Effect of benzodiazepines on NAD binding to synaptic membrane in the rat brain

The receptor protein solubilized from synaptic membranes specifically binds [14C] NAD (dissociation constant--0.75 microM, capacity of binding sites--0.0125 nmol of metaid per 1 mg of protein). All the studied benzodiazepines (phenazepam, nitrazepam, clonazepam, flunitrazepam) are able to displace [14C] NAD from its receptor sites, the mixed type of inhibition being manifested. An inhibition constant for flunitrazepam, a ligand of benzodiazepine receptors, equals 10 microM. GABA promotes an inhibiting effect of benzodiazepines. It is supposed that neurotropic action of NAD is realized through the GABA-benzodiazepine complex of neuronal membranes.     

Interaction of lipophorin with the plasma membrane of locust flight muscles

Binding of high-density lipophorin (HDLp) to a plasma membrane preparation of locust flight muscle tissue was studied using a radiolabelled ligand binding assay and ligand blotting techniques. Analysis at 33 degrees C of the concentration-dependent total binding of tritium-labelled HDLp ([3H]HDLp) to the membrane preparation revealed the presence of a single specific binding site with an equilibrium dissociation constant of Kd = 9 (+/- 2) X 10(-7) M and a maximal binding capacity of 84 (+/- 10) ng X (micrograms protein)-1. Unlabelled HDLp as well as unlabelled low-density lipophorin (LDLp) competed with [3H]HDLp for binding to the identified binding site. In addition, ligand blotting demonstrated that both HDLp and LDLp bind specifically to a 30-kDa protein in the plasma membrane preparation, suggesting the involvement of this protein in the binding of lipophorins to the isolated membranes. A possible relationship between the identified binding of lipophorins and the observed co-purification of lipophorin lipase activity with the plasma membranes is discussed.     

Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.     

Binding of nicotinamide adenine dinucleotide by the renal brush border membrane from rat kidney cortex

The characteristics of nicotinamide adenine dinucleotide (NAD) binding on brush border membranes prepared from rat renal cortex were investigated with the use of radioactively labelled NAD, [adenine-2,8-3H]NAD+, as a ligand. (1) We found that NAD binds on brush border membrane and that the extent of NAD binding is linearly proportional to the brush border membrane protein, and progressively increases with concentration of NAD in the medium. (2) The rate of NAD binding was dependent on temperature. At 20 degrees C, the equilibrium binding was obtained at 15 min, while NAD binding at 0 degree C was slower, but the final level of binding reached at 120 min was similar to that plateau of binding observed at 20 degrees C. Brush border membrane inactivated by heating at 95 degrees C for 3 min did not bind NAD. Binding of NAD on brush border membranes was reversed by simple dilution or by the addition of unlabelled NAD. Both alpha-NAD and beta-NAD stereoisomers displaced bound [3H]NAD. Reduced NAD (NADH) caused less displacement of bound NAD than oxidized NAD+. Adenine, nicotinamide, pyrophosphate, of 5'-AMP did not displace bound NAD. (3) The NAD binding to brush border membranes was nearly saturable, approximating saturation at 10(-4) M NAD. Kinetic analysis by Scatchard plot indicates two sets of NAD binding sites in brush border membranes: a high-affinity binding site (Kd = 1.9 . 10(-5) M) and a low-affinity binding site (Kd = 2.2 . 10(-3) M). (4) Unlike concentrative uptake of D-[14C]glucose by brush border membrane vesicles, binding of NAD was not dependent on the presence of an outside-in sodium gradient [Na+0 greater than Na+i], nor was it abolished by repeated freezing and thawing of brush border membranes. Unlike D-[14C]glucose uptake, NAD binding by brush border membranes did not change upon decrease of intravesicular volume in hypertonic media. These observations indicate that NAD association with brush border membranes is true binding rather than intravesicular uptake of this compound. (5) The presence of specific binding sites in renal brush border membrane capable of binding of NAD with a high degree of affinity suggests that such sites may be involved in previously observed (Kempson, S.A., Colon-Otero, G., Ou, S.L., Turner, S.T. and Dousa, T.P. (1981) J. Clin. Invest. 67, 1347) modulatory effect of NAD on sodium-gradient-dependent uptake of phosphate across luminal brush border membrane of proximal tubules.     

Characterization of binding sites for [3H]-DTG, a selective sigma receptor ligand, in the sheep pineal gland

Specific binding sites for [3H]-1,3 di-ortho-tolylguanidine ([3H]-DTG), a selective radiolabeled sigma receptor ligand, were detected and characterized in sheep pineal gland membranes. The binding of [3H]-DTG to sheep pineal membranes was rapid and reversible with a rate constant for association (K+1) at 25 degrees C of 0.0052 nM-1.min-1 and rate constant for dissociation (K-1) 0.0515 min-1, giving a Kd (K-1/K+1) of 9.9 nM. Saturation studies demonstrated that [3H]-DTG binds to a single class of sites with an affinity constant (Kd) of 27 +/- 3.4 nM, and a total binding capacity (Bmax) of 1.39 +/- 0.03 pmol/mg protein. Competition experiments showed that the relative order of potency of compounds for inhibition of [3H]-DTG binding to sheep pineal membranes was as follows: trifluoperazine = DTG greater than haloperidol greater than pentazocine greater than (+)-3-PPP greater than (+/-)SKF 10,047. Some steroids (testosterone, progesterone, deoxycorticosterone) previously reported to bind to the sigma site in brain membranes were very weak inhibitors of [3H]-DTG binding in the present study. The results indicate that [3H]-DTG binding sites having the characteristics of sigma receptors are present in sheep pineal gland. The physiological importance of these sites in regulating the synthesis of the pineal hormone melatonin awaits further study.     

Solubilization and various properties of NAD-binding receptor protein from rat brain synaptic membranes

The NAD-binding receptor protein has been solubilized from the synaptic membranes of the rat brain by different detergents. Digitanin proved to be the most effective detergent which exerted no action on the specific binding of [14C]NAD with the solubilized receptor protein. Kinetic parameters of the soluble ligand-receptor complex were studied. The affinity of the solubilized receptor protein to NAD did not change as compared to the protein of native membranes. The specific binding of [14C]NAD was saturated at Kd = 0.53 microM, Bmax = 0.011 nmol per 1 mg of protein. The molecular weight of the soluble NAD-receptor complex determined under native conditions was equal to 115 kDa.     

A method for determining binding kinetics applied to thiamine-binding protein

A rapid and simple method for assaying the binding activity of thiamine-binding protein is described. By this assay method, the binding characteristics of rice bran thiamine-binding protein have been evaluated with [14C]thiamine as ligand. Analysis of these data by Scatchard plot resulted in linear plots giving a dissociation constant (Kd) for thiamine of 0.55 microM and a maximum binding (Bmax) of 14.5 pmol of ligand bound/microgram of protein. Thiamine binding to the binding protein was time dependent and reached equilibrium at approximately 20 min. The Kob was 0.18 min-1 and the k1 was 1.25 X 10(5) min-1 M-1. Reversibility of thiamine binding at equilibrium was completed at 60 min with a k2 value of 0.052 min-1. The Kd calculated from the reverse rate constant was 0.42 microM. These results indicated that this binding assay method was substantially reliable and accurate.     

Reserpine binding to chromaffin granules suggests the existence of two conformations of the monoamine transporter

The binding of [3H]reserpine ([3H]RES) to purified bovine chromaffin granule membranes has been studied at low membrane concentration. Saturation isotherms indicated a dissociation equilibrium constant KD of 30 pM and a density of binding sites of 8 pmol/mg of protein at 30 degrees C. The association rate constant was 4.0 X 10(5) M-1 s-1, and the calculated dissociation rate constant was 1.2 X 10(-5) s-1, corresponding to a half-lifetime of about 16 h. Although this dissociation was too low to be measured directly, [3H]RES binding was indeed reversible since it was lost after addition of the detergent Triton X-100. Dihydrotetrabenazine (TBZOH) inhibited [3H]RES binding in a time-dependent manner, EC50 varying from 37 nM after a 1-h incubation to 600 nM after 16 h. On the contrary, [3H]RES binding inhibition by the substrate noradrenaline was time independent. It is proposed that the transporter exists in two different conformations which bind exclusively either tetrabenazine (TBZ) or RES and which are in equilibrium. The effects of detergents were consistent with this two-conformation model. The transporter solubilized by cholate bound [3H]TBZOH, but not [3H]RES. On the other hand, addition of cholate to membrane-bound [3H]RES solubilized the membrane without releasing the ligand from its binding site. It is proposed that the TBZ-binding conformation is obtained by solubilization with cholate and that RES stabilizes the RES-binding conformation, allowing its solubilization by this detergent.     

Different modes of ligand binding to the hepatic galactose/N-acetylgalactosamine lectin on the surface of rabbit hepatocytes

A study of the binding of three different 125I-labeled, galactose-terminated ligands to the hepatic galactose/N-acetylgalactosamine-specific lectin found on the surface of rabbit hepatocytes revealed that the different ligands manifest different physical parameters of binding. Asialoorosomucoid (125I-ASOR) binding was best described as involving two independent classes of binding sites on rabbit hepatocytes, with 161 000 sites/cell with a dissociation constant of 0.44 nM and 292 000 sites/cell with a Kd of 9.7 nM. Asialotriantennary glycopeptide purified from human alpha-1 protease inhibitor and modified with tyrosine at the N-terminus to permit radioiodination (TRI) [Lee, Y. C., Townsend, R. R., Hardy, M. R., L?nngren, J., Arnarp, J., Haraldsson, M., & L?nn, H. (1983) J. Biol. Chem. 258, 199-202] was also found to bind to two apparent classes of binding sites but with different binding parameters: 292 000 sites/cell of Kd = 1.47 nM and 982 000 sites/cell of Kd = 25.3 nM. A synthetic ligand, alpha,beta-diaspartamide of tris[(beta-lactosyloxy)methyl](6-aminohexanamido)methane (di-tris-lac) containing six nonreducing galactose residues [Lee, R. T., Lin, P., & Lee, Y. C. (1984) Biochemistry 23, 4255-4261], was found to bind to 817 000 sites/cell of Kd = 0.63 nM and 1.23 X 10(6) sites/cell of Kd = 25.3 nM. Thus, there were many more total binding sites for TRI or di-tris-lac on the surface of rabbit hepatocytes than there were for asialoorosomucoid, although the dissociation constants were similar for all three ligands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic evidence for differential agonist and antagonist binding to bovine hippocampal synaptic membrane opioid receptors

To examine the kinetics of opioid receptor binding, the agonists [D-Ala2-D-Leu5]enkephalin (DADL) and [D-Ala2-MePhe4-Gly-ol5]enkephalin (DAGO) and the antagonists diprenorphine and naltrexone were used with bovine hippocampal synaptic plasma membranes. By computer modeling of equilibrium binding displacement curves utilizing the LIGAND program, we found opioid peptides bind with high affinity to single populations of synaptic plasma membranes receptors, whereas opiate alkaloids bind to multiple sites. Initial kinetic experiments revealed that agonist rates of association were radioligand concentration-independent. Pseudo first-order rate constants for DADL, DAGO, diprenorphine, and naltrexone association were estimated to be 5.63 X 10(5), 5.08 X 10(5), 4.60 X 10(6), and 2.3 X 10(6) mol-1 X s-1, respectively. After preincubation of 0.2-1 nM radioligand for variable time intervals, dissociation was initiated by addition of 1 microM unlabeled ligand. If saturation binding was achieved before dissociation was initiated, then nearly monophasic dissociation of DADL, DAGO, and diprenorphine and a biphasic off-rate for naltrexone were observed. When association times were reduced to pre-equilibrium intervals, the kinetics of dissociation of agonists became biphasic and association time-dependent, but that for antagonists did not change significantly. Comparisons by both graphical methods and computerized nonlinear regression analyses of rate constants revealed that the fraction of the rapid component of agonist dissociation decreases and that of the slow component is elevated with increasing receptor occupancy. In the presence of 100 mM NaCl, DADL dissociation became association time-independent. These data are consistent with the idea that the Na+ effect is brought about by a change of receptor to an antagonist-like conformation. On the basis of both association and dissociation kinetic data, opioid agonists appear to interact in a multistep process in which a rapid, reversible association is followed by the formation of a more tightly bound complex.     

Stimulation of benzodiazepine binding to rat brain membranes and solubilized receptor complex by avermectin B1a and gamma-aminobutyric acid

The binding of [3H]flunitrazepam to benzodiazepine receptors in synaptic membranes and a digitonin-solubilized receptor fraction of rat brain is increased by avermectin B1a and gamma-aminobutyric acid (GABA). The effects of avermectin B1a and GABA are both sensitive to inhibition by (+)-bicuculline. Avermectin B1a and GABA both decrease the Kd and increase the Bmax of [3H]flunitrazepam binding to membranes. Kinetic analysis of the binding of [3H]flunitrazepam to rat brain membranes indicates that avermectin B1a and GABA reduce the rate constants of both association and dissociation between the ligand and the receptor. These results suggest a similar mechanism of modulation of benzodiazepine binding by avermectin B1a and GABA. This modulation may involve in interaction among the receptors for benzodiazepine, GABA and avermectin B1a.     

Somatostatin receptors on rat pancreatic acinar cells. Pharmacological and structural characterization and demonstration of down-regulation in streptozotocin diabetes

The binding of somatostatin-14 (S-14) to rat pancreatic acinar cell membranes was characterized using [125I-Tyr11]S-14 as the radioligand. Maximum binding was observed at pH 7.4 and was Ca2+-dependent. Such Ca2+ dependence of S-14 receptor binding was not observed in other tissues. Scatchard analysis of the competitive inhibition by S-14 of [125I-Tyr11]S-14 binding revealed a single class of high affinity sites (Kd = 0.5 +/- 0.07 nM) with a binding capacity (Bmax) of 266 +/- 22 fmol/mg of protein. [D-Trp8]S-14 and structural analogs with halogenated Trp moiety exhibited 2-32-fold greater binding affinity than S-14, [D-F5-Trp8]S-14 being the most potent. [Tyr11]S-14 was equipotent with S-14. The affinity of somatostatin-28 for binding to these receptors was 50% of that of S-14. Cholecystokinin octapeptide (CCK-8) inhibited the binding of [125I-Tyr11]S-14, but its inhibition curve was not parallel to that of S-14. In the presence of 1 nM CCK-8, the Bmax of S-14 receptors was reduced to 150 +/- 17 fmol/mg of protein. Dibutyryl cyclic GMP, a CCK receptor antagonist, partially reversed the inhibitory action of CCK-8, suggesting that CCK receptors mediate the inhibition of S-14 receptor binding. GDP, GTP, and guanyl-5'-yl imidodiphosphate inhibit S-14 receptor binding in this tissue. The inhibition was shown to be due to decrease in binding capacity and not due to change in affinity. Specifically bound [125I-Tyr11]S-14 cross-linked to the S-14 receptors was found associated with three proteins of approximate Mr = 200,000, 80,000, and 70,000 which could be detected under both reducing and nonreducing conditions. Finally, pancreatic acinar cell S-14 receptors were shown to be down-regulated by persistent hypersomatostatinemia 1 week after streptozotocin-induced diabetes characterized by decreased Bmax (105 +/- 13 fmol/mg of protein) without any change in affinity. We conclude that pancreatic acinar cell membrane S-14 receptors require Ca2+ for maximal binding and thus differ from S-14 receptors in other tissues, S-14 receptors in this tissue also exhibit selective ligand specificities, these receptors are regulated by CCK-8 and guanine nucleotides, three receptor proteins of apparent Mr = 200,000, 80,000, and 70,000 specifically bind S-14, and (v) these receptors are regulated by S-14 in vivo as evidenced by decreased binding in streptozotocin diabetic rats characterized by hypersomatostatinemia.     

Binding of N-[propionyl-3H]propionylated alpha-bungarotoxin and L-[benzilic-4,4'-3H] quinuclidinyl benzilate to CNS extracts of the cockroach Periplaneta americana

The nerve cord of the cockroach (Periplaneta americana) contains distinct saturable components of specific binding for the ligands N-[propionyl-3H]propionylated alpha-bungarotoxin and L-[benzilic-4,4'-3H]quinuclidinyl benzilate. N-[Propionyl-3H]propionylated alpha-bungarotoxin bound reversibly to homogenates with a Kd of 4.8 nM and Bmax of 910 fmol mg-1. The association rate constant (1.9 X 10(5) M-1 s-1) and dissociation rate constant (1.2 X 10(-4) s-1) yielded a Kd of 0.6 nM. Nicotinic ligands were found to displace toxin binding most effectively. The binding sites characterized in this way showed many similarities with the properties of the vertebrate neuronal alpha-bungarotoxin binding site. For a range of cholinergic ligands, inhibition constants calculated from toxin binding studies closely corresponded to their effectiveness in blocking the depolarizing response to acetylcholine recorded by electrophysiological methods from an identified cockroach motoneurone. The N-[propionyl-3H]propionylated alpha-bungarotoxin binding component therefore appears to be a constituent of a functional CNS acetylcholine receptor. Binding of L-[benzilic-4,4'-3H]quinuclidinyl benzilate was reversible with a Kd of 8 nM and Bmax of 138 fmol mg-1, determined from equilibrium binding experiments. The Kd calculated from the association rate constant (2.4 X 10(5) M-1 s-1) and dissociation rate constant (1.3 X 10(-4) s-1) was 1.9 nM. Muscarinic ligands were the most potent inhibitors of quinuclidinyl benzilate binding. The characteristics of this binding site resembled those of vertebrate CNS muscarinic cholinergic receptors. In contrast with vertebrate CNS, the nerve cord of Periplaneta americana contains more (approximately X 7) alpha-bungarotoxin binding sites than quinuclidinyl benzilate binding sites.     

Characterization of receptors for immune interferon in U937 cells with 32P-labeled human recombinant immune interferon

Recombinant human immune interferon (HuIFN-gamma) was labeled with [gamma-32P]ATP and cyclic-AMP-dependent protein kinase from bovine heart to a specific radioactivity of 11,000 Ci/mmol. At least two molecules of phosphate were incorporated per molecule of interferon. The binding of [32P]HuIFN-gamma to human U937 histiocytic lymphoma cells was time dependent, and displaceable by HuIFN-gamma but not by HuIFN-alpha A or HuIFN-beta. The specific binding was saturable with less than 10% nonspecific binding. The dissociation constant of [32P]HuIFN-gamma for U937 interferon receptors was calculated to be 1.5 X 10(-10) M with a total of 1,800 binding sites/cell. Dissociation of bound [32P]IFN-gamma at 24 degrees C exhibited two distinct rates. A fast dissociation with a specific rate constant of 0.141 min-1, and a slow dissociation with a specific rate constant of 0.0027 min-1. The Kd for [32P]HuIFN-gamma was calculated from kinetic constants to be 5.4 X 10(-10) M.     

Kinetic analyses demonstrate that the equilibrium assumption does not apply to [125I]endothelin-1 binding data.

The kinetics of [125I]Endothelin-1 ([125I]ET-1) binding were studied using membranes from rat heart, rat lung, rat brain, and porcine vascular smooth muscle at 37 degrees C in 0.05M Tris-HCl buffer (pH = 7.4). The dissociation half-life (t1/2, diss.) for bound [125I]ET-1 was in excess of 30 hours for each tissue studied. Equilibrium-time requirements for proper Scatchard analysis of [125I]ET-1 were also far in excess of 30 hours for each tissue. These data suggest that determination of dissociation constants, Kd, and receptor concentrations, Bmax, by conventional Scatchard analysis is not feasible with [125I]ET-1. Kinetic analyses may provide a more accurate means for determining [125I-ET-1] binding characteristics including Kd and Bmax.     

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.     

The formylpeptide chemotactic receptor on rabbit peritoneal neutrophils. I. Evidence for two binding sites with different affinities

F Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe binding to rabbit peritoneal neutrophils and purified membranes were measured at 4 degrees C silicone oil centrifugation assays, and the results were analyzed by the LIGAND computer program, which permits analysis of ligand binding to multiple classes of binding sites. LIGAND analysis of peptide binding to intact neutrophil indicated that both f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe detected two population of binding sites. The apparent Kd values for f Met-Leu-[3H]Phe binding were 1.6 +/- 1.0 X 10(-9) M and 2.2 +/- 0.9 X 10(-8) M, respectively, and 3.1 +/- 0.2 X 10(-9) M and 1.2 +/- 0.6 X 10(-7) M for f Nle-Leu-[3H]Phe. Furthermore, the higher affinity sites detected on whole cells comprised approximately 15 to 30% of the total sites. Two populations of binding sites were also detected on purified neutrophil plasma membranes by both radiolabeled chemotactic peptides. LIGAND analysis of peptide binding to purified membranes yielded apparent Kd values of 5.0 +/- 2.5 X 10(-10) M and 4.8 +/- 0.6 X 10(-8) M for f Met-Leu-[3H]Phe binding, and 4.7 +/- 4.2 X 10(-10) M and 3.0 +/- 1.3 X 10(-8) M for f Nle-Leu-[3H]Phe. The percentage of higher affinity sites detected by f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe on purified membranes were 1 to 5% of the total sites detected. These data are consistent either with the existence of two independent binding sites for formylpeptides on rabbit neutrophils or receptor negative cooperativity.     

Characterization and photoaffinity labeling of receptor sites for the Ca2+ channel inhibitors d-cis-diltiazem, (+/-)-bepridil, desmethoxyverapamil, and (+)-PN 200-110 in skeletal muscle transverse tubule membranes

In order to further understand the molecular nature of the voltage-sensitive Ca2+ channel in skeletal muscle, we have performed classical radioligand binding studies and photoaffinity labeling with different types of tritiated inhibitors of the Ca2+ channel. The equilibrium dissociation constants (KD) for (-)-[3H]desmethoxyverapamil, d-cis-[3H]diltiazem, and (+/-)-[3H]bepridil at their receptor sites in skeletal muscle transverse tubule membranes are: 1.5 +/- 0.5, 50 +/- 5, and 20 +/- 5 nM, respectively. Maximum binding capacities in picomoles/milligram of protein were: 70 +/- 10 for (-)-[3H]desmethoxyverapamil, 50 +/- 15 for d-cis-[3H]diltiazem, and 75 +/- 15 for (+/-)-[3H]bepridil. The kinetics of association at 10 degrees C for the three types of tritiated compounds were relatively slow (3 X 10(5) M-1 S-1 for (-)-[3H]desmethoxyverapamil, 8 X 10(3) M-1 S-1 for d-cis-[3H]diltiazem, and 4.2 X 10(5) M-1 S-1 for (+/-)-[3H]bepridil). The dissociation of (-)-[3H]desmethoxyverapamil and d-cis-[3H]diltiazem from their receptor sites was also a slow process with half-lives of dissociation of 33 and 36 min, respectively. Competition studies using the three tritiated ligands suggest that they bind to the same receptor site which appears to be in a 1:1 stoichiometry with the dihydropyridine receptor. Photoaffinity labeling with high intensity ultraviolet light in the presence of (+/-)-[3H]bepridil or d-cis[3H]diltiazem resulted in the specific covalent incorporation of radioactivity into a polypeptide of Mr 170,000 +/- 10,000. A polypeptide of Mr 170,000 was also specifically labeled in photoaffinity labeling experiments using the high affinity dihydropyridine derivative (+)-[3H]PN 200-100.     

Mechanism of agonist and antagonist binding to alpha 2 adrenergic receptors: evidence for a precoupled receptor-guanine nucleotide protein complex

The alpha 2 adrenergic receptor (AR) inhibits adenylate cyclase via an interaction with Ni, a guanine nucleotide binding protein. The early steps involved in the activation of the alpha 2 AR by agonists and the subsequent interaction with Ni are poorly understood. In order to better characterize these processes, we have studied the kinetics of ligand binding to the alpha 2 AR in human platelet membranes on the second time scale. Binding of the alpha 2 antagonist [3H]yohimbine was formally consistent with a simple bimolecular reaction mechanism with an association rate constant of 2.5 X 10(5) M-1 s-1 and a dissociation rate constant of 1.11 X 10(-3) s-1. The low association rate constant suggests that this is not a diffusion-limited reaction. Equilibrium binding of the alpha 2 adrenergic full agonist [3H]UK 14,304 was characterized by two binding affinities: Kd1 = 0.3-0.6 nM and Kd2 = 10 nM. The high-affinity binding corresponds to approximately 65% and the low-affinity binding to 35% of the total binding. The kinetics of binding of [3H]UK 14,304 were complex and not consistent with a mass action interaction at one or more independent binding sites. The dependence of the kinetics on [3H]UK 14,304 concentration revealed a fast phase with an apparent bimolecular reaction constant kappa + of 5 X 10(6) M-1 s-1. The rate constants and amplitudes of the slow phase of agonist binding were relatively independent of ligand concentration. These results were analyzed quantitatively according to several variants of the "ternary complex" binding mechanism. In the model which best accounted for the data, (1) approximately one-third of the alpha 2 adrenergic receptor binds agonist with low affinity and is unable to couple with a guanine nucleotide binding protein (N protein), (2) approximately one-third is coupled to the N protein prior to agonist binding, and (3) the remainder interacts by a diffusional coupling of the alpha 2 AR with the N protein or a slow, ligand-independent conformational change of the alpha 2 AR-N protein complex. The rates of interaction of liganded and unliganded receptor with N protein are estimated.     

Neurotrophic action of nicotinamide and nicotinoyl-GABA in rat brain in experimental Parkinsonism]

It was established, that the content of nicotinamide adenine dinucleotides and the binding of NAD by isolated brain cortex synaptic membranes under experimental parkinsonism are impaired. Treatment of the experimental results in the Scatchard plots for binding of [U-14C]NAD to the brain cortex synaptic membranes demonstrated that binding capacities lowered, without changes of affinities. NAD-glycohydrolase involved in development of this pathology. The modulative effect of in vivo administered NAm and nicotinoyl-GABA supposes that NAm acts via NAD which binds specifically with synaptic membranes. Thus, NAm and nicotinoyl-GABA are involved in the regulation of the processes in the brain under experimental parkinsonism.