protein domains G1, G2 and G3 [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333; Paulsson, Mörgelin, Wiedemann, Beardmore-Gray, Dunham, Hardingham, Heinegård, Timpl & Engel (1987) Biochem. J. 245, 763-772]. Fragments comprising the globular hyaluronate-binding region G1 form complexes with hyaluronate with an appearance of necklace-like structures, statistically interspaced by free hyaluronate strands. The closest centre-to-centre distance found between adjacent G1 domains was 12 nm. Another fragment comprising the binding region G1 and the adjacent second globular domain G2 attaches to hyaluronate only by one globule. Also, the core protein obtained by chondroitinase digestion of proteoglycan monomer binds only by domain G1, with domain G3 furthest removed from the hyaluronate. Globule G1 shows a statistical distribution along the hyaluronate strands. In contrast, when link protein is added, binding is no longer random, but instead uninterrupted densely packed aggregates are formed.

| S-EPMC1149272 | biostudies-other

Non-equilibrium repressor binding kinetics link DNA damage dose to transcriptional timing within the SOS gene network.

Project description:Biochemical pathways are often genetically encoded as simple transcription regulation networks, where one transcription factor regulates the expression of multiple genes in a pathway. The relative timing of each promoter's activation and shut-off within the network can impact physiology. In the DNA damage repair pathway (known as the SOS response) of Escherichia coli, approximately 40 genes are regulated by the LexA repressor. After a DNA damaging event, LexA degradation triggers SOS gene transcription, which is temporally separated into subsets of 'early', 'middle', and 'late' genes. Although this feature plays an important role in regulating the SOS response, both the range of this separation and its underlying mechanism are not experimentally defined. Here we show that, at low doses of DNA damage, the timing of promoter activities is not separated. Instead, timing differences only emerge at higher levels of DNA damage and increase as a function of DNA damage dose. To understand mechanism, we derived a series of synthetic SOS gene promoters which vary in LexA-operator binding kinetics, but are otherwise identical, and then studied their activity over a large dose-range of DNA damage. In distinction to established models based on rapid equilibrium assumptions, the data best fit a kinetic model of repressor occupancy at promoters, where the drop in cellular LexA levels associated with higher doses of DNA damage leads to non-equilibrium binding kinetics of LexA at operators. Operators with slow LexA binding kinetics achieve their minimal occupancy state at later times than operators with fast binding kinetics, resulting in a time separation of peak promoter activity between genes. These data provide insight into this remarkable feature of the SOS pathway by demonstrating how a single transcription factor can be employed to control the relative timing of each gene's transcription as a function of stimulus dose.

| S-EPMC5999292 | biostudies-literature

Large scale relative protein ligand binding affinities using non-equilibrium alchemy.

Project description:Ligand binding affinity calculations based on molecular dynamics (MD) simulations and non-physical (alchemical) thermodynamic cycles have shown great promise for structure-based drug design. However, their broad uptake and impact is held back by the notoriously complex setup of the calculations. Only a few tools other than the free energy perturbation approach by Schrödinger Inc. (referred to as FEP+) currently enable end-to-end application. Here, we present for the first time an approach based on the open-source software pmx that allows to easily set up and run alchemical calculations for diverse sets of small molecules using the GROMACS MD engine. The method relies on theoretically rigorous non-equilibrium thermodynamic integration (TI) foundations, and its flexibility allows calculations with multiple force fields. In this study, results from the Amber and Charmm force fields were combined to yield a consensus outcome performing on par with the commercial FEP+ approach. A large dataset of 482 perturbations from 13 different protein-ligand datasets led to an average unsigned error (AUE) of 3.64 ± 0.14 kJ mol-1, equivalent to Schrödinger's FEP+ AUE of 3.66 ± 0.14 kJ mol-1. For the first time, a setup is presented for overall high precision and high accuracy relative protein-ligand alchemical free energy calculations based on open-source software.

| S-EPMC8145179 | biostudies-literature

Glial hyaluronate-binding protein: a product of metalloproteinase digestion of versican?

Project description:Glial hyaluronate-binding protein (GHAP) is a 60 kDa glycoprotein with an amino acid sequence identical to that of the hyaluronate-binding region of versican, a large fibroblast aggregating proteoglycan found in the brain. Both GHAP and versican were identified by immunoblot in bovine brain extracts prepared only minutes after death. Human recombinant collagenase, stromelysin, mouse gelatinase and gelatinases isolated from human brain by affinity chromatography digest versican and give rise to a polypeptide with electrophoretic mobility identical to GHAP. Immunoblot analysis, peptide mapping and C-terminal amino acid sequencing indicate that the polypeptide generated by digestion with human brain gelatinases is identical to GHAP. We suggest that GHAP is a naturally occurring versican degradation product.

| S-EPMC1136273 | biostudies-other

Insights from free-energy calculations: protein conformational equilibrium, driving forces, and ligand-binding modes.

Project description:Accurate free-energy calculations provide mechanistic insights into molecular recognition and conformational equilibrium. In this work, we performed free-energy calculations to study the thermodynamic properties of different states of molecular systems in their equilibrium basin, and obtained accurate absolute binding free-energy calculations for protein-ligand binding using a newly developed M2 algorithm. We used a range of Asp-Phe-Gly (DFG)-in/out p38α mitogen-activated protein kinase inhibitors as our test cases. We also focused on the flexible DFG motif, which is closely connected to kinase activation and inhibitor binding. Our calculations explain the coexistence of DFG-in and DFG-out states of the loop and reveal different components (e.g., configurational entropy and enthalpy) that stabilize the apo p38α conformations. To study novel ligand-binding modes and the key driving forces behind them, we computed the absolute binding free energies of 30 p38α inhibitors, including analogs with unavailable experimental structures. The calculations revealed multiple stable, complex conformations and changes in p38α and inhibitor conformations, as well as balance in several energetic terms and configurational entropy loss. The results provide relevant physics that can aid in designing inhibitors and understanding protein conformational equilibrium. Our approach is fast for use with proteins that contain flexible regions for structure-based drug design.

| S-EPMC3400776 | biostudies-literature

The use of pseudo-equilibrium constant affords improved QSAR models of human plasma protein binding.

Project description:PURPOSE:To develop accurate in silico predictors of Plasma Protein Binding (PPB). METHODS:Experimental PPB data were compiled for over 1,200 compounds. Two endpoints have been considered: (1) fraction bound (%PPB); and (2) the logarithm of a pseudo binding constant (lnKa) derived from %PPB. The latter metric was employed because it reflects the PPB thermodynamics and the distribution of the transformed data is closer to normal. Quantitative Structure-Activity Relationship (QSAR) models were built with Dragon descriptors and three statistical methods. RESULTS:Five-fold external validation procedure resulted in models with the prediction accuracy (R²) of 0.67?±?0.04 and 0.66?±?0.04, respectively, and the mean absolute error (MAE) of 15.3?±?0.2% and 13.6?±?0.2%, respectively. Models were validated with two external datasets: 173 compounds from DrugBank, and 236 chemicals from the US EPA ToxCast project. Models built with lnKa were significantly more accurate (MAE of 6.2-10.7 %) than those built with %PPB (MAE of 11.9-17.6 %) for highly bound compounds both for the training and the external sets. CONCLUSIONS:The pseudo binding constant (lnKa) is more appropriate for characterizing PPB binding than conventional %PPB. Validated QSAR models developed herein can be applied as reliable tools in early drug development and in chemical risk assessment.

| S-EPMC4363124 | biostudies-literature

Oculopharyngeal muscular dystrophy mutations link the RNA-binding protein HNRNPQ to autophagosome biogenesis.

Project description:Autophagy is an intracellular degradative process with an important role in cellular homeostasis. Here, we show that the RNA binding protein (RBP), heterogeneous nuclear ribonucleoprotein Q (HNRNPQ)/SYNCRIP is required to stimulate early events in autophagosome biogenesis, in particular the induction of VPS34 kinase by ULK1-mediated beclin 1 phosphorylation. The RBPs HNRNPQ and poly(A) binding protein nuclear 1 (PABPN1) form a regulatory network that controls the turnover of distinct autophagy-related (ATG) proteins. We also show that oculopharyngeal muscular dystrophy (OPMD) mutations engender a switch from autophagosome stimulation to autophagosome inhibition by impairing PABPN1 and HNRNPQ control of the level of ULK1. The overexpression of HNRNPQ in OPMD patient-derived cells rescues the defective autophagy in these cells. Our data reveal a regulatory mechanism of autophagy induction that is compromised by PABPN1 disease mutations, and may thus further contribute to their deleterious effects.

| S-EPMC10577562 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data