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.

scholarly journals Validation of a High Throughput Scintillation Proximity Assay for 5-HydroxytryptaminelE Receptor Binding Activity

1997 ◽  
Vol 2 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Steven D. Kahl ◽  
Frederick R. Hubbard ◽  
G. Sitta Sittampalam ◽  
Joseph M. Zock
Keyword(s):  
High Throughput ◽  
Binding Capacity ◽  
Binding Activity ◽  
Proximity Effects ◽  
Gene Encoding ◽  
Scintillation Proximity Assay ◽  
Receptor Binding Activity ◽  
The Stability ◽  
Equilibrium Dissociation ◽  
Dissociation Constant Kd

Membranes prepared from stable transfected cells expressing the human gene encoding a functional 5-hydroxytryptaminelE (5HT1E) receptor were used to investigate high-affinity [3H]serotonin ([3H]5-HT) binding with scintillation proximity assay (SPA) technology. In this nonseparation format, membranes are captured by WGA-coated SPA fluoromicrospheres for detection of receptor-bound radioligand. Total binding observed was approximately 2000 cpm compared to a nonspecific signal of 100 cpm determined in the presence of 10,uM unlabeled 5-HT. Non-proximity effects (NPE) for the radiolabel and SPA beads averaged less than 100 cpm. Saturation binding analysis yielded an equilibrium dissociation constant (Kd) of 5.38 ± 0.43 nM and a maximum binding capacity (Bmax) of 6.42 + 0.15 pmol/mg protein. Competition with unlabeled serotonergic compounds demonstrated a specificity of the assay with rank potencies (5-HT > a-Me-5-HT > 2-Me-5-HT > 5-CT) similar to those observed using traditional fitration techniques. The variability of the assay and the stability of all reagents were investigated to validate the assay for extended use throughout a typical high throughput screen operation lasting several months.

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.

Androgen receptor in the Harderian gland of Rana esculenta

1991 ◽  
Vol 129 (2) ◽  
pp. 227-232 ◽  
Author(s):  
M. d'Istria ◽  
G. Chieffi-Baccari ◽  
L. Di Matteo ◽  
S. Minucci ◽  
B. Varriale ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Binding Capacity ◽  
Specific Binding ◽  
Secretory Activity ◽  
Harderian Gland ◽  
Nuclear Extract ◽  
Rana Esculenta ◽  
Binding Activity ◽  
Single Class ◽  
Nuclear Extracts

ABSTRACT An androgen receptor has been identified in the cytosolic and nuclear extracts of the Harderian gland of the frog, Rana esculenta. A single class of high-affinity binding sites was found: Kd = 1·9±1·3 (s.d.) nmol/l (n = 26) for the cytosolic extract and Kd = 0·9±0·8 nmol/l (n = 15) for the nuclear extract. The presence of binding activity in both nuclear and cytosolic extracts and the low rate of ligand-receptor dissociation are characteristics that distinguish this receptor from a steroid-binding protein. The Kd did not show any sex difference and did not exhibit any secretory activity-related change. Binding in both cytosolic and nuclear extracts was specific for androgens (testosterone = 5α-dihydrotestosterone); oestradiol-17β showed a 30% cross-reaction; moreover, specific binding of [3H]oestradiol-17β was not detectable. The binding capacity of the Harderian gland increased progressively in both fractions from October to December, reaching a peak in May, and decreased suddenly during July to August. The lack of any morphological sex-related difference in the Harderian gland of the green frog might be accounted for by the high amount of circulating androgens as well as a similar concentration of androgen receptor in both sexes. Journal of Endocrinology (1991) 129, 227–232

scholarly journals Characterization of the Binding Specificity of K88ac and K88ad Fimbriae of Enterotoxigenic Escherichia coli by Constructing K88ac/K88ad Chimeric FaeG Major Subunits

2008 ◽  
Vol 77 (2) ◽  
pp. 699-706 ◽  
Author(s):  
Weiping Zhang ◽  
Ying Fang ◽  
David H. Francis
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Binding Capacity ◽  
Specific Binding ◽  
Binding Specificity ◽  
Binding Activity ◽  
Enterotoxigenic Escherichia Coli ◽  
Single Amino Acid ◽  
Fimbrial Subunit ◽  
K88 Fimbriae

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 (F4) fimbriae are the major cause of diarrhea in young pigs. Three antigenic variants of K88 fimbriae (K88ab, K88ac, and K88ad) have been identified among porcine ETEC strains. Each K88 fimbrial variant shows a unique pattern in binding to different receptors on porcine enterocytes. Such variant specificity in fimbrial binding is believed to be controlled by the major subunit (FaeG) of the K88 fimbriae, because the genes coding for the only other fimbrial subunit are identical among the three variants. Uniqueness in binding to host receptors may be responsible for differences in the virulence levels of porcine diarrhea disease caused by K88 ETEC strains. To better understand the relationships between the structure of FaeG proteins and fimbrial binding function, and perhaps virulence in disease, we constructed and expressed various K88ac/K88ad faeG gene chimeras and characterized the binding activity of each K88 chimeric fimbria. After verifying biosynthesis of the chimeric fimbriae, we examined their binding specificities in bacterial adherence assays by using porcine brush border vesicles that are specific to either the K88ac or K88ad fimbria. Results showed that each fimbria switched binding specificity to that of the reciprocal type when a peptide comprising amino acids 125 to 163 was exchanged with that of its counterpart. Substitutions of a single amino acid within this region negatively affected the binding capacity of each fimbria. These data indicate that the peptide including amino acids 125 to 163 of the FaeG subunit is essential for K88 variant-specific binding.

Free and bound androgens in the female guinea pig during gestation and after parturition and in the offsprings during the perinatal period

1980 ◽  
Vol 95 (1) ◽  
pp. 101-109 ◽  
Author(s):  
N. Rigaudière ◽  
P. Pradier ◽  
P. Delost
Keyword(s):  
Binding Capacity ◽  
Specific Binding ◽  
Equilibrium Dialysis ◽  
Plasma Concentrations ◽  
Perinatal Period ◽  
Maternal Plasma ◽  
Binding Activity ◽  
Low Concentrations ◽  
High Binding Affinity ◽  
Newborn Animals

Abstract. Total amounts of testosterone (T) and dihydrotestosterone (DHT) and their distribution between non-protein-bound (free), albumin-bound and PBG-(progesterone binding globulin)-bound fractions were determined by radioimmunoassay and equilibrium dialysis from plasma samples. The samples were taken from male guinea pigs during the perinatal period and from pregnant females during gestation and after parturition. Plasma proteins of the foetus and newborn animals appeared to have no high binding affinity for androgens. On the other hand albumin having a binding capacity of 56% for testosterone and 75% for DTH irrespective of the total androgen concentration may be considered to be an important low affinity binding protein. Maternal plasma developed a specific binding activity with the appearance of PBG in early pregnancy. Alterations in the binding capacity of PBG for T or DHT paralleled changes in plasma concentrations of both androgens, the highest values being observed on day 48 of pregnancy, with a prompt return to normal after parturition. Irrespective of the total androgen concentration, it was evident that PBG was capable of maintaining the free T and DHT at low concentrations (about 0.20 and 0.17 ng/10 ml, respectively). Besides the specific binding due to PBG a non-specific binding due to albumin was observed. The competition which exists between these two binding systems for T and DHT was evident when the quantity of bound androgens was expressed as a percentage. Neither the sex, nor the number of the foetuses, nor the interaction between the two, was found to have any significant effect in the maternal androgens, whether total or free hormone was considered.

Erythropoietin binding sites in human foetal tissues

1987 ◽  
Vol 116 (4) ◽  
pp. 561-567 ◽  
Author(s):  
F. Pekonen ◽  
K. Rosenlöf ◽  
E.-M. Rutanen ◽  
F. Fyhrquist
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Recombinant Dna ◽  
Binding Capacity ◽  
Specific Binding ◽  
First Trimester ◽  
Binding Activity ◽  
Renin Substrate ◽  
Human Erythropoietin ◽  
Foetal Liver

Abstract. Using 125I labelled recombinant DNA human erythropoietin (EP), we have explored the presence and properties of EP binding sites in foetal human tissues. The EP binding site is present in the foetal liver already during the first trimester of pregnancy. The binding site has an equilibrium association constant of 4.1–6.2 × 109 1/mol and is specific for EP. The cross-reactivities of FSH, TSH, hCG, insulin and renin substrate were less than 0.01%. The EP binding capacity of foetal liver was 5.4–16 fmol/mg membrane protein. In foetal lung tissue, a slight EP binding activity was observed, whereas foetal spleen, muscle, brain, thyroid and placental tissues were virtually devoid of EP binding capacity. The same level of binding was reached at 37°C in 1 h and at 4°C in 24 h. The binding was pH-dependent with maximal specific binding at pH 7.7. SDS-PAGE gel electrophoresis analysis of covalently cross-linked 125I-EP to foetal liver membranes suggested that the EP binding site was composed of two subunits with an apparent mol wt of 41000 and 86000 dalton, respectively.

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.

Gastric oxyntic cell tubulin: characterization and possible significance

1981 ◽  
Vol 240 (4) ◽  
pp. G317-G323
Author(s):  
H. E. Stewart ◽  
D. K. Kasbekar
Keyword(s):  
Binding Capacity ◽  
Specific Binding ◽  
Sodium Dodecyl ◽  
Binding Constants ◽  
Electrophoretic Analysis ◽  
Binding Activity ◽  
Gastric Epithelium ◽  
Temperature Sensitive ◽  
Major Band ◽  
Oxyntic Cell

Colchicine-binding activity in the homogenate fractions of the whole gastric epithelium and oxynein tissue has been studied to determine if tubulin is present in the bullfrog stomach. Most of the specific binding activity is found in the 100,000-g soluble fraction and is enriched in the oxynein tissue. Binding is proportional to protein concentration, linear up to 30 min, and half saturable at 16 microM colchicine concentration. Scatchard and direct linear plot analysis yields the following values for binding constants: Kd = 12 microM, Ka = 0.08 X 10(6) M-1, maximal binding capacity (Bmax) = 0.19 X 10(-6) M, and n = 84 pmol/mg protein. Fiftyfold excess lumicolchicine does not affect colchicine binding. The binding activity has been partially purified by the temperature-sensitive cycling technique. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the product shows a major band corresponding to tubulin in the 55,000-dalton region. Tubulin in isolated oxyntic cells can be visualized by indirect immunofluorescence. These data and the observed increase in colchicine-binding activity in the burimamide- over the histamine-treated tissue, possibly due to changes in the levels of tubulin polymerization, suggest a likely involvement of the cytoskeleton in gastric secretion.

Sign in / Sign up

Export Citation Format

Share Document