Photoaffinity labeling of the adenosine cyclic 3',5'-monophosphate receptor protein of Escherichia coli with 8-azidoadenosine 3',5'-monophosphate

Photoaffinity labeling of the cAMP receptor protein (CRP) of Escherichia coli with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) has been demonstrated. 8-N3cAMP is able to support the binding of (3H)d(I-C)n by CRP, indicating that it is a functional cAMP analogue. Following irradiation at 254 nm, (32P)-8-N3cAMP is photocross-linked to CRP. Photolabeling of CRP by (32P)-8-N3cAMP is inhibited by cAMP but not by 5'AMP. The data indicate that (32P)-8-N3cAMP is covalently incorporated following binding at the cAMP binding site of CRP. The (32P)-8-N3cAMP-CRP digested with chymotrypsin was analyzed by NaDodSO4-polyacrylamide gel electrophoresis. Of the incorporated label, one-third remains associated with the amino-proximal alpha core region of CRP [Eilen, E., Pampeno, C., & Krakow, J.S. (1978) Biochemistry 17, 2469] which contains the cAMP binding domain; the remaining two-thirds of the label associated with the beta region are digested. Limited proteolysis of the (32P)-8-N3cAMP-CRP by chymotrypsin in the presence of NaDodSO4 shows the radioactivity to be distributed between the molecular weight 9500 (amino-proximal) and 13,000 (carboxyl-proximal) fragments produced. These results suggest that a part of the carboxyl-proximal region is folded over and close enough to the cAMP binding site to be cross-linked by the photoactivated (32P)-8-N3cAMP bound at the cAMP binding site.     

Additional evidence for a proform to tropoelastin from chick aorta.

Evidence is presented for the presence of precursor to tropoelastin in chick arterial extracts. The precursor is approx. 100 000 daltons in size. It is suggested to be a precursor to tropoelastin (72 000 daltons). This protein may be observed in culture in vitro if appropriate precautions are taken to inhibit proteolysis. Once synthesized, it appears to be converted into tropoelastin within 10--20 min. The protein may also be detected in vivo. When 1-day-old cockerels were fed on a copper-deficient diet (less than 1 p.p.m. to inhibit cross-linking) containing epsilon-aminohexanoic acid (0.2%) to retard proteolysis and then injected wiht [3H]valine, extraction of arterial proteins 12h after injection resulted in detection of two major peaks of [3H]valine-labelled protein with pI values of pH 7.0 and 5.0 respectively. The protein that focused at pH 7.0 was estimated to be about 100 000 daltons in size and could be shown to be converted into a more basic protein with the properties of tropoelastin. It is speculated that the protein with pI 5.0 may be yet another extension peptide. The data appear to be in keeping with similar observations by ourselves and others that a proform of tropoelastin exists, and, in at least one step before conversion into tropoelastin, exists as a 100 000-dalton protein subunit.     

Structural analysis of the hepatic glucagon receptor. Identification of a guanine nucleotide-sensitive hormone-binding region

[125I-Tyr10]Monoiodoglucagon [( 125I]MIG) was cross-linked to liver membrane glucagon receptors with hydroxysuccinimidyl-p-azidobenzoate, and the products were analyzed by sodium dodecyl sulfate-gel electrophoresis. Autoradiograms of the gel obtained after a 24-h exposure showed one major band at Mr = 63,000 that was sensitive to GTP and excess unlabeled glucagon. Exposure for 7 days showed labeling of an additional Mr = 33,000 species that was also sensitive to excess unlabeled glucagon. The Mr = 33,000 peptide can be obtained by subtilisin, trypsin, elastase, or Staphylococcus aureus V8 protease treatment of the [125I]MIG-occupied receptor in the membrane or in Lubrol-PX solution. In contrast, limited proteolysis of membranes containing vacant receptors results in labeling of a Mr = 24,000 peptide. The Mr = 24,000 peptide specifically binds [125I]MIG in a GTP-sensitive manner. The Mr = 33,000 peptide also retains GTP sensitivity since it releases bound [125I]MIG upon addition of GTP. Elastase treatment of the electroeluted Mr = 33,000 peptide yields the Mr = 24,000 and 15,000 fragments. The Mr = 15,000 peptide is the smallest fragment of the receptor as yet identified. Treatment of the Mr = 63,000 receptor with [125I]MIG cross-linked to it with endo-beta-N-acetylglucosaminidase F results in four distinct fragments with Mr values of 61,000, 56,000, 51,000, and 45,000; prolonged treatment resulted in the accumulation of the last two. Neither the Mr = 33,000 nor the Mr = 24,000 fragment appeared to be substrates for endo-beta-N-acetylglucosaminidase F. These data indicate that glucagon receptor is a glycoprotein of approximately 60,000 daltons which contains at least four N-linked glycans accounting for 18,000 daltons of its mass. Both its glucagon binding function and its capacity to interact with the stimulatory regulator of adenylyl cyclase are contained within a fragment of only approximately 21,000 daltons that does not contain any N-linked glycans. Hormone occupancy of the receptor results in a conformational change so as to expose a region that is susceptible to proteolysis by proteases of varying specificities to yield a peptide of approximately 30,000 daltons that also does not contain N-linked glycans.     

Reduction of Muscarinic Acetylcholine Receptor Number and Affinity by an Endogenous Substance

We examined the soluble fraction from homogenates of 12-day embryonic chick heart for the presence of an endogenous modulator of muscarinic acetylcholine receptors (mAChR). Homogenates were separated into 100,000 g soluble and crude membrane fractions by differential centrifugation. Aliquots of membranes were incubated in the presence or absence of the soluble fraction and the muscarinic antagonist, [3H]quinuclidinyl benzilate ( [3H]QNB), and the data subjected to Scatchard analysis. In the presence of the soluble fraction, mAChR number decreased up to 70% and the affinity for [3H]QNB decreased six- to eightfold. These results suggested that an endogenous soluble factor (ESF) affected cholinergic ligand binding to the receptor. The amount of ESF extracted from less than 10 mg of brain was sufficient to reduce by 50% [3H]QNB binding to 50 fmol mAChR. ESF activity was partially purified by heat and acid treatment. The loss of receptors was dependent upon the amount of ESF added and was time dependent. QNB protected some receptors from loss due to ESF. The change in mAChR affinity for [3H]QNB was observed only if ESF was present continuously during the [3H]QNB binding assay. Ultrafiltration and gel filtration showed that ESF was less than 10,000 daltons and probably less than 700 daltons. ESF activity was blocked by EDTA. However, ESF was not a divalent cation since it was base labile, and removal of divalent cations with Chelex-100 did not inhibit ESF activity. ESF activity was also blocked by catechol, catecholamines, ascorbate, and dithiothreitol. ESF was present in embryonic but not in adult heart.     

Characterization of cyclic AMP-binding proteins in rat Sertoli cells using a photoaffinity ligand

Summary Protein-bound cyclic AMP (cAMP) levels in cultured rat Sertoli cells have been determined after exposure to follicle-stimulating hormone (FSH) and agents which elevate intracellular cAMP or mimic cAMP action. Changes in the content of protein-bound cAMP were correlated with changes in receptor availability determined by measuring [³H] cAMP binding. Using the photoaffinity analog of cAMP, 8-N₃ [³²P] cAMP, two major cAMP-binding proteins in Sertoli cell cytosol, with molecular weights of 47 000 and 53 000 daltons, were identified as regulatory subunits of type I and type II cAMP-dependent protein kinases, respectively. Densitometric analysis of autoradiograms demonstrated differential activation of the two isozymes in response to treatment with FSH and other agents. Results of this study demonstrate the value of measuring changes in protein-bound cAMP and the utility of the photoaffinity labeling technique in correlating hormone-dependent processes in which activation of cAMP-dependent protein kinase occurs.     

Covalent cross-linking of prostaglandin E receptor from bovine adrenal medulla with a pertussis toxin-insensitive guanine nucleotide-binding protein

Prostaglandin E2 (PGE2) specifically bound to 100,000 X g pellet prepared from bovine adrenal medulla, and [3H]PGE2-bound proteins were solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The dissociation of bound [3H]PGE2 from the proteins was enhanced by GTP. [3H]PGE2-specifically bound proteins were adsorbed onto a wheat germ agglutinin column and GTP treatment decreased the amount of [3H]PGE2 retained on the column. When [3H]PGE2-bound proteins were cross-linked in the membrane by dithiobis(succinimidyl propionate) and solubilized, bound [3H]PGE2 was no longer dissociated by GTP treatment, suggesting that cross-linking produced a stable and high-affinity complex of PGE receptor with a GTP-binding protein. Covalent cross-linking of the complex was attested by adsorption of dithiobis(succinimidyl propionate)-treated [3H]PGE2-bound proteins to GTP-Sepharose, and co-elution of [35S]guanosine 5'-O-(3-thiotriphosphate) binding activity and immunoreactivities of alpha o and beta subunits of a GTP-binding protein. The cross-linked [3H]PGE2-bound complex was eluted as an apparently single radioactive peak at the position of Mr = 200,000 by gel filtration. These results have demonstrated that PGE receptor is a glycoprotein with an approximate Mr of 110,000, assuming that the Mr of the GTP-binding protein is 90,000. PGE2 neither activated nor inhibited adenylate cyclase activity, and pertussis toxin (islet-activating protein) did not affect PGE2 binding and its GTP sensitivity. These results suggest that the PGE receptor may be functionally associated with a pertussis toxin-insensitive GTP-binding protein and is not coupled to the adenylate cyclase system in bovine adrenal medulla.     

Cooperative non-specific DNA binding of the N-terminal core of the cyclic AMP receptor protein of Escherichia coli and its modulation by cyclic AMP

The non-specific DNA binding of CRP and its N-terminal core, alpha CRP, to a 298 base pair DNA fragment, in the presence and absence of cAMP, has been studied using the nitrocellulose filter binding technique and analysed quantitatively using the theory of Clore et al. [J. Mol. Biol. (1982) 155, 447-466]. It is shown that both CRP and alpha CRP bind cooperatively to DNA. At an ionic strength of 100 mM and pH 7.5, the intrinsic equilibrium association constant for the binding of alpha CRP to DNA is approximately 10-times smaller than that for CRP, but the cooperativity parameter is approximately 17-times larger for alpha CRP than CRP. cAMP exerts its effect solely on the intrinsic equilibrium constant and does not alter the cooperativity. In the case of alpha CRP, cAMP reduces the intrinsic equilibrium association constant by a factor of 3, in contrast to the case of CRP where cAMP increases it by a factor of 3. The possible location of the DNA binding site present in the N-terminal core of CRP is discussed in the light of crystallographic data on the cAMP . CRP complex [McKay et al. (1982) J. Biol. Chem. 257, 9518-9524].     

Cross-linking of actin to myosin subfragment 1 in the presence of nucleotides

Chemical cross-linking of actin to the 20K and 50K fragments of tryptically cleaved myosin subfragment 1 (S-1) by the zero-length cross-linking reagent 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide (EDC) was used as a probe of the acto-S-1 interface in the presence of nucleotides. The course of the two reactions was monitored by measuring on sodium dodecyl sulfate (SDS)-polyacrylamide gels the time-dependent formation of the 20K-actin and 50K-actin cross-linked products. Both reactions were inhibited somewhat in the presence of MgADP, were slowed 3-4-fold in the presence of magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP), and proceeded at least 7-fold slower with N,N'-p-phenylenedimaleimide (pPDM) modified S-1, as compared to the respective rates in the absence of nucleotides. However, neither the binding of the nucleotides MgADP and MgAMPPNP to S-1 nor the modification of S-1 by pPDM significantly changed the ratio of the cross-linking rates of actin to the 20K and 50K fragments. Similar to what was previously observed in the absence of nucleotides [Chen, T., Applegate, D., & Reisler, E. (1985) Biochemistry 24, 137-144], actin was cross-linked at an approximately 3-fold faster rate to the 20K fragment than to the 50K fragment under all reaction conditions tested. Thus, irrespective of the extent of acto-S-1 dissociation or the binding of nucleotides to acto-S-1, the 20K fragment remains the preferred cross-linking site for actin. These results show that the interaction of actin with each of the cross-linking sites on S-1 is not under selective or preferential control by nucleotides.     

Specificity of cGMP binding to a purified cGMP-stimulated phosphodiesterase from bovine adrenal tissue

The binding of [3H]cGMP (guanosine 3',5'-monophosphate) to purified bovine adrenal cGMP-stimulated phosphodiesterase was measured by Millipore filtration on cellulose ester filter. [3H]cGMP-binding activity was enhanced when the assay was terminated in buffer containing 70% of saturated ammonium sulfate to dilute the enzyme and wash the filters. The cGMP-binding activity was co-purified with the phosphodiesterase activity. The binding of [3H]cGMP to purified enzyme was measured in the presence or absence of the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine. 1-Methyl-3-isobutylxanthine showed linear competitive inhibition with respect to cGMP as substrate in the phosphodiesterase reaction but stimulated the [3H]cGMP-binding activity in the binding assay. The stimulatory effect appeared not to be the result of preservation from [3H]cGMP hydrolysis; no cGMP phosphodiesterase activity has been measured under the cGMP-binding assay conditions, in the absence or presence of the inhibitor. Half-maximal stimulation by 1-methyl-3-isobutylxanthine occurred in the 5-7 microM concentration range. The specificity of binding of [3H]cGMP was investigated by adding increasing concentration of unlabeled analogs of cAMP (adenosine 3',5'-monophosphate) and cGMP. The binding of [3H]cGMP (50 nM) was displaced by unlabeled cGMP and cAMP with the following potency: 50% displacement was reached at the 0.1 microM cGMP range and only at a fiftyfold higher cAMP concentration. Our data with comparative series of analogs (e.g. 5'-amino-5'-deoxyguanosine 3',5'-monophosphate and 3'-amino-3'-deoxyguanosine 3',5'-monophosphate) showed that the potencies of stimulation of cAMP phosphodiesterase activity parallels displacement curves or [3H]cGMP binding to purified enzyme with no correlation with phosphodiesterase inhibition sequences. Those experiments suggest that the cGMP-binding activity is directly related to the non-catalytic (allosteric) cGMP-binding site.     

Purification and characterization of an inactive form of cAMP-dependent protein kinase containing bound cAMP

By a new procedure, the holoenzyme of bovine heart type II cAMP-dependent protein kinase was purified to homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A high performance liquid chromatography-DEAE purification step resolved two distinct peaks of protein kinase activity, which were designated Peak 1 and Peak 2 based on their order of elution. The two peaks exhibited similar Stokes radii and sedimentation coefficients. They had similar ratios of regulatory to catalytic subunits both by densitometric scanning of SDS-PAGE bands and by the ratios of equilibrium [3H]cAMP binding to maximal kinase activity. These results suggested that the holoenzyme of each peak contained two regulatory subunits and two catalytic subunits, although a subpopulation of holoenzyme lacking one catalytic subunit also appeared to be present in Peak 2. Assays of cAMP indicated that the Peak 1 holoenzyme was cAMP-free, but half of the Peak 2 holoenzyme cAMP binding sites contained cAMP. Determination of [3H]cAMP dissociation rates showed that the cAMP was equally distributed in binding Site 1 and Site 2 of Peak 2. Although SDS-PAGE analysis ruled out conversions by proteolysis or autophosphorylation-dephosphorylation, Peak 1 could be partially converted to Peak 2 by the addition of subsaturating amounts of cAMP. Interconvertibility of the two holoenzyme peaks strongly suggested that the difference between the two peaks was caused by the presence of cAMP in Peak 2. Peak 2 holoenzyme, as compared to Peak 1, had enhanced binding in nonequilibrium [3H]cIMP and [3H]cAMP binding assays, as was expected due to the presence of cAMP and to the known positive cooperativity in binding of cyclic nucleotides to the kinase. The positive cooperativity in kinase activation, as indicated by the Hill coefficient, was greater for Peak 2 than Peak 1, but the cAMP concentration required for half-maximal activation (Ka) of each of the two peaks was very similar. In conclusion, Peak 2 is an inactive ternary complex of cAMP, regulatory subunit, and catalytic subunit, and Peak 1 is a cAMP-free holoenzyme. The cAMP-bound form may represent a major cellular form of the enzyme which is primed for activation.     

Binding of [3H]SKF 38393 to Dopamine D-l Receptors in Rat Striatum In Vitro; Estimation of Receptor Molecular Size by Radiation Inactivation

[3H]SKF 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine) binds with high affinity to 3,4-dihydroxyphenylethylamine (dopamine) D-1 receptors in rat striatum in vitro (KD = 7 and 14 nM in nonfrozen and frozen striatum, respectively). The number of binding sites (Bmax) was approximately 80.0 pmol/g of original tissue, a value similar to the Bmax for the dopamine D-1 antagonist SCH 23390. Nondisplaceable [3H]SKF 38393 binding was approximately 45% of total binding. Irradiation (0-4 Mrad) of frozen whole striata decreased the number of [3H]SKF 38393 binding sites monoexponentially without changing the binding affinity. The functional molecular mass for the agonist dopamine D-1 binding site was 132,800 daltons, which is higher than the functional molecular mass of the antagonist dopamine D-1 binding site (approximately 80,000 daltons).     

Demonstration of receptor heterogeneity and affinity modulation by nonequilibrium binding experiments. The cell surface cAMP receptor of Dictyostelium discoideum

The binding of [3H]cAMP to Dictyostelium discoideum cells was analyzed on a seconds time scale under both equilibrium and nonequilibrium conditions. The binding of [3H]cAMP increases rapidly to a maximum obtained at about 6 s, which is followed by a decrease to an equilibrium value reached at about 45 s. This decrease of [3H]cAMP binding is not the result of ligand degradation or isotope dilution by cAMP secretion but is due to a transition of high-affinity binding to low-affinity binding. Analysis of the dissociation rate of [3H]cAMP from the binding sites indicates that these high- and low-affinity binding sites are both fast dissociating with a half-life of about 1 s. In addition, these dissociation experiments reveal a third binding type which is slowly dissociating with a half-life of about 15 s. The number and affinity of these slowly dissociating sites does not change during the incubation with [3H]cAMP. The drugs caffeine and chlorpromazine do not change the total number of binding sites, but they change the ratio of the three binding types. In the presence of 10 mM caffeine almost all binding sites are in the low affinity conformation, while in the presence of 0.1 mM chlorpromazine the ratio is shifted to both the high-affinity type and slowly dissociating type. The results indicate that the cAMP-binding activity of D. discoideum cells is heterogeneous. In the absence of cAMP about 4% of the sites are slowly dissociating with Kd = 12.5 nM, about 40% are fast dissociating with high affinity (Kd = 60 nM), and about 60% are fast dissociating with low affinity (Kd = 450 nM). During the binding reaction the number of slowly dissociating sites does not change. The number of high-affinity sites decreases to a minimum of about 10% with a concomitant increase of low-affinity sites to about 90%. This transition of binding types shows first-order kinetics with a half-life of about 9 s. A half-maximal transition is induced by 12.5 nM cAMP.     

A prostaglandin E receptor coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rabbit cortical collecting tubule cells

At different concentrations, prostaglandin E2 (PGE2) can either stimulate or inhibit cAMP formation in freshly isolated rabbit cortical collecting tubule (RCCT) cells, but in cultured RCCT cells PGE2 can only stimulate cAMP synthesis (Sonnenburg, W. K., and Smith W. L. (1989) J. Biol. Chem. 263, 6155-6160). Here, we report characteristics of [3H]PGE2 binding to membrane receptor preparations from both freshly isolated and cultured RCCT cells. [3H]PGE2 binding to membranes from freshly isolated RCCT cells was saturable and partially reversible. Equilibrium binding analyses indicated that in the absence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) there is a single class of PGE2 binding sites (KD = 4.2 +/- 0.4 nM; Bmax = 583 +/- 28 fmol/mg); in the presence of 100 microM GTP gamma S, there is also only one class of binding sites but with a somewhat lower KD = 1.2 +/- 0.5 nM (Bmax = 370 +/- 40 fmol/mg). This stimulatory effect of GTP gamma S was blocked by pretreatment of the freshly isolated RCCT cells with pertussis toxin. The relative affinities of prostanoids for the [3H]PGE2-binding site were determined to be 17,18,19,20-tetranor-16-phenoxy-PGE2-methylsulfonylamide (sulprostone) approximately PGE2 approximately PGE1 approximately 16,16-dimethyl-PGE2 greater than carbacyclin approximately PGF2 alpha greater than PGD2. This is the order of potency with which prostaglandins inhibit arginine vasopressin-induced cAMP formation in fresh RCCT cells. Interestingly, [3H]PGE2 binding to membranes from cultured cells, which, unlike fresh cells, fail to show an inhibitory response to PGE2, was only 10-20% of that observed with membranes from fresh cells; moreover, binding of [3H]PGE2 to membranes from cultured cells was neither stimulated by GTP gamma S nor inhibited by sulprostone. The prostanoid binding specificities and the unusual pertussis toxin-sensitive, stimulatory effect of GTP gamma S on binding of [3H]PGE2 to membranes from freshly isolated RCCT cells are characteristics shared by a Gi-linked PGE receptor from renal medulla (Watanabe, T., Umegaki, K., and Smith, W. L. (1986) J. Biol. Chem. 261, 14340-14349). Our results suggest that the [3H]PGE2 binding site of freshly isolated RCCT cells is the PGE receptor which is coupled to a Gi to attenuate arginine vasopressin-induced cAMP synthesis in the renal collecting tubule.     

Two different intrachain cAMP binding sites of cAMP-dependent protein kinases

The regulatory subunits of both isozymes of cAMP-dependent protein kinase bind 2 mol of cAMP/mol of monomer. cAMP dissociation studies indicate similar cAMP binding behavior for each isozyme. Each has two different intrachain cAMP binding components present in approximately equal amounts and the rate of cAMP dissociation is 5- to 10-fold slower from one site (Site 1) than from the other (Site 2). Equilibrium [3H]cAMP binding is inhibited by several competing cyclic nucleotides. Following equilibrium binding using saturating [3H]cAMP in the presence of competing nucleotide, the pattern of release of [3H]cAMP, monitored in the presence of an excess of nonradioactive cAMP, suggests site-specific selectivity of some of the cyclic nucleotides. As compared with cAMP, cIMP prefers Site 2 for both regulatory subunits, whereas N6, O2-dibutyryl-cAMP shows a similar preference only with isozyme II regulatory subunit. 8-Bromo-cAMP, 8-bromo-cGMP, and 8-azido-cAMP prefer Site 1 of both proteins. The results indicate that for each isozyme the two intrachain binding sites have different analogue specificities and cAMP dissociation rates. Site 1 or Site 2 of one isozyme has a similar but not identical cyclic nucleotide specificity and cAMP dissociation rate to the corresponding site of the other isozyme.     

Cyclic AMP-mediated intersubunit disulfide crosslinking of the cyclic AMP receptor protein of Escherichia coli.

The protomeric form of the cyclic AMP receptor protein (CRP) of Escherichia coli is composed of two identical subunits of molecular weight 22,500 and contains two buried and two available cysteine residues. Titration of the two available cysteines with DTNB⁴ eliminates cyclic AMP-dependent DNA binding activity which is regenerated by incubating the modified protein with β-mercaptoethanol. In the absence of cAMP, the formation of the TNB anion from DTNB and the incorporation of [¹⁴C]TNB into CRP are approximately stoichiometric. In the presence of cAMP, there is an increase in the rate of formation of the TNB anion while the incorporation of [¹⁴C]TNB into CRP is markedly inhibited. These observations are reconciled by the observation that cAMP induces DTNB-mediated disulfide crosslinking of the two available sulfhydryls to produce a species migrating as a 45,000 molecular weight subunit on SDS-polyacrylamide gels. A mechanism is suggested by which an intersubunit, intraprotomer disulfide bond is produced by secondary disulfide interchange after the incorporation of the initial TNB group. Based on the observation of cAMP-mediated disulfide crosslinking, the available cysteines of the DNA binding region are proposed to reside in close proximity as part of an antiparallel β-sheet structure formed by the two carboxyl proximal polypeptides when CRP is in the DNA binding conformation.     

Solubilization and photoaffinity labeling of renal membrane cyclic AMP receptors.

Renal cortical plasma membranes were solubilized with sodium deoxycholate. The membrane-bound cyclic AMP receptors retained biologic activity in the detergent-dispersed state exhibiting the properties of high affinity for cyclic AMP, saturability and specificity. Half-maximal binding of cycle [3H]-AMP to these receptors was found to occur at 0.06 muM and 1.5 pmol of cyclic [3H]AMP was bound per mg membrane protein at saturation (0.5 muM cyclic [3H]AMP). Sodium deoxycholate-solubilized membrane proteins were chromatographed on Biogel A-5m. Cyclic [3H]AMP receptors eluted in the internal volume at positions equivalent to molecular sizes of 50 000 and 20 000 daltons and in the void volume at molecular size greater than 450 000. After photoaffinity labeling the renal membrane receptors with cyclic [3H]AMP, we found peaks of tritium radioactivity which eluted at similar molecular size positions on this Bogel A-5m column. Further treatment of photoaffinity labeled membranes with sodium dodecyl sulfate, mercaptoethanol and urea, followed by polyacrylamide gel electrophoresis, showed bands of tritium-labeled receptor protein with relative mobilities corresponding to molecular sizes of 26 000 and 21 000 daltons. This study shows that porcine renal cortical membranes contain at least two molecular species of cyclic AMP receptors which may be associated with regulation of the membrane-bound cyclic AMP-dependent protein kinase.     

Affinity Labelling and Identification of the High-Affinity Choline Carrier from Synaptic Membranes of Torpedo Electromotor Nerve Terminals with [3H]Choline Mustard

The physiological mechanisms regulating activity of the sodium-dependent, high-affinity choline transporter and the molecular events in the translocation process remain unclear; the protein has not been purified or characterized biochemically. In the present study, [3H]choline mustard aziridinium ion [( 3H]ChM Az), a nitrogen mustard analogue of choline, bound irreversibly to presynaptic plasma membranes from Torpedo electric organ in a hemicholinium-sensitive, and sodium-, time-, and temperature-dependent manner. Specific binding of this ligand was greatest when it was incubated with membranes in the presence of sodium at 30 degrees C. Separation of the 3H-labelled membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the radiolabel was associated with a polypeptide of apparent molecular mass of approximately 42,000 daltons; labelling of this species was abolished in membranes incubated with ligand in the presence of HC-3. Two other 3H-labelled polypeptides were detected, with apparent molecular masses of approximately 58,000 and 90,000 daltons; radiolabelling of the former was also HC-3 sensitive. [3H]ChM Az may be a useful affinity ligand in the purification of the choline carrier from cholinergic neurons.     

Photoaffinity labelling of Ca2+ channels with [3H]azidopine

A 1,4-dihydroypyridine arylazide photoaffinity ligand, [3H]azidopine (50.6 Ci/mmol), has been synthesized. [3H]Azidopine binds reversibly with a Kd of 350 pM to guinea-pig skeletal muscle membranes in the absence of ultraviolet light. The reversible [3H]azidopine binding is inhibited steroselectively by 1,4-dihydropyridines, phenylalkylamine Ca2+ channel blockers and La3+. Covalent incorporation into membrane proteins after photolysis was investigated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. [3H]Azidopine is photoincorporated specifically into a protein of Mr approximately 145 000. The covalent labelling of the Mr approximately 145 000 band is inhibited stereoselectively by drugs and cations which block the reversible [3H]azidopine binding. It is suggested that [3H]azidopine is photoincorporated into a subunit of the putative Ca2+ channel.     

Mineralocorticosteroid receptor of the chick intestine. Oligomeric structure and transformation

The binding of [3H]aldosterone in the chick intestine cytosol was analyzed in terms of affinity and specificity. In this tissue, aldosterone binds to the mineralocorticosteroid receptor, with a high affinity (Kd approximately 0.3 nM) and low capacity (approximately 50 fmol/mg protein), and to the glucocorticosteroid receptor. The selective labeling of the mineralocorticosteroid receptor was achieved by incubating the cytosol with [3H]aldosterone in the presence of RU 486. This synthetic steroid completely inhibited the binding of [3H]aldosterone to the glucocorticosteroid receptor and did not bind to the mineralocorticosteroid receptor. The oligomeric structure of the mineralocorticosteroid receptor was studied by using BF4, a monoclonal antibody which reacts with the 90-kDa heat shock protein (hsp 90), a nonhormone-binding component of nontransformed steroid receptors. The mineralocorticosteroid receptor sedimented at 8.5 +/- 0.4 S (n = 8) in a 15-40% glycerol gradient. This peak was shifted to 11.2 +/- 0.6 S (n = 5) after incubation with BF4, indicating that, in the cytosol, hsp 90 was associated with the mineralocorticosteroid receptor. Dissociation of the complex was observed on gradients containing 0.4 M KCl, as judged by the absence of displacement by BF4 of the 4.3 +/- 0.4 S (n = 10) peak. The effect of molybdate and tungstate ions, and of dimethyl pimelimidate, an irreversible cross-linking agent, on the stability of the hsp 90-receptor complex was investigated. Complexes recovered in the presence of 20 mM molybdate ions dissociated on gradients containing 0.4 M KCl (5.2 +/- 0.6 S (n = 4), whereas complexes prepared in the presence of 20 mM tungstate ions sedimented at 8.5 +/- 0.4 S (n = 7). Similarly, complexes prepared in the presence of molybdate ions dissociated during high pressure liquid chromatography (HPLC) gel filtration analysis performed in 0.4 M KCl (RS (Stokes radius) = 3.9 +/- 0.5 nm (n = 3) versus 7.3 +/- 0.2 nm (n = 3) in the presence of 20 mM molybdate ions), whereas complexes prepared in the presence of tungstate ions did not dissociate (RS = 6.9 +/- 0.2 nm (n = 3]. As observed for the tungstate-stabilized receptor, the cross-linked receptor dissociated neither on gradient containing 0.4 M KCl (9.5 +/- 0.1 S (n = 3] nor during HPLC performed in 0.4 M KCl (RS = 6.5 +/- 0.3 (n = 4]. Furthermore, the cross-linked receptor was more resistant to the inactivating effect of urea on aldosterone binding than the noncross-linked receptor prepared in the presence of either molybdate or tungstate ions.