Increase in the amount of adenylate cyclase in rat gastrocnemius muscle after denervation

After section of the sciatic nerve, the basal adenylate cyclase (AC) activity in rat gastrocnemius muscle increased 6-7 times per membrane protein and about 2 times per whole muscle in the following 30 or 40 days. The AC activity in the muscle 30 days after denervation was increased about 4 times by forskolin. Calcitonin gene-related peptide (CGRP) also increased the adenylate cyclase activity in the denervated muscle. The binding of [3H]-forskolin (10nM) to cells isolated from gastrocnemius muscle was examined to determine the amount of AC molecules. Inhibition of [3H]-forskolin binding by increasing amounts of unlabeled forskolin gave a sigmoid curve with a IC50 value of 3 x 10(-7) M. Results showed that the number of [3H]-forskolin binding sites per cell was higher on the denervated side than on the control side, like the basal AC activity. The IC50 values for inhibition by unlabeled forskolin of binding of [3H]-forskolin were similar to muscles on the control and denervated sides. These results suggest that an increase in the AC activity induced by denervation was due to an increase in the numbers of AC molecules in the muscle.     

Characterization of [3H]-forskolin binding sites in young and adult rat brain cortex: identification of suramin as a competitive inhibitor of [3H]-forskolin binding

Little is know about forskolin binding in the rat brain during ontogenetic development. For this paper, we have characterized specific binding sites for [3H]-forskolin in cerebrocortical membranes from young (12-day-old) and adult (90-day-old) rats. High-affinity, as well as super-high-affinity, [3H]-forskolin binding sites were detected in samples from both age groups tested, and the binding parameters of these sites differed significantly. Whereas the number of high-affinity [3H]-forskolin binding sites was higher by about 50% in adult than in young rats, their affinity was markedly (about 4 times) lower. In the presence of AlF4-, the number high-affinity [3H]-forskolin binding sites in samples from young rats rose to the level determined in samples from adult animals, and the number of super-high-affinity sites considerably increased in both age groups. The different characteristics of [3H]-forskolin binding found in cerebrocortical membranes from young and adult rats may be closely related to markedly diminished adenyl cyclase activity in preparations from adult animals. Results of our experiments with suramin indicated that this drug may act as a competitive inhibitor of [3H]-forskolin binding.     

Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of [3H]forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited [3H]forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP gamma S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolin but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP gamma S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.     

Binding of [3H]forskolin to solubilized preparations of adenylate cyclase

The binding of [3H]forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating [3H]forskolin bound to protein from free [3H]forskolin by rapid filtration. The Kd for [3H]forskolin binding to solubilized proteins was 14 nM which was similar to that for [3H]forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for [3H]forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. [3H]forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmol/mg protein which increased to 94 fmol/mg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on [3H]forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmol/mg/min which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmol/mg/min which was not stimulated by GppNHp or forskolin. Thus, the number of high affinity binding sites for [3H]forskolin in solubilized preparations correlated with the activation of adenylate cyclase by GppNHp via the guanine nucleotide binding protein (GS).     

Presence of basic fibroblast growth factor receptors in bovine brain membranes

We described a protocol for purification of bovine brain membranes suitable to study the binding of iodinated basic fibroblast growth factor (FGF) to bovine brain membrane preparation. The binding of 125I basic FGF to brain membranes reached equilibrium within 30 min at 20 degrees C, was reversible, and displaced by an excess of unlabeled basic FGF. Scatchard analysis of the data revealed that two classes of binding sites could be detected with an apparent Kd of 30 pM and a capacity of 0.24 pmol/mg of membrane proteins for the high affinity binding site and Kd of 3 nM with a capacity of 51 pmol/mg of membrane proteins for the low affinity binding site. Cross-linking experiments of labeled basic FGF to brain membrane receptor yield the formation of a single major complex with an apparent molecular mass of 170 kDa which is similar to the value obtained for the high affinity binding site for basic FGF on target cells in tissue culture. Hence these data present the first biochemical evidence suggesting that membrane purified from bovine brain contain two classes of specific binding sites for basic FGF and confirm results described with cells grown in vitro.     

Evidence for vasoactive intestinal peptide receptors in apical membranes from tracheal epithelium

[125I]VIP (vasoactive intestinal peptide) bound to apical membranes isolated from the bovine tracheal epithelium with a half maximal inhibition by unlabeled VIP (IC50) of 0.6 x 10(-9)M and binding was reversible. Glucagon did not affect [125I]VIP binding to the membranes. [125I]VIP was covalently cross-linked to tracheal membrane proteins using disuccinimidyl suberate. SDS-polyacrylamide gel electrophoresis of labeled tracheal membranes revealed one major [125I]-receptor complex of Mr = 71,000 to which binding of [125I]VIP was inhibited by 10 microM unlabeled VIP. These results are consistent with the presence of a specific, high-affinity receptor for VIP, with a Mr = 71,000, in apical membrane vesicles isolated from the bovine tracheal epithelium.     

Catecholamine binding to plasma membrane enriched fractions of heart and skeletal muscle.

Binding of [3H]epinephrine to plasma membrane enriched fractions from guinea pig heart and rabbit skeletal muscle was investigated using the micropore filtration technique. [3H]Epinephrine and [3H]norepinephrine were found to be degraded rapidly in aqueous buffer at pH 7.6 and 37 degrees C. Deterioration of the compounds could be prevented by low concentrations of dithiothreitol. Binding of [3H]epinephrine to both membrane preparations was a slow process requiring 60 min to approach equilibrium in the case of cardiac membranes at 37 degrees C, and 20 min for skeletal muscle membranes at O degrees C. Binding was antagonized by the unlabeled beta-agonists, isopropylnorepinephrine, epinephrine, and norepinephrine but all were equipotent. A variety of catechol compounds were as effective antagonists of binding as the catecholamines. The beta-adrenergic antagonists propranolol, pronethalol, and dichloroisoproterenol were not effective in inhibiting binding to either membrane preparation. D-Norepinephrine and L-norepinephrine were equi-effective in antagonizing binding of [3H]norephinephrine to skeletal muscle membranes. It was concluded that binding of labeled catecholamine to isolated tissue membranes using the micropore filtration technique does not represent interaction with the specific beta-adrenergic receptor, but more likely reflects a less specific binding of compounds having one or more hydroxyl groups on a ring.     

Characterization of soluble and particulate parathyroid hormone receptors using a biotinylated bioactive hormone analog

A bioactive biotin-containing derivative of the synthetic bovine parathyroid hormone analog [Nle8,Nle18,Tyr34]bovine parathyroid hormone-(1-34) (bPTH-(1-34] amide was prepared by reacting the peptide with N-biotinyl-epsilon-aminocaproic acid N-hydroxysuccinimide ester. The derivative was incubated with particulate renal plasma membranes or with detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) extracts of renal cortical membranes, and two membrane components were identified. Labeling of these components was competitively inhibited by underivatized bPTH-(1-34) or bPTH-(3-34) but not by insulin, adrenocorticotropin, or oxidized rat PTH-(1-34). PTH-binding components that were immobilized on nitrocellulose could be detected by incubating the membrane with biotinyl-bPTH-(1-34). Binding components of apparent molecular mass 68, 70, and 150 kDa were specifically labeled in plasma membranes derived from canine, human, and porcine renal cortex, rat liver, and human fibroblasts. The 68-kDa binding protein was found to be consistently more acidic than the 70-kDa binding protein in human, porcine, and canine renal membranes analyzed by two-dimensional electrophoresis. The 68-70-kDa receptor doublet could be specifically isolated by streptavidin-agarose chromatography of solubilized membrane extracts that had first been incubated with biotinyl-BPTH-(1-34). Biotinyl-bPTH-(1-34) should be useful as a tool for further characterization and purification of the PTH receptor.     

Effect of chronic administration of antidepressants on the binding of 3H-imipramine with mouse brain synaptic membranes

Subchronic (14 days) administration of chlorimipramine and zimelidine, and a new morpholine derivative produces different effects on 3H-imipramine binding with synaptic membranes of the mouse brain. Chlorimipramine increases the concentration of the binding sites (Bmax), zimelidine raises both the Bmax and the dissociation constant, while the new morpholine derivative does not significantly change these characteristics. It has been demonstrated that the changes in the Bmax that occur during prolonged administration of chlorimipramine depend on the method for the obtaining of membrane brain preparations.     

Studies of binding of parathyroid hormone to a detergent-dispersed preparation from bovine kidney cortex plasma membranes.

Bovine kidney plasma membranes containing parathyroid hormone-sensitive adenylate cyclase activity were dispersed with 1% Triton X-100 and centrifuged at 150,000 X g for 2 h. Approximately 40% of the total membrane protein was extracted by this procedure. The extraction greatly reduces the fluoride-stimulated and the parathyroid hormone-sensitive adenylate cyclase activity of the membranes and yields a supernatnat which binds biologically active, tritiated parathyroid hormone. Hormone binding is stable for up to 15 h and has a linear dependence on protein concentration in the extract. Binding of the labeled hormone at concentrations of 5 to 10 nM is inhibited by preincubation with unlabeled min, and displays a dependence on temperature, time, and pH. Binding specificity is maximal at physiological pH, being inhibited by only the native hormone or its synthetic 1-34 NH2-terminal, biologically active fragment. Binding increases dramatically at pH 6.0, but is nonspecific in character. Half-maximal inhibition of the binding was achieved at 3.2 X 10(-7) M concentrations of the native hormone and 5.0 X 10(-7) M concentrations of the synthetic 1-34 NH2-terminal fragment. Calcium does not inhibit either total or specific binding. Inhibition, kinetic, and pH dependence data suggest that the extracted component(s) represent the parathyroid hormone binding protein(s) formerly identified in particulate membrane preparations.     

Stimulation of Postsynaptic and Inhibition of Presynaptic Adenylyl Cyclase Activity by Metabotropic Glutamate: Receptor Activation

Abstract: To determine the subcellular distribution of cyclic AMP-coupled metabotropic glutamate receptors (mGluRs), the effects of glutamate agonists on adenylyl cyclase activity were examined using two hippocampal membrane preparations. These were synaptosomes (SY), which are composed of presynaptic terminals, and synaptoneurosomes (SN), which are composed of both pre-and postsynaptic elements. In SY, a water-soluble analogue of forskolin (7β-forskolin) increased enzyme activity ˜ 10-fold at the highest concentration tested. The selective metabotropic receptor agonist (1S,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3 R -ACPD) inhibited enzyme activity as did glutamate and quisqualate. l -Amino-4-phosphobutanoate ( l -AP4) had no effect on enzyme activity at any concentration tested. The metabotropic receptor antagonist l -2-amino-3-phosphopropionic acid ( l -AP3) was not effective in the SY in antagonizing the agonist-induced decreases in adenylyl cyclase activity by glutamate or 1S,3 R -ACPD. It was, however, effective at antagonizing quisqualate-induced decreases in enzyme activity. In SN, at the highest concentration tested, 7β-forskolin produced a 60-fold increase in adenylyl cyclase activity. As was observed in SY, glutamate decreased adenylyl cyclase activity in SN. In contrast, 1S,3 R -ACPD, quisqualate, and l -AP4 increased adenylyl cyclase activity. In the SN, l -AP3 was ineffective in antagonizing any agonist-induced increases (1S,3 R -ACPD, l -AP4, and quisqualate) or decreases (glutamate) in adenylyl cyclase activity. The data suggest that postsynaptic metabotropic glutamate receptor activation results in stimulation of adenylyl cyclase activity, whereas inhibition of this enzyme appears to be mediated at least partly through presynaptic mechanisms.     

Affinity cross-linking of atrial natriuretic factor to its receptor in bovine adrenal zona glomerulosa

125I-Labeled atrial natriuretic factor (ANF) was covalently cross-linked to its binding sites in bovine adrenal zona glomerulosa membranes using the hydrophilic cross-linker bis(sulfosuccinimidyl) suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol revealed that two protein bands with apparent Mr 68,000 and 114,000 were specifically labeled. The labeling of the two bands was prevented in a dose-dependent fashion by unlabeled ANF with a significant inhibition observed at 10(-10) M. High concentrations of angiotensin II and adrenocorticotropic hormone did not compete with 125I-ANF for binding and cross-linking. The dose-response curve for inhibition of covalent cross-linking of 125I-ANF by unlabeled ANF coincided with the dose-response curve for inhibition of binding to the receptor. No radioactive bands were observed in liver membranes. Experiments in which adrenal membranes were prepared and incubated in the presence of protease inhibitors showed no difference in the labeling pattern. Electrophoresis in the absence of reductant showed that the affinity-labeled species are not derived from larger disulfide-linked components. The apparent molecular weight of the two labeled species was not affected by a 100-fold variation in cross-linker concentration, and the labeling of both species increased in parallel. Possible relationships between the two labeled species are discussed.     

Binding to opioid receptors of enkephalin derivatives taking alpha-helical conformation and its dimer.

The opioid receptor binding of [Leu]enkephalin derivatives with extended address segment to the C-terminal was studied. The extension peptide is designed to take an amphiphilic helical structure in order to evaluate effects of helical conformation and membrane affinity of enkephalin moiety of the derivatives on receptor binding. In the delta-receptor-selective binding assay, Tyr-Gly-Gly-Phe-Leu-Lys-Aib-Leu-Aib-OH (1) showed the same affinity as enkephalinamide, whereas in the mu-receptor-selective binding assay, a 7-fold reduction in affinity was observed. On the other hand, Tyr-Gly-Gly-Phe-Leu-(Lys-Aib-Leu-Aib)2-OH (2) showed 51- and 96-fold decreases in affinities for delta- and mu-receptors, respectively, compared with enkephalinamide. The low receptor affinity of derivative 2 is considered due to alpha-helical conformation, which might not be compatible with topological requirements of delta- and mu-receptors. A dimer, Tyr-Gly-Gly-Leu-Phe-(Lys-Aib-Leu-Aib)2-Lys(X)-Aib-OCH3 (X = Tyr-Gly-Gly-Phe-Leu-, (4], showed 2.5- and 3.0-fold increases in affinities respectively for delta- and mu-receptors compared with the monovalent derivative 2, possibly due to cross-linking of neighboring receptors. The Hill plot of the binding of the dimer to bovine brain membranes was composed of two phases, although such a heterogeneity of receptors was not observed in the presence of naloxone or in the binding to NG108-15 cell membranes. These findings indicate the presence of the bivalent-ligand-induced interactions between delta- and mu-receptors in bovine brain membranes.     

Saturable binding to cell membranes of the presynaptic neurotoxin, beta-bungarotoxin.

Brief exposure to the protein neurotoxin, beta-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using 125I-labelled toxin. At low membrane protein concentration 125I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration 125I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes. In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of 125I-labeled toxin binding sites on brain mitochondrial preparations (3.3 +/- 0.3 pmol/mg) exceeded by a factor of ten the density on liver mitochondrial preparations (0.3 +/- 0.05 pmol/mg). It is also shown that labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude that beta-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.     

Characterization of canine renal receptors for the parathyroid hormone-like protein associated with humoral hypercalcemia of malignancy

Parathyroid hormone-like proteins (PTHLP) display actions in the kidney which are similar to those of parathyroid hormone (PTH). We compared the binding properties of PTHLP and PTH in canine renal cortical membranes to determine if they interacted with the same or different receptors. Radioiodination to high specific activity (greater than 400 microCi/micrograms) of [Nle8,18,Tyr34]human PTH-(1-34)amide and [Tyr36]PTHLP-(1-36)amide was performed using the lactoperoxidase method. Complete enzymatic digestion of both radioligands demonstrated that the peptides were monoiodinated. Both radioligands retained full biological activity in the renal adenylate cyclase assay, and neither was significantly degraded during incubation with highly purified canine renal membranes under binding assays conditions. Specific binding reached equilibrium by 20 min at 20 degrees C. Competition binding studies using unlabeled [Nle8,18,Tyr34]human PTH-(1-34)amide, [Tyr36] PTHLP-(1-36)amide, and bovine PTH-(1-34) with either radioligand revealed similar binding affinities for all three peptides. Biologically inactive PTHLP fragments did not show significant displacement. In contrast to its similar binding affinity, [Tyr36]PTHLP-(1-36)amide was 6-15-fold less potent than bovine PTH-(1-34) in the renal adenylate cyclase assay, suggesting less efficient receptor-effector coupling. Photoaffinity cross-linking using either radioligand in canine renal membrane labeled indistinguishable 70,000-dalton proteins. In the presence of multiple protease inhibitors, binding to an 85-kDa component was observed. Labeling of both receptor forms was specifically abolished by an excess of either cold peptide and dose-response curves using affinity cross-linked membranes corroborated the apparent binding affinities determined by conventional radioligand binding assays. We conclude that PTHLP-(1-36) and amino-terminal PTH analogues bind to indistinguishable receptors in canine renal cortical membranes, but display differential coupling to post-receptor events.     

Angiotensin II binding to mammalian brain membranes.

High affinity binding sites for angiotensin II in bovine and rat brain membranes have been identified and characterized using monoiodinated Ile5-angiotensin II of high specific radioactivity. Degradation of labeled and unlabeled peptide by washed brain particulate fractions was prevented by adding glucagon to the final incubation medium and including a proteolytic enzyme inhibitor (phenylmethylsulfonyl fluoride) in preincubation and incubation procedures. 125I-Angiotensin II binding can be studied using either centrifugation or filtration techniques to separate tissue-bound radioactivity. 125I-Angiotensin II binding to calf brain membranes is saturable and reversible, with a dissociation binding constant of 0.2 nM at 37 degrees. A similar binding constant is found in rat brain membranes. Analogues and fragments of angiotensin II compete for these brain binding sites with potencies which correlate with both their in vivo potencies and their binding inhibition protencies at adrenal cortex angiotensin II receptors. Angiotensin I is 1 to 2 orders of magnitude weaker than angiotensin II; the 3-8 hexapeptide and 4-8 pentapeptide are much weaker still. (desAsp1) angiotensin II (angiotensin III) is slightly more potent than angiotensin II, as are several antagonists of angiotensin II with aliphatic amino acids substituted at position 8. In calf brain 125I-angiotensin II binding is restricted almost exclusively to the cerebellum (cortex and deep nuclei). In rat brain, angiotensin II binding is highest in the thalamus-hypothalamus, midbrain, and brainstem, areas which are believed to be involved in mediating angiotensin II-induced central effects. These findings illustrate the presence of high affinity specific binding sites for angiotensin II in rat and bovine brain and suggest a physiological role for angiotensin peptides in the central nervous system.     

Insulin receptor interaction in human placental plasma membranes

Insulin-receptor interaction in partially purified preparations of human placental plasma membranes from normal mothers at term of pregnancy has been characterized. 125I-insulin became rapidly and reversibly bound to plasma membranes, being time and temperature dependent. The binding readily appeared at 1.0 ng/ml insulin concentration which falls within the physiological range of peripheral blood. Low levels of unlabeled insulin inhibited binding; 20 ng/ml insulin produced fifty per cent inhibition. Scatchard plots of data from competitive insulin binding proved to be curvilinear. The insulin greater ability for binding observed in this preparation can be explained by the purification degree achieved at the plasma membranes. 125I-insulin was less degraded by partially purified placental plasma membranes than by a microsomal-membrane preparation obtained without differential centrifugation in sucrose linear gradient. All these properties strongly suggest that the insulin-binding sites characterized in the plasma membrane fraction of the placenta represent biologically important receptors to hormone.     

Levels of Gsα(short and long), Gα(olf) and Gβ(common) subunits, and calcium-sensitive adenylyl cyclase isoforms (1, 5/6, 8) in post-mortem human brain caudate and cortical membranes: comparison with rat brain membranes and potential stoichiometric relationships

The levels of expression of Gsα(short and long), Gα(olf) and Gβ(common) subunits, and calcium-sensitive adenylyl cyclases isoforms (AC1, 5/6, and 8) in human brain cortical and caudate membranes were quantified by western blot analysis in order to establish their contribution to the patterns of AC functioning. Both areas expressed Gsα(long) (52 kDa) with values ranging from about 1400 ng/mg of membrane protein in cerebral cortex to close to 600 ng/mg of membrane protein in caudate nucleus. In contrast, Gsα(short) and Gsα(olf) were expressed separately, Gsα(short) in cortical membranes with values around 500 ng/mg of membrane protein and Gα(olf) in caudate membranes with values around 1300 ng/mg of membrane protein. Quantitative measurements of Gβ, revealed a similar expression level in cortical and caudate membranes (5444±732 versus 5511±394 ng/mg protein; p=0.966). The B(max) values of GTPγS-dependent [(3)H]-forskolin binding show the following descending order: rat striatal membranes>rat cortical membranes=human caudate membranes>human cortical membranes. Therefore, as measured immunochemically and by [(3)H]-forskolin binding, there seems to be a vast excess of Gsα subunits over catalytic units of AC. The highest levels of AC5/6 expression were detected in caudate membranes. AC8 was little expressed, and there were no significant differences in the relative values between both human brain regions. Finally, the levels of the AC1 isoform were significantly lower in caudate than in cortical membranes. It is concluded that these stoichiometric data contribute nonetheless to explain the significant differences observed in signalling capacities through the AC system in both human brain regions.     

Characterization of Membrane-Bound and Solubilized High-Affinity Binding Sites for 5'-N-Ethylcarboxamido[3H]adenosine from Bovine Cerebral Cortex

Abstract: A high-affinity binding site for 5'- N -ethylcarboxamido[³H]adenosine ([³H]NECA) from bovine cerebral cortex has been characterized in its membrane-bound and solubilized state after gel filtration on Sepharose CL-6B. For detection of this site in membranes, it was necessary to remove metabolites with high affinities for this site enzymatically, e.g., adenosine by addition of adenosine deaminase and inosine by addition of nucleoside phosphorylase. The pore-forming peptide antibiotic alamethicin further enhanced binding of [³H]NECA to this site in membranes. In contrast to adenosine receptors and the adenotin-like low-affinity binding protein, this novel site was extremely sensitive against treatment with the sulfhydryl alkylating agent N -ethylmaleimide. In competition experiments, this site could be differentiated from adenosine receptors by its high affinity for adenine nucleotides and its lack of affinity for adenosine receptor antagonists. Inosine and its derivative S -(4-nitrobenzyl)-6-thioinosine were relatively potent ligands with K _i values in the high nano- and low micromolar range, respectively. We conclude that the high-affinity NECA binding site described previously in bovine striatum is not exclusively located in the striatum, but can also be detected in membrane preparations and soluble extracts of bovine brain cortex.     

Binding of tritiated bovine parathyroid hormone to plasma membranes from bovine kidney cortex.

A membrane fraction enriched in parathyroid hormone (PTH)-sensitive adenylate cyclase and sodium and potassium ion-activated (Na+, K+)-ATPase was prepared from bovine kidney. Tritiated PTH binding to this membrane fraction was dependent on both hormone and membrane protein concentration. Both total and specific binding of the hormone decreased significantly after 5 to 10 min of incubation at 22 degrees. PTH binding was highly specific, being sensitive to inhibition only with active forms of unlabeled hormone (native and 1-34 PTH). Specific binding showed a pH optimum of 7.3 to 7.5. Inhibition of binding of tritiated hormone by unlabeled PTH was also highly effective at pH 6.0, but this apparently specific binding was also inhibited by adrenocorticotropic hormone, insulin, glucagon, and vasopressin. Dissociation of bound hormone was demonstrated, and an apparent dissociation constant of 4.6 X 10(-2) min-1 was obtained. Specific binding was eliminated by pretreatment of the membranes with trypsin. The concentration dependence for inhibition of binding with unlabeled PTH was identical to that for activation of adenylate cyclase in this membrane preparation, and binding was also inhibited by concentrations of calcium in the 0.5 to 2 mM range.