共查询到20条相似文献,搜索用时 31 毫秒
1.
E. Fabbri A. Capuzzo A. Gambarotta T.W. Moon 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1995,112(4):643-651
Epinephrine (EPI) is thought to act by stimulating adenylyl cyclase (ACase) and cAMP production through β-adrenoceptors in the liver of more primitive vertebrates. Recent observations, however, point to an involvement of α1-adrenoceptors in EPI action, at least in some fish species. The role of the α1- and β-adrenergic transduction pathways has been investigated in rainbow trout (Oncorhynchus mykiss) hepatic tissue. Radioligand-binding assays with the β-adrenergic antagonist 3H-CGP-12177 using hepatic membranes purified on a discontinuous sucrose gradient confirmed the presence of β-adrenoceptors (Kd0.36 nM, Bmax 8.61 fmol · mg−1 protein). We provide the first demonstration of α1-adrenoceptors in these same membranes; analysis of binding data with the α1-adrenergic antagonist 3H-prazosin demonstrated a single class of binding sites with a Kdof 15.4 nM and a Bmax of 75.2 fmol · mg−1 protein. There is a straight correlation between β-adrenoceptor occupancy, ACase activation and cAMP production. On the contrary, the role of inositol 1,4,5-trisphosphate (IP3) has to be elucidated; in fact, despite the presence of specific microsomal binding sites for IP3 (Kd 6.03 nM, Bmax 90.2 fmol · mg−1 protein), its cytosolic concentration was not modulated by EPI. On the other hand, we have previously shown in American eel and bullhead hepatocytes that α1-adrenergic agonists are able to increase intracellular concentrations of IP3 and Ca2+ and to activate glycogenolysis. These data suggest a marked variation in the liver of different fish both in terms of α1-binding sites affinity and of α1-adrenoceptor/IP3/Ca2+ transduction systems. 相似文献
2.
F León-Velarde J-P Richalet J-C Chavez R Kacimi M Rivera-Chira J-A Palacios D Clark 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1998,119(4):819-823
The heart rate response to isoproterenol (HR-Iso), density and affinity (kd) of β-adrenergic (β-AR) and muscarinic (M2) receptors were compared among three rodents with different generation-life histories of confinement and of high altitude exposure. The European guinea pig (Cavia porcellus) (EGp), a laboratory animal that arrived in Europe after the Spanish Conquest of South America and the Peruvian guinea pig (C. porcellus) (PGp), a semi-wild animal that came from the altiplano to sea level at least 25 generations ago, were used for intra-species comparison. Wistar rats (WR) were used for inter-species comparison as representative of a typical sea level laboratory animal. The HR-Iso was lower in EGp than in the PGp. The PGp showed the highest β-AR density (P<0.0005) and the highest β-AR kd values (P<0.0005) when compared to both EGp and WR groups (β-AR Bmax (fmol mg−1 prot), WR, 19±4; Egp, 34±10; PGp, 74±15. β-AR kd (pM), WR, 24±10; Egp, 17±7; PGp, 39±14). In contrast, PGp showed lower M2 receptor density values than the EGp (P<0.0005). The WR had the highest M2 receptor densities (M2 Bmax (fmol mg−1 prot), WR, 188±15; Egp, 147±9; PGp, 118±6 and M2 kd (pM), WR, 65±12; Egp, 67±6; PGp, 92±2). The inter and intra-species differences found may be related to their respective history of confinement rather than to their history of exposure to high altitude. 相似文献
3.
E.C.K. Liu A. Hedberg H.J. Goldenberg D.N. Harris M.L. Webb 《Prostaglandins & other lipid mediators》1992,44(2)
DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A2/prostaglandin H2 (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [3H]SQ 29,548 (5 nM) in human platelets with kd/slope factor values of 9.6±1.4 μM/1.1±0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [3H]SQ 29,548 binding in platelets (Kd/slope factor:200 μM/0.86). [3H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (Kd: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (Bmax: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC50=46 μM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors. 相似文献
4.
B. N. Manukhin L. A. Nesterova 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2011,5(4):350-354
Activation and inhibition of muscarinic cholinoceptors by atropine and carbachol are shown to exert allosteric effects on
the binding of specific nonselective α2-adrenoceptor antagonist [3H]RX821002 in rat brain cortex membranes. The ligand-receptor interaction for α2-adrenoceptors corresponded to the model suggesting the presence of one homogeneous pool of receptors with two specific binding
sites. The parameters of the [3H]RX821002 binding were as follows: [3H]RX821002 -K
d = 1.94 ± 0.08 nM, B
max = 13.4 ± 1.8 fmol/mg protein, n = 2. The inhibition of muscarinic cholinoceptors by atropine induced an increase of affinity (K
d = 1.36 ± 0.12 nM) and a decrease of the α2-adrenoceptor density (B
max = 10.18 ± 0.48 fmol/mg protein). The muscarinic cholinoceptor agonist carbachol induced an increase of the affinity (K
d = 1.56 ± 0.05 nM) and quantity of binding sites (B
max = 16.61 ± 0.29 fmol/mg protein). As a result, under the influence of atropine and carbachol, the efficiency of binding (E = B
max/2K
d) increased from 3.50 ± 0.40 to 5.60 ± 0.79 and 6.86 ± 0.20 fmol/mg protein/nM, respectively. The data suggest that α2-adrenoceptors exist in rat brain cortex as homodimers. 相似文献
5.
Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6
glioma cells. In membranes, saturation experiment performed with [3H]DPCPX, selective A1R antagonist, revealed a single binding site with a K
D = 9.4 ± 1.4 nM and B
max = 62.7 ± 8.6 fmol/mg protein. Binding of [3H]DPCPX in intact cell revealed a K
D = 17.7 ± 1.3 nM and B
max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [3H]ZM241385 binding experiments revealed a single binding site population of receptors with K
D = 16.5 ± 1.3 nM and B
max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K
D = 4.7 ± 0.6 nM and B
max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A2A receptor in C6 cells. A1, A2A, A2B and A3 adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR
assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A1R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic
activity in C6 cells. These results suggest that C6 glioma cells endogenously express A1 and A2 receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a
model to study the role of adenosine receptors in tumoral cells. 相似文献
6.
Abd-Elfattah Anwar-Saad A. Hoehner Jeff Wechsler Andrew S. 《Molecular and cellular biochemistry》1998,180(1-2):105-110
The role of nucleoside transport in ischemia-reperfusion injury and arrhythmias has been well documented in various animal models using selective blockers. However, clinical application of nucleoside transport inhibitors remains to be demonstrated in humans. It is not known whether human heart has nucleoside transport similar to that of animals. The aim of this study is to pharmacologically identify the presence of nucleoside transport binding sites in the human myocardium compared to animals.Myocardial tissue was obtained from guinea pig left and right ventricle, canine left ventricle, human intraoperative right atrium and human cadaveric right atrium and right and left ventricles. Myocardial preparations were obtained from tissue samples after homogenized and a differential centrifugation.Equilibrium binding assays were performed using [3H]-p-nitrobenzylthioinosine (NBMPR) at room temperature in the presence or absence of non-radioactive NBMPR or other nucleoside transport blockers such as p-nitrobenzylthioguanosine dipyridamole, lidoflazine, papaverin, adenosine and doxorubcine. From saturation curves and inhibition kinetics, we determined the relative maximal binding (Bmax) and dissociation constant (Kd) of [3H]-NBMPR binding of human myocardial preparations.Results demonstrated that the fresh human myocardial preparations have a specific binding site for NBMPR with a Bmax of 283 ± 32 fmol/mg protein and Kd of 0.56 ± 0.12 nM. These values are lower than those obtained from guinea pigs (Bmax = 1440 ± 187 fmol/mg protein and Kd = 0.21 ± 0.03 nM) and canine atrium (Bmax 594 ± 73 fmol/mg protein, and Kd = 1.12 ± 0.22 nM).Displacement kinetics studies revealed the relative potencies (of certain unrelated drugs as follow: p-nitrobenzylthioguanosine > dipyridamole > lidoflazine > pavaverine > Diltazam > adenosine > doxyrubicin. It is concluded that human myocardium contains an active nucleoside transport site which may play a crucial role in post-ischemic reperfusion-mediated injury in a wide spectrum of ischemic syndromes. 相似文献
7.
The presence and specificity of insulin receptors was investigated in cultured cells obtained from 15–16 days old embryonic mouse cerebra. Developmental studies suggested that the maximum insulin binding occurred at about 11 days in vitro (DIV). Scatchard analysis of binding data revealed two types of binding sites. One type of receptor was the high affinity type (K
d=7.77×10–9 M; number of receptor sites,B
max=350 fmol/mg protein) while the other type was of low affinity type (K
d=5.75×10–8 M;B
max=1150 fmol/mg protein). The specificity of receptors for insulin was also confirmed by showing that [125I]insulin was displaced by non-radioactive insulin but not by glucagon or growth hormone. Insulin displayed a clear dose-dependent stimulation of thymidine incorporation. It also stimulated the activity of the enzyme 2,3-cyclic nucleotide phosphohydrolase (CNPase), which is specifically associated with myelin produced from oligodendroglia. Thus insulin has a positive influence on the proliferation and differentiation of brain cells. 相似文献
8.
Subchronic treatment with MAP (4.6 mg/kg, i.p., once daily for 11 days) significantly decreased the Kd, but not Bmax, values of [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) binding to adenosine A1 receptors in the prefrontal cortex and hippocampus, but not striatum, of rat brain. However, subchronic treatment with PCP (10 mg/kg, i.p., once daily for 11 days) did not alter the Kd and Bmax values of [3H]DPCPX binding to adenosine A1 receptors in these three regions. Subchronic treatment with MAP or PCP did not alter the Bmax and Kd values of [3H]2-p-(2-carboxyehyl)phenethylamino-5-N-ethylcarboxyamidoadenosine ([3H]CGS21680) binding to adenosine A2A receptors in the striatum. Furthermore, subchronic treatment with MAP or PCP significantly decreased the specific binding of [3H]CGS21680 to adenosine A2A receptors in the hippocampus, but not in the prefrontal cortex. Thus, these results suggest that MAP and PCP may produce differential effects on the adenosine A2A receptors, but not adenosine A1 receptors in rat brain. 相似文献
9.
Thomas Bauknecht 《Human genetics》1977,39(3):321-328
Summary Cultured human fibroblasts and amniotic fluid cells (AF cells) were examined for the presence of steroid hormone receptors. In both cell types, the androgen (DHT) or glucocorticoid (dexamethasone) receptor was detected, but not the estrogen receptor. Binding parameters in fibroblasts were: for androgen: KD=3.7×10-9M, Bmax=13 fmol/mg; for dexamethasone: KD=4.5×10-9M, Bmax=120 fmol/mg. Binding parameters in AF cells were: for androgen: KD=4×10-9M, Bmax=8 fmol/mg; for dexamethasone: KD=1.9×10-8M, Bmax=375 fmol/mg. Cultured cells derived from the gonads (of a patient with 17-ketosteroid reductase deficiency) seem to have more receptors than cells from extragenital body parts (Bmax=21 fmol). With the aid of gel chromatography, the molecular weight of the androgen receptor was estimated to be 30–40 000D. 相似文献
10.
Sándor Benyhe Géza Tóth Judit Kevei Mária Szücs Anna Borsodi Katalin Di Gléria Judit Szécsi Helga Süli-Vargha Kálmán Medzihradszky 《Neurochemical research》1985,10(5):627-635
[Tyr-3,5-3H]1,d-Ala2, Leu5-enkephalin ([3H]DALA) was used for labeling the opioid receptors of rat brain plasma membranes. The labeled ligand was prepared from [Tyr-3,5-diiodo]1,d-Ala2, Leu5-enkephalin by catalytic reductive dehalogenation in the presence of Pd catalyst. The resulting [Tyr-3,5-3H]1,d-Ala2, Leu5-enkephalin had a specific activity of 37.3 Ci/mmol. In the binding experiments steady-state level was reached at 24°C within 45 min. The pseudo first order association rate constant was 0.1 min–1. The dissociation of the receptor-ligand complex was biphasic with k–1-s of 0.009 and 0.025 min–1. The existence of two binding sites was proved by equilibrium studies. The high affinity site showed aK
D=0.7 nM andB
max=60 fmol/mg protein; the low affinity site had aK
D=5 nM andB
max=160 fmol/mg protein. A series of opioid peptides inhibited [3H]DALA binding more efficiently than morphine-like drugs suggesting that labeled ligand binds preferentially to the subtype of opioid receptors. Modification of the original peptides either at the C or N terminal ends of the molecules resulted in a decrease in their affinity. 相似文献
11.
Gamma amino butyric acid is a major inhibitory neurotransmitter in the central nervous system. In the present study we have
investigated the alteration of GABA receptors in the brain stem of rats during pancreatic regeneration. Three groups of rats
were used for the study: sham operated, 72 h and 7 days partially pancreatectomised. GABA was quantified by [3H]GABA receptor displacement method. GABA receptor kinetic parameters were studied by using the binding of [3H]GABA as ligand to the Triton X-100 treated membranes and displacement with unlabelled GABA. GABAA receptor activity was studied by using the [3H]bicuculline and displacement with unlabelled bicuculline. GABA content significantly decreased (P < 0.001) in the brain stem during the regeneration of pancreas. The high affinity GABA receptor binding showed a significant
decrease in B
max (P < 0.01) and K
d (P < 0.05) in 72 h and 7 days after partial pancreatectomy. [3H]bicuculline binding showed a significant decrease in B
max and K
d (P < 0.001) in 72 h pancreatectomised rats when compared with sham where as B
max and K
d reversed to near sham after 7 days of pancreatectomy. The results suggest that GABA through GABA receptors in brain stem
has a regulatory role during active regeneration of pancreas which will have immense clinical significance in the treatment
of diabetes. 相似文献
12.
Isolated, intact rat liver nuclei have high-affiity (Kd=10−9 M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [3H]tamoxifen binding capacity is thermolabile, being most stable at 4°C and rapidly lost at 37°C. More [3H]tamoxifen, however, is specifically bound at incubation temperatures of 25°C and 37°C than at 4°C although prewarming nuclei has no effect, suggesting exchange of [3H]tamoxifen for an unidentified endogenous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (Kd=10−9 M) [3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78±0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000×g, 30 min) contains high-capacity (955±405 (S.D.) fmol/mg protein), low-affinity (Kd=10.9±4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000×g, 60 min) contains low-capacity (46±15 (S.D.) fmol/mg protein), high-affinity (Kd=0.61± 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%–3.2%, and nuclear sites less than 0.5% of total sites. 相似文献
13.
Konopska B Gburek J Gołab K Warwas M 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2007,146(4):482-488
Chicken cystatin, a homologue of human cystatin C, like other low-molecular-weight proteins is metabolized by renal proximal tubule cells. However, the precise mechanism(s) of this process has not been elucidated yet. To characterize chicken cystatin binding to renal brush-border membranes, the incubation of fluorescein labelled protein with rat cortical homogenate was performed. Saturation-dependent and reversible binding with low affinity (Kd = 3.67–4.07 μM) and high capacity (Bmax = 2.32–2.79 nmol/mg) was observed. Bovine albumin was the most potent competitor (Ki = 0.7 μM) among other megalin/cubilin ligands tested. The presence of Ca+ 2 ions was necessary to effective cystatin binding by brush-border membranes. Obtained data strongly support the hypothesis that chicken cystatin is a novel ligand for megalin/cubilin receptors tandem on proximal tubular cells. 相似文献
14.
L. A. Nesterova B. N. Manukhin 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2011,5(1):85-91
The influence of isoprenaline- and propranolole-induced activation and inhibition of β-adrenoreceptors on the specific nonselective
α2-antagonist [3H]RX821002 binding was studied on rat cerebral cortex subcellular membrane fractions. It was shown that the ligand-receptor
interaction for α2-adrenoreceptors corresponded to the model that assumed the presence of one receptor pool and binding of two ligand molecules
to a receptor dimer. The following parameters were determined for [3H]RX821002 binding to α2-adrenoreceptors: K
d1 = 1.57 ± 0.27 nM, B
max = 7.24 ± 1.63 fmol/mg of protein, n = 2. In the case of isoprenaline-induced activation of β-adrenoreceptors the binding of radiolabeled ligand to α2-adrenoreceptors was described by the same model. The affinity of α2-adrenoreceptors for [3H]RX821002 decreased more than twofold (K
d = 3.55 ± 0.02 nM) and the quantity of active receptors increased by 69% (B
max = 12.24 ± 0.06 fmol/mg of protein). Propranolole changed the model of ligand binding, and two pools of receptors were detected
with the following parameters: K
d1 = 0.61 ± 0.02 nM, K
d2 = 3.41 ± 0.13 nM, B
ml = 1.88 ± 0.028 fmol/mg of protein, B
m2 = 9.27 ± 0.08 fmol/mg of protein, n = 2. The data suggest that α2-adrenoreceptors in subcellular membrane fractions from rat cerebral cortex exist in dimeric form. Isoprenaline and propranolole
exhibit modulating effect on the specific antagonist binding to α2-adrenoreceptors, which results in the inhibition and alteration of [3H]RX821002 binding parameters. 相似文献
15.
Harada N Himeno A Shigematsu K Sumikawa K Niwa M 《Cellular and molecular neurobiology》2002,22(2):207-226
1. Interaction in the recognition of endothelin-1 (ET-1), a typical bivalent ET receptor-ligand, between ETA and ETB receptors was investigated in the rat anterior pituitary gland, using our quantitative receptor autoradiographic method with tissue sections preserving the cell-membrane structure and ET receptor-related compounds.2. In saturation binding studies with increasing concentrations (0.77–200 pM) of 125I-ET-1 (nonselective bivalent radioligand), 125I-ET-1 binding to the rat anterior pituitary gland was saturable and single with a K
D of 71 pM and a B
max of 120 fmol mg–1. When 1.0 M BQ-123 (ETA antagonist) was added to the incubation buffer, binding parameters were 8.3 pM of K
D and 8.0 fmol mg–1 of B
max, whereas 10 nM sarafotoxin S6c (ETB agonist) exerted little change in these binding parameters (K
D, 72 pM; B
max, 110 fmol mg–1).3. Competition binding studies with a fixed amount (3.8 pM) of 125I-ET-1 revealed that when 1.0 M BQ-123 was present in the incubation buffer, ETB receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620 (ETB agonist), and BQ-788 (ETB antagonist) competitively inhibited 125I-ET-1 binding with K
is of 140, 18, 350 pM, and 14 nM, respectively, however, these compounds were not significant competitors for 125I-ET-1 binding in the case of absence of BQ-123.4. In cold-ligand saturation studies with a fixed amount (390 pM) of 125I-IRL 1620 (ETB radioligand), IRL1620 bound to a single population of the ETB receptor, and no change was observed in binding characteristics in the presence of 1.0 M BQ-123. 125I-IRL1620 binding was competitively inhibited by ET-1 and ET-3 in the absence of BQ-123, with K
is of 20 and 29 pM, respectively, the affinities being much the same as those of 29 nM, in the presence of 1.0 M BQ-123.5. Two nonbivalent ETA antagonists, BQ-123 and PD151242, were highly sensitive and full competitors for 125I-ET-1 binding (5.0 pM), in the presence of 10 nM sarafotoxin S6c.6. Taken together with the present finding that mRNAs encoding the rat ETA and the ETB receptors are expressed in the anterior pituitary gland, we tentatively conclude that although there are ETA and ETB receptors with a functional binding capability for ET receptor-ligands, the ETB receptor does not independently recognize ET-1 without the aid of the ETA receptor. If this thesis is tenable, then ET-1 can bridge between the two receptors to form an ETA–ETB receptor heterodimer. 相似文献
16.
Randall K Packer Jeffrey L Garvin 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1998,120(4):777-783
We measured Na+/K+ ATPase activity in homogenates of gill tissue prepared from field caught, winter and summer acclimatized yellow perch, Perca flavescens. Water temperatures were 2–4°C in winter and 19–22°C in summer. Na+/K+ ATPase activity was measured at 8, 17, 25, and 37°C. Vmax values for winter fish increased from 0.48±0.07 μmol P mg−1 protein h−1 at 8°C to 7.21±0.79 μmol P mg−1 protein h−1 at 37°C. In summer fish it ranged from 0.46±0.08 (8°C) to 3.86±0.50 (37°C) μmol P mg−1 protein h−1. The Km for ATP and for Na+ at 8°C was ≈1.6 and 10 mM, respectively and did not vary significantly with assay temperature in homogenates from summer fish. The activation energy for Na+/K+ ATPase from summer fish was 10 309 (μmol P mg−1 h−1) K−1. In winter fish, the Km for ATP and Na+ increased from 0.59±0.08 mM and 9.56±1.18 mM at 8°C to 1.49±0.11 and 17.88±2.64 mM at 17°C. The Km values for ATP and Na did not vary from 17 to 37°C. A single activation energy could not be calculated for Na/K ATPase from winter fish. The observed differences in enzyme activities and affinities could be due to seasonal changes in membrane lipids, differences in the amount of enzyme, or changes in isozyme expression. 相似文献
17.
L. A. Nesterova B. N. Manukhin 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2010,4(2):206-211
The effects of activation and inhibition of muscarinic cholinoceptors by carbachol and atropine on the binding of specific
nonselective α1-antagonist [3H]prazosine in synaptosomal membranes of rat cerebral cortex have been studied. It has been shown that the ligand-receptor
interaction of α1-adrenoceptors corresponds to the model suggesting the presence of a single receptor pool and the binding of two ligand molecules
to the receptor. The parameters of [3H]prazosine binding to α1-adrenoceptors were as follows: K
d = 1.56 ± 0.17 nM, B
max = 30.25 ± 1.78 fmol/mg protein, n = 2. Upon inhibition of muscarinic cholinoceptors by atropine or their activation by carbachol, the radiolabelled ligand
is bound to α1-adrenoceptors according to the same model but at n = 1. In the presence of atropine, the sensitivity of α1-adrenoceptors to [3H]prazosine decreases more than twofold (K
d = 3.52 ± 0.36 nM) and the concentration of the active receptors is 36% lower (B
max = 19.45 ± 1.46 fmol/mg protein). Carbachol does not reduce the affinity of adrenoceptors to the ligand, while the concentration
of active receptors decreases like in the case of atropine. It is supposed that α1-adrenoceptors in the membranes of rat cerebral cortex exist as dimers. The modulating effects of atropine and carbachol on
the binding of specific antagonist by α1-adrenoceptors are exhibited as changes in the general character of binding (monomerization of α1-adrenoceptors) and as inhibitory effect on the [3H]prazosine binding parameters. 相似文献
18.
L. A. Nesterova B. N. Manukhin 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2013,7(1):45-51
The effects of activation and inhibition of serotonin receptors by serotonin (5-HT) and mianserin on the specific nonselective α1-antagonist [3H]prazosine binding in rat cerebral cortex membranes was studied. It was shown that the ligand-receptor interaction of α1-adrenoceptors corresponded to the model suggesting the presence of one pool of receptors and the binding of two ligand molecules to the receptor. The parameters of [3H]prazosine binding to α1-adrenoceptors were as follows: K d =1.85 ± 0.16 nM, B max = 31.1 ± 0.3 fmol/mg protein, n = 2. In case of activation of 5HT-receptors by serotonin, the character of ligand binding was different: two pools of receptors were detected with the parameters K d1 = 0.61 ± 0.04, K d2 = 3.82 ± 0.15 nM, B m1 = 6.6 ± 0.7, B m2 = 25.6 ± 0.4 fmol/mg protein, n = 2. The sensitivity of the high-affinity pool increased threefold and the sensitivity of the low-affinity pool decreased twofold as compared to the control. The value of maximal reaction (B max) did not change. In the case of inhibition of 5HT-receptors by mianserin, radioactive ligand is bound to α1-adrenoceptors according to the same model as in the control conditions. The affinity of α1-adrenoceptors to [3H]prazosine decreases twofold and the concentration increases (K d = 3.97 ± 0.12 nM, B max = 40.0 ± 0.5 fmol/mg protein). The data suggest that α1-adrenoceptors in rat cerebral cortex exist as a dimer. The modulatory effects of serotonin and mianserin on the specific binding of [3H]prazosine to α1-adrenoceptors was detected, manifesting itself as changes in the binding parameters and in the general character of ligand-receptor interactions. 相似文献
19.
Digitalis lanata 《Plant Physiology and Biochemistry》2000,38(12):929
A cardenolide-hydrolysing β-D-glucosidase was isolated from young leaves of Digitalis lanata. Since this enzyme differs from the cardenolide glucohydrolase (CGH) described and characterised previously, it was termed cardenolide glucohydrolase II (CGH II). CGH II was detected in various Digitalis tissue cultures as well as in young leaves of D. lanata. The latter source was used as the starting material for the isolation and purification of CGH II. The specific enzyme activity reached about 15 pkat·mg–1 protein in buffered leaf extracts. Optimal CGH II activity was seen at around pH 6.0 and 50 °C. CGH II was purified about 600-fold by anion exchange chromatography, size exclusion chromatography and hydroxyapatite chromatography. The apparent molecular mass of CGH II was 65 kDa as determined by SDS-PAGE. CGH II exhibited a high substrate specificity towards cardenolide disaccharides, especially to those with a 1-4-β-linked glucose-digitoxose moiety such as glucoevatromonoside. The Km- and Vmax-values for this particular substrate were calculated to be 101 μM and 19.8 nkat·mg–1 protein, respectively. 相似文献
20.
Seth R. Hootman 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,716(3):341-347
Chronic administration of DL-isoproterenol, a β-adrenergic agonist, to male Sprague-Dawley rats increased submandibular gland weights by 3 to 4-fold. This increase resulted from a combination of hyperplasia and hypertrophy of secretory cells. Possible effects of this drug regimen on submandibular gland muscarinic acetylcholine receptors were examined by analysis of the binding of the cholinergic antagonist, L-quinuclidinyl [3H]benzilate, to receptors in gland homogenates. Parallel investigations of receptors in exorbital lacrimal glands, an organ that is not grossly affected by chronic isoproterenol treatment, were also carried out. [3H]QNB bound to submandibular receptors with a Kd of 37.8±6.3 pM in control rats and 41.0±4.0 pM in isoproterenol-treated animals, a non-significant difference (P > 0.05). In contrast, the maximal binding level (Bmax) is isoproterenol-treated rats, 1.52±0.10 fmol/μg DNA, was depressed by approx. 30% (P<0.05) from that of 2.22±0.16 fmol/μg DNA in control animals. In lacrimal glands, both Kd (61.3±5.3 vs. 53.2±4.0 pM) and Bmax (1.74±0.24 vs. 1.78±0.17 fmol/μg DNA) were unchanged by isoproterenol treatment. The affinity of glandular muscarinic receptors for cholinergic agonists was also examined by competition experiments using carbachol. This agonist inhibited [3H]QNB binding to receptors in homogenates from both glands in a dose-dependent fashion. Inhibition constant (Ki) for this interaction were similar in control and isoproterenol-treated lacrimal glands; 53.6±5.4 μM and 66.6±7.9 μM, respectively (P>0.05). In submandibular glands, isoproterenol treatment elicited a highly significant (P < 0.01) shift in Ki from 17.3±1.4 μM to 68.3±5.2 μM. These results demonstrate that chronic administration of isoproterenol to rats results in a reduction in receptor numbers and a decrease in their sensitivity to cholinergic agonists in submandibular, but not lacrimal, glands. 相似文献