scholarly journals Molecular-size-dependent variations in the proportions of chains with high binding affinities for antithrombin in rat skin heparin proteoglycans

1989 ◽  
Vol 262 (3) ◽  
pp. 953-958
Author(s):  
A A Horner
Keyword(s):  
Binding Site ◽  
Gel Filtration ◽  
Molecular Size ◽  
Binding Affinities ◽  
Rat Skin ◽  
Size Dependent ◽  
High Affinity ◽  
High Binding ◽  
Site Density ◽  
High Degree

Approximately half of all rat skin heparin proteoglycans have polysaccharide chains that have no sites with high binding affinity for antithrombin. The rest have chains with high-affinity antithrombin-binding-site densities ranging from zero to five sites per chain, with a high degree of variation. Proteoglycans vary in size because of diversity in the number of chains per molecule; the relationship between proteoglycan size and high-affinity antithrombin-binding-site density has not been studied previously. Polydisperse heparin proteoglycans from rat skin, labelled biosynthetically with 35S, were fractionated by gel filtration on Bio-Gel A-150m and arbitrarily divided into five fractions of decreasing average molecular size. Fractionation of these products on antithrombin-agarose showed that the proportion of proteoglycans with high affinity for antithrombin decreased from 39% to 25% as molecular size decreased. However, as the molecular size of high-affinity proteoglycans decreased, the proportion of their chains that had high affinity increased from 29% to 59%. Therefore molecular size is a significant factor in determining the proportion of high-affinity chains in heparin proteoglycans. A model of heparin biosynthesis is proposed in which areas of specific enzyme activity that control the synthesis of the antithrombin-binding-site sequence are sparsely and nonrandomly distributed on mast-cell Golgi membranes. It is postulated that the likelihood of a developing proteoglycan encountering one of these hypothetical areas is molecular-size-dependent.

scholarly journals Determination of the range in binding-site densities of rat skin heparin chains with high binding affinities for antithrombin

1988 ◽  
Vol 251 (1) ◽  
pp. 141-145 ◽  
Author(s):  
A A Horner ◽  
M Kusche ◽  
U Lindahl ◽  
C B Peterson
Keyword(s):  
Chemical Analysis ◽  
Binding Site ◽  
Binding Affinity ◽  
Intrinsic Fluorescence ◽  
Binding Affinities ◽  
Rat Skin ◽  
High Binding ◽  
Site Density ◽  
High Binding Affinity

Rat skin heparin proteoglycans vary markedly in the proportions of their constituent polysaccharide chains that have high binding affinity for antithrombin. As the proportion of such chains in a proteoglycan rises, their degree of affinity for antithrombin also increases [Horner (1987) Biochem. J. 244, 693-698]. The antithrombin-binding-site densities of such chains have now been determined, by measuring heparin-induced enhancement of the intrinsic fluorescence of antithrombin and by chemical analysis for the disaccharide sequence glucuronosyl-N-sulphoglucosaminyl (3,6-di-O-sulphate), which is unique to this site in heparin [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555]. Antithrombin-binding-site density ranged from one to five sites per chain.

scholarly journals Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO

2019 ◽  
Vol 20 (6) ◽  
pp. 1444 ◽  
Author(s):  
Soria Iatmanen-Harbi ◽  
lucile Senicourt ◽  
Vassilios Papadopoulos ◽  
Olivier Lequin ◽  
Jean-Jacques Lacapere
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Changes ◽  
Protoporphyrin Ix ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Translocator Protein ◽  
Binding Affinities ◽  
Ligand Selectivity ◽  
High Affinity

The optimization of translocator protein (TSPO) ligands for Positron Emission Tomography as well as for the modulation of neurosteroids is a critical necessity for the development of TSPO-based diagnostics and therapeutics of neuropsychiatrics and neurodegenerative disorders. Structural hints on the interaction site and ligand binding mechanism are essential for the development of efficient TSPO ligands. Recently published atomic structures of recombinant mammalian and bacterial TSPO1, bound with either the high-affinity drug ligand PK 11195 or protoporphyrin IX, have revealed the membrane protein topology and the ligand binding pocket. The ligand is surrounded by amino acids from the five transmembrane helices as well as the cytosolic loops. However, the precise mechanism of ligand binding remains unknown. Previous biochemical studies had suggested that ligand selectivity and binding was governed by these loops. We performed site-directed mutagenesis to further test this hypothesis and measured the binding affinities. We show that aromatic residues (Y34 and F100) from the cytosolic loops contribute to PK 11195 access to its binding site. Limited proteolytic digestion, circular dichroism and solution two-dimensional (2-D) NMR using selective amino acid labelling provide information on the intramolecular flexibility and conformational changes in the TSPO structure upon PK 11195 binding. We also discuss the differences in the PK 11195 binding affinities and the primary structure between TSPO (TSPO1) and its paralogous gene product TSPO2.

Identification of high-affinity calmodulin-binding proteins in rat liver

1987 ◽  
Vol 252 (3) ◽  
pp. C277-C284 ◽  
Author(s):  
R. M. Hanley ◽  
J. R. Dedman ◽  
S. Shenolikar
Keyword(s):  
Rat Liver ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Apparent Molecular Weight ◽  
Convincing Evidence ◽  
Calmodulin Binding ◽  
High Affinity

The Ca2+-dependent binding of [125I]calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or “acceptor” proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised greater than 80% of the Ca2+-dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding “subunit” of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca2+ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver.

Flunitrazepam binding sites in rat diaphragm. Receptors for direct neuromuscular effects of benzodiazepines?

1982 ◽  
Vol 60 (7) ◽  
pp. 1003-1005 ◽  
Author(s):  
M. Wilkinson ◽  
Dale Grovestine ◽  
J. T. Hamilton
Keyword(s):  
Binding Site ◽  
Muscle Relaxant ◽  
Binding Sites ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
Rat Diaphragm ◽  
High Affinity Binding ◽  
High Affinity ◽  
Site Density ◽  
Peripheral Type

The evidence for direct muscle relaxant effects of benzodiazepines is controversial. We now show that a crude membrane preparation of rat diaphragm possesses binding sites for [3H]flunitrazepam (FNZ). Scatchard analysis gave a binding site density of 1689 ± 143 fmol/mg protein (Kd = 25.6 ± 2.6 nM). These sites are of the "peripheral" type since clonazepam fails to displace [3H]FNZ as effectively as R05-4864 (IC50 values: 7.5 × 10 6 M and 8 × 10−9 M, respectively). Diazepam is almost as effective as R05-4864 and potently displaces [3H]FNZ binding (IC50 = 3 × 10−8 M). We propose that the previously described effects of diazepam on rat diaphragm are mediated through high-affinity binding sites.

Stimulation of β-(l → 6)-glucanase production by oxidized pustulan

1972 ◽  
Vol 18 (10) ◽  
pp. 1543-1550 ◽  
Author(s):  
Robert G. Brown
Keyword(s):  
Enzyme Activity ◽  
Isoelectric Focusing ◽  
Gel Filtration ◽  
Tween 80 ◽  
Sole Source ◽  
Cellulase Production ◽  
Low Degree ◽  
Degree Of Oxidation ◽  
High Degree ◽  
Stimulation Of

A strain of Penicillium lilacinum, isolated from soil, produced pustulanase, β-(1 → 3)-glucanase, (EC. 3.2.1.6) and cellulase (EC.3.2.1.4) when cultivated on a medium containing pustulan as the sole source of carbon. If pustulan was replaced by ketopustulan, the production of pustulanase was stimulated about 10-fold although the amount of stimulation was dependent on the degree of oxidation of pustulan. β-(1 → 3)-Glucanase production was stimulated slightly by ketopustulan; however, the degree of oxidation did not affect significantly the yield of this enzyme. Cellulase production was either unaffected by the oxidized polymer, or at higher degrees of oxidation, decreased. Tween 80 stimulated the production of the three enzymes in media containing ketopustulan with a low degree of oxidation but was inhibitory to pustulanase and cellulase production in media containing ketopustulan with a high degree of oxidation. A combination of gel filtration and isoelectric focusing revealed that each enzyme activity was attributable to at least two proteins.

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