scholarly journals Identification of a heparin-releasable hepatic lipase binding protein from rat liver

1998 ◽  
Vol 330 (2) ◽  
pp. 785-789 ◽  
Author(s):  
Belinda BREEDVELD ◽  
Kees SCHOONDERWOERD ◽  
Hans JANSEN
Keyword(s):  
Rat Liver ◽  
Binding Assay ◽  
Solid Phase ◽  
Binding Capacity ◽  
Specific Binding ◽  
Hepatic Lipase ◽  
Binding Activity ◽  
Cross Linking ◽  
Major Protein ◽  
Slot Blot

Hepatic lipase (HL) plays a key role in the metabolism of several lipoproteins. Metabolically active HL is bound in liver parenchymal cells to specific binding sites. We studied the nature of the HL binding in rat liver. Rat livers were perfused with heparin, which lead to a loss of 80% of the HL binding capacity of the liver. The heparin-containing perfusates possessed HL binding capacity, determined by slot-blot assay. The perfusates were loaded on to a heparin-Sepharose column and eluted with a linear salt gradient (0.2-1 M). HL binding activity, assessed by a slot-blot binding assay, eluted both at 0.3 M and at 0.8 M NaCl. A 0.5 M NaCl eluate was used to further characterize the HL binding activity. In this fraction the major protein had a molecular mass of 70 kDa. The fraction showed saturable HL binding in a solid-phase binding assay. Cross-linking of the 0.5 M NaCl fraction to 125I-labelled HL yielded a complex of 130 kDa, suggesting the cross-linking of the 57 kDa 125I-labelled HL to a protein of about 73 kDa. We concluded that heparin releases a protein of about 73 kDa from rat liver, which associates with HL. This protein may represent the HL binding site in liver.

Characterization of the binding of 3,3′-di-iodo-l-thyronine to rat liver mitochondria

1997 ◽  
Vol 154 (1) ◽  
pp. 119-124
Author(s):  
A Lombardi ◽  
M Moreno ◽  
C Horst ◽  
F Goglia ◽  
A Lanni
Keyword(s):  
Rat Liver ◽  
Binding Capacity ◽  
Specific Binding ◽  
Local Environment ◽  
Liver Mitochondria ◽  
Ion Concentration ◽  
Affinity Constant ◽  
Rat Liver Mitochondria ◽  
Inner Mitochondrial Membrane ◽  
Thyroid State

Abstract The binding of labelled 3,3′-di-iodo-l-thyronine (3,3′-T2) to isolated rat liver mitochondria has been characterized. Specific binding could be detected only in the inner mitochondrial membrane, not in other mitochondrial subfractions. The composition of the incubation medium influenced the binding capacity, the best combination of high specific binding and low non-specific binding being observed in phosphate buffer, pH 6·4. The specific binding of 3,3′-T2 to mitochondria requires low ionic strength: concentrations of K+ and Na+ higher than 10 mmol/l and 0·1 mmol/l respectively resulted in a decreased binding capacity. The optimal calcium ion concentration was in the range 0·01–1·0 mmol/l. Varying magnesium ion, over the range of concentrations used (0·1–100 mmol/l), had no effect. Both ADP and ATP, at over 1 mmol/l, resulted in an inhibition of the specific binding. Incubation with protease resulted in a decrease in specific binding and an increase in non-specific binding, thus indicating the proteic nature of the binding sites. In addition to the above factors in the local environment the thyroid state of the animal might influence the 3,3′-T2-binding capacity. In fact, the thyroid state of the animal seemed not to have an influence on the affinity constant, but it did affect binding capacity. Journal of Endocrinology (1997) 154, 119–124

scholarly journals Hepatic lipase is localized at the parenchymal cell microvilli in rat liver

1997 ◽  
Vol 321 (2) ◽  
pp. 425-430 ◽  
Author(s):  
Belinda BREEDVELD ◽  
Kees SCHOONDERWOERD ◽  
Adrie J. M. VERHOEVEN ◽  
Rob WILLEMSEN ◽  
Hans JANSEN
Keyword(s):  
Endothelial Cells ◽  
Rat Liver ◽  
Kupffer Cells ◽  
Binding Capacity ◽  
Hepatic Lipase ◽  
Parenchymal Cell ◽  
Liver Cells ◽  
Minor Amount ◽  
Parenchymal Liver ◽  
Parenchymal Cells

Hepatic lipase (HL) is thought to be located at the vascular endothelium in the liver. However, it has also been implicated in the binding and internalization of chylomicron remnants in the parenchymal cells. In view of this apparent discrepancy between localization and function, we re-investigated the localization of HL in rat liver using biochemical and immunohistochemical techniques. The binding of HL to endothelial cells was studied in primary cultures of rat liver endothelial cells. Endothelial cells bound HL in a saturable manner with high affinity. However, the binding capacity accounted for at most 1% of the total HL activity present in the whole liver. These results contrasted with earlier studies, in which non-parenchymal cell (NPC) preparations had been found to bind HL with a high capacity. To study HL binding to the different components of the NPC preparations, we separated endothelial cells, Kupffer cells and blebs by counterflow elutriation. Kupffer cells and endothelial cells showed a relatively low HL-binding capacity. In contrast, the blebs, representing parenchymal-cell-derived material, had a high HL-binding capacity (33 m-units/mg of protein) and accounted for more than 80% of the total HL binding in the NPC preparation. In contrast with endothelial and Kupffer cells, the HL-binding capacity of parenchymal cells could account for almost all the HL activity found in the whole liver. These data strongly suggest that HL binding occurs at parenchymal liver cells. To confirm this conclusion in situ, we studied HL localization by immunocytochemical techniques. Using immunofluorescence, we confirmed the sinusoidal localization of HL. Immunoelectron microscopy demonstrated that virtually all HL was located at the microvilli of parenchymal liver cells, with a minor amount at the endothelium. We conclude that, in rat liver, HL is localized at the microvilli of parenchymal cells.

Inactivation of the β-adrenergic receptor in cardiac muscle by dithiols

1985 ◽  
Vol 63 (8) ◽  
pp. 932-936 ◽  
Author(s):  
Trevor I. Prior ◽  
Vandana Patel ◽  
G. I. Drummond
Keyword(s):  
Adrenergic Receptor ◽  
Reduced Glutathione ◽  
Binding Capacity ◽  
Specific Binding ◽  
Binding Activity ◽  
Ventricular Muscle ◽  
Adrenergic Antagonist ◽  
Equilibrium Dissociation ◽  
Rabbit Ventricular Muscle ◽  
Membrane Binding Sites

The effect of sulfhydryl reagents on binding of the β-adrenergic antagonist (−)-[3H]dihydroalprenolol hydrochloride ((−)-[3H]DHA) to a microsomal fraction of rabbit ventricular muscle was studied. Incubation with the disulfide reducing agents dithiothreitol (DTT), 2-mercaptocthanol, and reduced glutathione resulted in loss of (−)-[3H]DHA binding. At 500 μM DTT, less than 50% of specific binding activity remained; at 100 mM, binding was completely eliminated. 2-Mercaptoethanol and reduced glutathione were less effective than DTT at inhibiting binding activity. The total binding capacity (Bmax) decreased from 155.4 fmol mg−1 of protein, in the absence of DTT, to 92.4 and 77.5 fmol mg−1 at 0.25 and 0.7 mM DTT, respectively. The equilibrium dissociation constant (KD) increased from 7.6 nM, in the absence of DTT, to 10.3 nM at 0.25 mM DTT and to 20.8 nM at 0.7 mM DTT. Thus, DTT-induced decline in (−)-[3H]DHA binding results from a decrease in both the number and affinity of membrane binding sites for the tracer. Receptors could be protected from DTT inactivation by preincubation with β-adrenergic ligands. Oxidants could not reverse inactivation, with the exception of o-iodosobenzoate which was only partially effective. Thus, the β-adrenergic receptor of rabbit ventricular muscle contains essential disulfide moietie(s) which can be inactivated by reducing thiols.

The Effect of Human Serum on the Binding Activity of Radiolabelled Monoclonal Antibodies

1987 ◽  
Vol 73 (6) ◽  
pp. 547-554
Author(s):  
Silvia Camagni ◽  
Silvana Canevari ◽  
Marina Ripamonti ◽  
Delia Mezzanzanica ◽  
Rosaria Orlandi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Blood ◽  
Solid Phase ◽  
Binding Capacity ◽  
Binding Activity ◽  
Ovarian Carcinomas ◽  
Radiolabelled Monoclonal Antibodies ◽  
In Vitro Stability ◽  
Murine Monoclonal Antibodies

Three murine monoclonal antibodies (MoAbs), MBrl and MOv2 of IgM isotype and MOv8 of IgG isotype, with restricted reactivity for breast or ovarian carcinomas, were labelled with 125I in the perspective of obtaining specific and stable radioimmunopharmaceutical reagents. The radiolabeled MoAbs were analyzed for their « in vitro » stability in human blood. They were incubated at 37 °C for various lengths of time in human or, as a control, in murine blood and their binding capacity was evaluated by solid-phase RIA and compared with that obtained after incubation with buffer. In human blood, serum and plasma, but not with other components such as erythrocytes, leukocytes, HSA and IgG, the MoAbs revealed a loss of binding reactivity which was marked and constant for the IgM MoAbs, and only occasional for the IgG MoAb. In murine serum the decrease was not so rapid. The same change in the binding capacity was observed when the MoAbs were labelled with 3H or 35S, excluding the involvement of dehalogenating mechanisms. In the perspective of using MoAbs for intracavity therapy the effect of ascitic or pleural fluids on their binding activity was also evaluated. The inhibition of the binding reactivity was not as evident and was not related to the MoAb isotype.

The galactose-specific receptor system in rat liver during development

Development ◽  
1987 ◽  
Vol 100 (1) ◽  
pp. 13-22
Author(s):  
L. Dini ◽  
L. Conti-Devirgiliis ◽  
S. Russo-Caia
Keyword(s):  
Endothelial Cells ◽  
Ligand Binding ◽  
Rat Liver ◽  
Binding Capacity ◽  
Specific Binding ◽  
Cell Types ◽  
Receptor System ◽  
In Situ Experiments

The number and distribution of galactose-specific binding sites were investigated in rat liver cells during perinatal development. Ligand binding to hepatocytes, macrophages and endothelial cells was followed with in vitro and in situ experiments by electron microscopy, using lactosylated bovine serum albumin adsorbed onto 5 nm colloidal gold particles as ligand. Binding capacity, starting at a late stage of fetal development, is very low both on the hepatocyte and on the macrophage surface, which show single particles statistically distributed. By contrast, bound particles are absent from fetal endothelial cells, which also lack the typical coated regions. In vivo, experiments at 37 degrees C show that endocytosis occurs to some extent in prenatal life. These results indicate that the expression of galactose-specific receptors' activity on the different liver cell types follows different developmental patterns, which are independently modulated.

scholarly journals Studies on membrane proteins involved in ribosome binding on the rough endoplasmic reticulum. Ribophorins have no ribosome-binding activity

1987 ◽  
Vol 245 (3) ◽  
pp. 811-819 ◽  
Author(s):  
H Yoshida ◽  
N Tondokoro ◽  
Y Asano ◽  
K Mizusawa ◽  
R Yamagishi ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Rat Liver ◽  
Concanavalin A ◽  
Protein Fraction ◽  
Proteolytic Enzymes ◽  
Binding Capacity ◽  
Liver Microsomes ◽  
Binding Activity ◽  
Rat Liver Microsomes ◽  
Ribosome Binding

A membrane protein fraction showing affinity for ribosomes was isolated from rat liver microsomes (microsomal fractions) in association with ribosomes by treatment of the microsomes with Emulgen 913 and then solubilized from the ribosomes with sodium deoxycholate. This protein fraction was separated into two fractions, glycoproteins, including ribophorins I and II, and non-glycoproteins, virtually free from ribophorins I and II, on concanavalin A-Sepharose columns. The two fractions were each reconstituted into liposomes to determine their ribosome-binding activities. The specific binding activity of the non-glycoprotein fraction was approx. 2.3-fold higher than that of the glycoprotein fraction. The recovery of ribosome-binding capacity of the two fractions was about 85% of the total binding capacity of the material applied to a concanavalin A-Sepharose column, and about 90% of it was found in the non-glycoprotein fraction. The affinity constants of the ribosomes for the reconstituted liposomes were somewhat higher than those for stripped rough microsomes. The mode of ribosome binding to the reconstituted liposomes was very similar to that to the stripped rough microsomes, in its sensitivity to proteolytic enzymes and its strong inhibition by increasing KCl concentration. These results support the idea that ribosome binding to rat liver microsomes is not directly mediated by ribophorins I and II, but that another unidentified membrane protein(s) plays a role in ribosome binding.

A specific binding moiety for 1,25-dihydroxyvitamin D3 in basal lateral membranes of carp enterocytes

2000 ◽  
Vol 279 (3) ◽  
pp. E614-E621 ◽  
Author(s):  
Ilka Nemere ◽  
Dennis Larsson ◽  
Kristina Sundell
Keyword(s):  
Vitamin D ◽  
Gadus Morhua ◽  
Atlantic Cod ◽  
Binding Capacity ◽  
Specific Binding ◽  
Signal Transduction Pathway ◽  
Binding Activity ◽  
Protein Receptor ◽  
High Calcium ◽  
Plasma Membrane Receptor

Carp (Cyprinus carpio), a freshwater fish that lives in a low-calcium environment, and Atlantic cod (Gadus morhua), a seawater fish that lives in a high-calcium environment, were studied for the presence of a novel membrane binding protein (“receptor”) for the vitamin D metabolite, 1,25-dihydroxyvitamin D3[1,25(OH)2D3]. Basal lateral membranes from enterocytes of either species were prepared and analyzed for specific saturable binding. Membranes from carp revealed a dissociation constant of 1.23 nM with a maximal binding capacity of 212 fmol/mg protein. In comparison, membranes from Atlantic cod enterocytes revealed very low and nonsignificant levels of specific binding. The [3H]1,25(OH)2D3 binding activity in carp was further characterized for protein dependence, detergent extractability, and competition for binding with the metabolites 25(OH)D3 and 24 R,25(OH)2D3. Finally, introduction of 1,25(OH)2D3 to isolated carp enterocytes enhanced protein kinase C activity within 5 min, whereas intracellular Ca2+ concentrations were unaffected by a range of 1,25(OH)2D3 concentrations, as judged by fura 2 fluorescence. Thus the binding moiety may be a putative plasma membrane receptor for vitamin D, because it is functionally coupled to at least one signal transduction pathway.

scholarly journals Human somatotropin binding to rabbit kidney microsomal fraction

1981 ◽  
Vol 200 (2) ◽  
pp. 257-264 ◽  
Author(s):  
L P Roguin ◽  
S H Sánchez ◽  
J S Bonifacino ◽  
A C Paladini
Keyword(s):  
Binding Capacity ◽  
Specific Binding ◽  
Binding Activity ◽  
Rabbit Kidney ◽  
Scatchard Analysis ◽  
Temperature Dependent ◽  
Binding Reaction ◽  
Dissociation Equilibrium ◽  
Microsomal Membranes ◽  
Kidney Microsomes

Specific binding of 125I-labelled human somatotropin was demonstrated in microsomal membranes (microsomes) from rat and rabbit kidneys. Female rabbit kidney microsomes showed the highest binding activity and were used for further study. The association of 125I-labelled human somatotropin was time- and temperature-dependent and the binding reaction was reversible. Scatchard analysis of saturation data indicated a dissociation equilibrium constant, KD, of 56 pM and a binding capacity of 37 fmol per mg of protein. Similar results were obtained from competition experiments. Binding of 125I-labelled human somatotropin to the microsomes was specifically inhibited by hormones with lactogenic activity. The binding sites, as well as 125I-labelled human somatotropin, were not inactivated on incubation. Treatment of the microsomes with trypsin and chymotrypsin decreased the specific binding by over 90%. Preheating of the microsomes at 55 degrees C for 15 min abolished 50% of the specific binding activity.

Development of a Homogeneous High Throughput Fluorescence Polarization Assay for G Protein-Coupled Receptor Binding

2000 ◽  
Vol 5 (6) ◽  
pp. 415-419 ◽  
Author(s):  
Paul H. Lee ◽  
Debra J. Bevis
Keyword(s):  
G Protein ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
Binding Assay ◽  
Solid Phase ◽  
Binding Activity ◽  
G Protein Coupled Receptor ◽  
Separation Step ◽  
Fluorescence Polarization Assay ◽  
G Protein Coupled

Traditional methods that follow receptor ligand interactions are competitive assays in which the test compound displaces a radiolabeled molecule. These assays require either a time-consuming step for separation of free ligands from bound ligands or immobilization of receptors and the scintillant on a solid-phase support. In this report, we describe the development of a homogeneous binding assay for a G protein-coupled receptor in the fluorescence polarization format. This homogeneous fluorescence polarization binding assay format is superior to the traditional binding methods because no radioisotope, separation step, or solid-phase support is required. The elimination of the separation step enhances detection of low-affinity ligands and enables a real-time, continuous readout of the binding activity in a high throughput 384-well microplate format.

scholarly journals Binding of Aβ to α- and β-synucleins: identification of segments in α-synuclein/NAC precursor that bind Aβ and NAC

1997 ◽  
Vol 323 (2) ◽  
pp. 539-546 ◽  
Author(s):  
Poul H. JENSEN ◽  
Peter HØJRUP ◽  
Henrik HAGER ◽  
Morten S. NIELSEN ◽  
Linda JACOBSEN ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Solid Phase ◽  
Amyloid Plaques ◽  
Binding Activity ◽  
Cross Linking ◽  
Spectrometric Analysis ◽  
Binding Domains ◽  
Aβ Fibrils

NAC, a 35-residue peptide derived from the neuronal protein α-synuclein/NAC precursor, is tightly associated with Aβ fibrils in Alzheimer's disease amyloid, and α-synuclein has recently been shown to bind Aβ in vitro. We have studied the interaction between Aβ and synucleins, aiming at determining segments in α-synuclein that can account for the binding, as well as identifying a possible interaction between Aβ and the β-type synuclein. We report that Aβ binds to native and recombinant α-synuclein, and to β-synuclein in an SDS-sensitive interaction (IC50 approx. 20 μM), as determined by chemical cross-linking and solid-phase binding assays. α-Synuclein and β-synuclein were found to stimulate Aβ-aggregation in vitro to the same extent. The synucleins also displayed Aβ-inhibitable binding of NAC and they were capable of forming dimers. Using proteolytic fragmentation of α-synuclein and cross-linking to 125I-Aβ, we identified two consecutive binding domains (residues 1–56 and 57–97) by Edman degradation and mass spectrometric analysis, and a synthetic peptide comprising residues 32–57 possessed Aβ-binding activity. To test further the possible significance in pathology, α-synuclein was biotinylated and shown to bind specifically to amyloid plaques in a brain with Alzheimer's disease. It is proposed that the multiple Aβ-binding sites in α-synuclein are involved in the development of amyloid plaques.

Sign in / Sign up

Export Citation Format

Share Document