Demonstration of two distinct tachykinin receptors in rat brain cortex

Eledoisin and substance P are members of a class of peptides termed tachykinins. They share a similar spectrum of biological activities but their relative potencies in various pharmacological assays differ. We have investigated whether there is more than one receptor for these tachykinins in rat brain cortex membranes. 125I-Bolton Hunter-conjugated eledoisin specifically binds to rat brain cortex membranes with high affinity. The binding is inhibited over 95% by unlabeled eledoisin (6.6 microM). Scatchard analysis of the binding of this ligand is curvilinear suggesting that there are two binding sites with KD values of 0.9 +/- 0.7 nM and 20 +/- 10 nM. We tested various analogs and fragments of substance P and eledoisin for their ability to inhibit the binding of 125I-Bolton Hunter-conjugated eledoisin and 125I-Bolton Hunter-conjugated substance P to these membranes. The following peptides are more potent as inhibitors of the 125I-Bolton Hunter-conjugated eledoisin binding site than of the 125I-Bolton Hunter-conjugated substance P binding site: nonradioactive Bolton Hunter-conjugated eledoisin (greater than 100-fold), eledoisin (12-fold), kassinin (22-fold), neuromedin K (greater than 58-fold), and pyroglutamyl substance P(6-11)hexapeptide (4-fold). In contrast, substance P (21-fold), physalaemin (8-fold), and substance P methyl ester (1200-fold) were more potent as inhibitors of 125I-Bolton Hunter-conjugated substance P binding. These results suggest that these two ligands may bind to distinct receptors. 125I-Bolton Hunter-conjugated substance P binds specifically to rat parotid cell receptors, but 125I-Bolton Hunter-conjugated eledoisin does not, indicating that parotid cells contain only one of the receptor subtypes. The cortex membrane binding of both ligands is stimulated by low concentrations of MnCl2 (ED50 = 0.05 mM) and is inhibited by guanylyl-5'-(beta, gamma-imido)diphosphate (IC50 = 0.5 microM).     

Specific substance P binding sites in rat thymus and spleen: in vitro autoradiographic study

Substance P binding sites were localized in rat thymus and spleen by incubation of tissue sections with [125I]Bolton-Hunter substance P, [3H]Ultrofilm autoradiography with image analysis coupled to computerized microdensitometry and comparison with 125I standards. The tissue localization of the binding sites was determined with emulsion autoradiography. A single type of specific, saturable, high affinity binding sites was found associated with the vasculature in the medulla of the thymus and the marginal sinus of the spleen, with a Kd of 0.10 and 0.14 nM, respectively. Of all the unlabeled tachykinins tested (substance P, physalaemin, substance K, eledoisin, kassinin, and neuromedin K) substance P was the most potent inhibitor of [125I]Bolton-Hunter substance P binding, with an IC50 of approximately 0.5 nM, indicating the presence of substance P-P binding sites. Our results support the hypothesis of a role for substance P in the modulation of the immune system.     

Binding sites for tachykinin peptides in the brain and stomach of the dogfish, Scyliorhinus canicula.

In membranes of dogfish brain and stomach, two binding sites for tachykinins were identified. One site specifically bound [125I]-Bolton-Hunter substance P (BH-SP) and the rank potency of tachykinins to compete for BH-SP binding revealed similarities with the rank potency of an NK1 receptor. The pharmacology of the other site, which specifically bound [125I]-Bolton-Hunter scyliorhinin II (BH-Scy II), did not resemble any of the mammalian tachykinin receptors. The rank potency to inhibit BH-Scy II binding to this second site was: scyliorhinin II approximately scyliorhinin I greater than eledoisin approximately substance P approximately neurokinin A greater than phyllomedusin approximately physalaemin greater than [Sar9Met(O2)11]substance P. Neurokinin B and senktide did not displace BH-Scy II binding. In addition, nucleotide analogues inhibited BH-SP binding but not BH-Scy II binding. Our binding data suggest the existence of a mammalian-like NK1 receptor and of a nonmammalian tachykinin receptor in the dogfish.     

Characterization of the substance P receptor in rat brain cortex membranes and the inhibition of radioligand binding by guanine nucleotides

Rat brain cortex membranes bind to a conjugate of substance P and 125I-labeled Bolton-Hunter reagent, and this binding can be inhibited by a low concentration of substance P (Kd = 1.2 +/- 0.4 X 10(-8) M). This binding is reversible and saturable (0.5 +/- 0.1 pmol of binding sites/mg of protein). Fragments of substance P as small as the carboxyl-terminal hexapeptide can inhibit the binding although their potency decreases with the decrease in the length of the peptides. The binding affinities of smaller peptides or peptides in which the carboxyl-terminal amide or amino acids are removed are drastically reduced. Biologically active analogs of substance P, physalaemin, eledoisin, substance P methyl ester, [D-Ala0]hepta(5-11)substance P, kassinin, and the eledoisin-related hexapeptide also can inhibit the binding. However, the binding is not inhibited by polypeptides structurally unrelated to substance P or by amine hormones/neurotransmitters. The binding affinities of biologically active peptides to rat brain cortex membranes are almost identical with their affinities for rat parotid cells which we previously determined. Furthermore, the recently described substance P antagonist, [D-Pro, D-Trp]substance P, inhibits the binding of the 125I-labeled substance P derivative to brain cortex membranes and to parotid cells equally well. These results suggest that the substance P receptors in the brain cortex and the parotid gland are similar. The brain cortex membrane binding of the 125I-labeled substance P derivative can be inhibited by micromolar concentrations of GTP, GDP, and their analogs. ITP and IDP were less active. Adenine and pyridine nucleotides were inactive.     

Specific binding of an immunoreactive and biologically active 125I-labeled N(1)acylated substance P derivative to parotid cells

A biologically active ¹²⁵I-substance P derivative (I¹²⁵-BH-substance P), prepared by conjugation of substance P with [^125I]Bolton-Hunter reagent, binds specifically to isolated rat parotid cells. The Kd is 4 nM for I-BH-substance P, 5 nM for substance P, 0.18 μM for substance P octa(4–11)peptide, and 1.6 μM for substance P [pyroglutamyl⁶]hexa(6–11)peptide. Substance P free acid and substance P penta(7–11)peptide are much weaker competitors and the C-terminal tri(9–11)peptide has no effect at 30 μM. The binding is also inhibited by 1 μM physalaemin, eledoisin and substance P methyl ester, but not by unrelated peptides. The selective inhibition of the binding by the biologically active analogs and fragments of substance P indicates that the ¹²⁵I-labeled N(1)acylated substance P derivative may interact with a substance P receptor on parotid cells.     

[3H]Neurokinin B and 125I-Bolton Hunter Eledoisin Label Identical Tachykinin Binding Sites in the Rat Brain

[3H]Neurokinin B ([3H]NKB) of high specific activity (75 Ci/mmol) was synthesized for study of its binding to crude synaptosomes from the rat cerebral cortex. The specific binding of [3H]NKB (75% of total binding) was temperature dependent, saturable, and reversible. Scatchard analyses and Hill plots showed the existence of a single population of noninteracting binding sites (KD = 4.3 nM; Bmax = 123 fmol/mg of protein). Competition studies indicated the following rank order of potencies among tachykinins: NKB greater than eledoisin (E) greater than kassinin greater than physalaemin greater than neurokinin A (NKA) greater than substance P (SP), a result suggesting that NKB might be the endogenous ligand for [3H]NKB binding sites. It is of interest that 127I-Bolton Hunter (BH) NKA (127I-BHNKA) was much more potent than NKA in inhibiting the specific binding of [3H]NKB, which raises certain questions concerning the use of 125I-BHNKA as a ligand for NKA binding sites in the brain. These results, as well as those obtained with different SP analogues, show a close similarity to those obtained previously with 125I-BHE binding to cortical synaptosomes. This suggested that the two ligands labeled identical binding sites. In addition, using either [3H]NKB or 125I-BHE as ligands, similar displacement curves were obtained with increasing concentrations of NKB and 127I-BHE. The similarity of the [3H]NKB and 125I-BHE binding sites was further confirmed by comparison of their localization on rat brain sections by autoradiography. The distribution of binding sites for [3H]NKB and 125I-BHE was identical throughout the brain, and the highest density of binding sites for the two ligands was found in layers IV and V of the cerebral cortex, the paraventricular nucleus of the hypothalamus (magnocellular part), and the ventral tegmental area.     

125I]-Bolton-Hunter scyliorhinin II: a novel, selective radioligand for the tachykinin NK3 receptor in rat brain

The cyclic tachykinin scyliorhinin II (SCYII) has high affinity for the [neurokinin B (NKB)-preferring] NK3 receptor. SCYII was iodinated using [125I]-Bolton-Hunter reagent and the product BHSCYII purified using reverse phase HPLC. In rat brain membranes, binding of BHSCYII and of the relatively unselective radioligand [125I]-Bolton-Hunter eledoisin (BHELE) was saturable, reversible and to an NK3 site. In competition studies, the rank order of potency in inhibiting binding of BHSCYII and BHELE was: SCYII greater than or equal to [MePhe7]-NKB approximately senktide greater than NKB greater than or equal to kassinin greater than or equal to eledoisin greater than [Pro7]-NKB greater than neurokinin A greater than neuropeptide K greater than or equal to substance P greater than [Sar9, Met(O2)11]-substance P. In "cold" saturation experiments, binding of BHELE occurred to a single class of high affinity sites (KD, 18.6 +/- 0.91 nM). Binding of BHSCYII was of greater affinity than for BHELE and could be resolved into a high (KD, 1.33 +/- 0.98 nM; 27% of sites) and low affinity (KD, 9.84 +/- 2.75; 73% of sites) component. The total number of binding sites was similar for both radioligands (BHSCYII, 8.27 +/- 0.98; BHELE, 7.94 +/- 0.32 fmol/mg wet weight). In vitro autoradiography in slide-mounted sections of rat brain showed identical binding patterns for both radioligands (100 pM), with dense binding localized predominantly to the cortex, Ammon's horn field 1, premammillary nuclei and interpeduncular nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)     

The inhibitory effect of [D-Arg1,D-Phe,D-Try7,9,Leu11] substance P on endothelin-1 binding sites in rat cardiac membranes.

The specific binding of [125I]ET-1 to rat cardiac membrane fragments was inhibited by [D-Arg1,D-Phe, D-Try7,9,Leu11] substance P [substance P(D)], a potent bombesin antagonist. This inhibitory effect required high concentrations (greater than 3X10(-6)M) of substance P(D) and was accompanied by a steep increase in non-specific binding, and not a decrease in total binding. Such results indicate that substance P(D) does not competitively inhibit the specific binding of [125I]ET-1 to rat cardiac membrane fragments.     

Pharmacologic characterization and autoradiographic distribution of binding sites for iodinated tachykinins in the rat central nervous system

P-type, E-type, and K-type tachykinin binding sites have been identified in the mammalian CNS. These sites may be tachykinin receptors for which the mammalian neuropeptides substance P, neuromedin K, and substance K are the preferred natural agonists, respectively. In the present investigation, we have compared the pharmacology and the autoradiographic distribution of CNS binding sites for the iodinated (¹²⁵I-Bolton-Hunter reagent) tachykinins substance P, eledoisin, neuromedin K, and substance K. Iodinated eledoisin and neuromedin K exhibited an E-type binding pattern in cortical membranes. Iodinated eledoisin, neuromedin K, and substance K each labeled sites that had a similar distribution but one that was considerably different from that of sites labeled by iodinated substance P. CNS regions where there were detectable densities of binding sites for iodinated eledoisin, neuromedin K, and substance K and few or no sites for iodinated substance P included cortical layers IV–VI, mediolateral septum, supraoptic and paraventricular nuclei, interpeduncular nucleus, ventral tegmental area, and substantia nigra pars compacta. Binding sites for SP were generally more widespread in the CNS. CNS regions where there was a substantial density of binding sites for iodinated substance P and few or no sites for iodinated eledoisin, neuromedin K, and substance K included cortical layers I and II, olfactory tubercle, nucleus accumbens, caudate-putamen, globus pallidus, medial and lateral septum, endopiriform nucleus, rostral thalamus, medial and lateral preoptic nuclei, arcuate nucleus, dorsal raphe nucleus, dorsal parabrachial nucleus, parabigeminal nucleus, cerebellum, inferior olive, nucleus ambiguus, retrofacial and reticular nuclei, and spinal cord autonomic and somatic motor nuclei. In the brainstem, iodinated substance P labeled sites in both sensory and motor nuclei whereas iodinated eledoisin, neuromedin K, and substance K labeled primarily sensory nuclei. Our results are consistent with either of two alternatives: (1) that iodinated eledoisin, neuromedin K, and substance K bind to the same receptor site in the rat CNS, or (2) that they bind to multiple types of receptor sites with very similar distribution.     

Radioactive labels for Protein A: evaluation in the indirect immunoradiometric assay (IRMA) for Bacillus anthracis spores

Staphylococcus aureus Protein A (SpA) labelled with [125I] by the Bolton & Hunter (1973) method performed about as well as labelled sheep anti-rabbit globulin (SAR) in an indirect immunoradiometric assay (IRMA) for Bacillus anthracis spores immobilized on multispot microscope slides. SpA labelled with [3H] by propionylation also performed well but would be expensive to use. SpA labelled with [3H] fluorodinitrobenzene, or labelled with [125I] by the chloramine T reaction gave erratic assay results, high noise values and low signal-to-noise ratios, indicating substantial direct binding of labelled SpA to the slide surface and to the bacterial preparation. The uptake of radioactively labelled SpA in the IRMA was compared with the fluorescence intensity of individual spores in a microfluorometric immunofluorescence (IF) test involving dual labelled fluorescein-[125I]-SpA. The maximum number of SAR molecules bound to the mixture of spores and cell-free antigens in the B. anthracis IRMA was about twice the maximum number of radioactively labelled SpA molecules bound. The SAR:SpA saturation binding ratio on the surface of the spores, however, was approximately the inverse of this. It is concluded that radioactively-labelled SpA is not recommended in preference to anti-species antibody reagents in bacterial IRMA tests but fluorescein-conjugated SpA deserves further consideration for use in microscope-based IF tests for bacterial antigens.     

Interaction of the catecholamine release-inhibitory peptide catestatin (human chromogranin A(352-372)) with the chromaffin cell surface and Torpedo electroplax: implications for nicotinic cholinergic antagonism

The catecholamine release-inhibitory chromogranin A fragment catestatin (chromogranin A(344-364)) exhibits non-competitive antagonism of nicotinic cholinergic signaling in chromaffin cells. A previous homology model of catestatin's likely structure suggested a mode of interaction of the peptide with the nicotinic receptor, but direct evidence has been lacking. Here we found that [125I]-catestatin binds to the surface of intact PC12 and bovine chromaffin cells with high affinity (K(D)=15.2+/-1.53 nM) and specificity (lack of displacement by another [N-terminal] fragment of chromogranin A). Nicotinic agonist (carbamylcholine) did not displace [125I]-catestatin from chromaffin cells, nor did catestatin displace the nicotinic agonist [3H]-epibatidine; these observations indicate a catestatin binding site separate from the agonist binding pocket on the nicotinic receptor, a finding consistent with catestatin's non-competitive nicotinic mechanism. [125I]-catestatin could be displaced from chromaffin cells by substance P (IC(50) approximately 5 microM), though at far lower potency than displacement by catestatin itself (IC(50) approximately 350-380 nM), suggesting that catestatin and substance P occupy an identical or overlapping non-competitive site on the nicotinic receptor, at different affinities (catestatin > substance P). Small, non-peptide non-competitive nicotinic antagonists (hexamethonium or clonidine) did not diminish [125I]-catestatin binding, suggesting distinct non-competitive binding sites on the nicotinic receptor for peptide and non-peptide antagonists. Similar binding and inhibitory profiles for [125I]-catestatin were observed on chromaffin cells as well as nicotinic receptor-enriched Torpedo membranes. Covalent cross-linking of [125I]-catestatin to Torpedo membranes suggested specific contacts of [125I]-catestatin with the delta, gamma, and beta subunits of the nicotinic receptor, a finding consistent with prior homology modeling of the interaction of catestatin with the extracellular face of the nicotinic heteropentamer. We conclude that catestatin occludes the nicotinic cation pore by interacting with multiple nicotinic subunits at the pore vestibule. Such binding provides a physical explanation for non-competitive antagonism of the peptide at the nicotinic receptor.     

Interaction of substance P antagonists with substance P receptors on dispersed pancreatic acini

In the present study we examined the abilities of three analogs of substance P, [D-Pro2-, D-Phe7-, D-Trp9]-substance P, [D-Pro2-, D- Trp7 ,9]-substance P and [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to alter substance P-induced changes in pancreatic acinar cell function and to occupy substance P receptors. At 30 microM, each analog of substance P lacked agonist activity and inhibited amylase secretion stimulated by substance P receptor agonists. The inhibition was reversible and specific for peptides that interact with substance P receptors (physalaemin, substance P, eledoisin, kassinin ). The analogs of substance P did not inhibit the actions of cholecystokinin, caerulein, gastrin, carbamylcholine, secretin, vasoactive intestinal peptide, PHI, ionophore A23187 or 8Br -cAMP. At high concentrations, [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P, but not [D-Pro2-, D- Trp7 ,9]-substance P or [D-Pro2-, D-Phe7-, D-Trp9]-substance P, caused a small but significant inhibition of bombesin-stimulated amylase release. For each analog of substance P, the inhibition was competitive in nature in that there was a rightward shift of the dose-response curve for physalaemin-stimulated amylase secretion with no change in efficacy. From Schild plots of the ability of [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to inhibit either substance p- or physalaemin-stimulated amylase release, the slopes were not different from unity. For each analog of substance P, there was a close correlation between its ability to inhibit substance P- or physalaemin-stimulated amylase release and its ability to inhibit binding of 125I-labeled substance P or 125I-labeled physalaemin. [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P was 2-fold more potent than [D-Pro2-, D- Trp7 ,9]-substance P which was 4-fold more potent than [D-Pro2-, D-Phe7-, D-Trp9]-substance P, (i.e., pA2 6.1, 5.9, and 5.2, respectively). For each analog, the dose-response curve for its ability to inhibit physalaemin-stimulated amylase release was superimpossible on the dose-response curve for its ability to inhibit binding of 125I-labeled physalaemin. These results indicate that each of these analogs of substance P is a specific competitive inhibitor of the action of the substance P on dispersed acini from guinea-pig pancreas, and that their abilities to inhibit substance P-induced changes in acinar cell function can be accounted for by their abilities to occupy the substance P receptor.     

The rat submaxillary gland contains predominantly P-type tachykinin binding sites

The specific binding of the ¹²⁵I-Bolton-Hunter labeled tachykinins substance K (BHSK), eledoisin (BHE), and substance P (BHSP) was examined in crude membrane suspensions and by autoradiography in rat submaxillary gland. All three ligands at 0.1 nM concentrations exhibited binding that was inhibited by tachykinins in a potency rank order of substance P > physalaemin > substance K > eledoisin > kassinin > neuromedin K with slope factors essentially equal to unity. All tachykinins were 5 to 10 times more potent in inhibiting BHSK and BHE binding compared to BHSP binding. Autoradiographic visualization of BHSK and BHSP binding sites in the gland revealed extensive labeling of mucous and serous acini. The intensity of labeling was much less for BHSK than for BHSP. The results indicate that the rat submaxillary gland contains predominantly P-type tachykinin binding sites.     

Radioactive labels for Protein A: evaluation in the indirect immunoradiometric assay (IRMA) for Bacillus anthracis spores

P hillips , A.P. & M artin , K.L. 1984. Radioactive labels for Protein A: evaluation in the indirect immunoradiometric assay (IRMA) for Bacillus anthracis spores. Journal of Applied Bacteriology 56 , 449–456.
Staphylococcus aureus Protein A (SpA) labelled with [¹²⁵I] by the Bolton & Hunter (1973) method performed about as well as labelled sheep anti-rabbit globulin (SAR) in an indirect immunoradiometric assay (IRMA) for Bacillus anthracis spores immobilized on multispot microscope slides. SpA labelled with [³H] by propi-onylation also performed well but would be expensive to use. SpA labelled with [³H] fluorodinitrobenzene, or labelled with [¹²⁵I] by the chloramine T reaction gave erratic assay results, high noise values and low signal-to-noise ratios, indicating substantial direct binding of labelled SpA to the slide surface and to the bacterial preparation. The uptake of radioactively labelled SpA in the IRMA was compared with the fluorescence intensity of individual spores in a micro-fluorometric immunofluorescence (IF) test involving dual labelled fluorescein-[¹²⁵I]-SpA. The maximum number of SAR molecules bound to the mixture of spores and cell-free antigens in the B. anthracis IRMA was about twice the maximum number of radioactively labelled SpA molecules bound. The SAR : SpA saturation binding ratio on the surface of the spores, however, was approximately the inverse of this. It is concluded that radioactively-labelled SpA is not recommended in preference to anti-species antibody reagents in bacterial IRMA tests but fiuorescein-conjugated SpA deserves further consideration for use in microscope-based IF tests for bacterial antigens.     

Characterization of functional melanotropin receptors in lacrimal glands of the rat

The specific melanotropin (MSH) binding sites of rat lacrimal glands were characterized with respect to anatomic distribution, peptide specificity and selectivity, and coupling to a biological response. Tissue distribution of MSH binding sites was determined by autoradiography following in situ binding of a radiolabeled, biologically active preparation of a superpotent alpha-MSH analog, [125I]-[Nle4,D-Phe7]-alpha-MSH ([125I]-NDP-MSH). Intense, specific (i.e., alpha-MSH-displaceable) [125I]-NDP-MSH binding was observed throughout lacrimal acinar tissue, but not in ducts or stroma. In freshly isolated lacrimal acinar cells, specific binding of [125I]-NDP-MSH was maximal within 30 min and rapidly reversible, with a dissociation half-time of about 15 min. A number of melanotropins [alpha-MSH, [N,O-diacetyl-Ser1]-alpha-MSH, [des-acetyl-Ser1]-alpha-MSH, beta-MSH, ACTH(1-24) and ACTH(1-39)] were recognized by these binding sites, as assessed by their inhibition of [125I]-NDP-MSH binding; NDP-MSH was the most potent (IC50 = 1.3 x 10(-9) M). In contrast, other peptides, including ACTH(4-10) and the nonmelanotropic peptides VIP, substance P, somatostatin, and ACTH(18-39) (CLIP), had no effects on tracer binding. In isolated lacrimal acinar cells, alpha-MSH and NDP-MSH stimulated intracellular cyclic AMP accumulation. We conclude that lacrimal acinar cells express functional receptors recognizing melanotropins, suggesting that the lacrimal gland may be a target for physiological regulation by endogenous melanotropins.     

Contractile response to substance P in isolated smooth muscle strips from the intestinal bulb of the carp (Cyprinus carpio)

1. The effect of substance P on the mechanical activity of carp intestinal bulb smooth muscle was investigated in vitro. 2. Bath-applied substance P (1 nM-1 microM) caused concentration-dependent contraction of the smooth muscle. The EC50 value was 20 +/- 3 nM (N = 13). 3. Pretreatment with tetrodotoxin (780 nM) or atropine (500 nM) partially decreased the contractile response to substance P, while methysergide (3 microM) did not decrease the response. 4. The contractile response to substance P was not decreased by [D-Pro2, D-Trp7.9]-substance P or [D-Pro4, D-Trp7.9]-substance P (4-11) pretreatment (10 microM for 5 min). 5. Exposure of the intestinal bulb to substance P (100 nM and 1 microM for 15 min) decreased the response to subsequent application of substance P, physalaemin and eledoisin in a concentration dependent manner, while the contractile response to acetylcholine or methionine-enkephalin was not affected. 6. Exposure of the intestinal bulb to physalaemin and eledoisin (100 nM for 15 min) decreased the response to subsequent application of substance P. 7. The above results indicate that substance P causes the contraction of the carp intestinal bulb smooth muscle through its direct action on the smooth muscle and its indirect action through enteric cholinergic nerves. Long-term exposure to substance P causes desensitization of the preparation to substance P, physalaemin and eledoisin at the receptor level.     

Radioiodinated [D-Ala2, Met5] enkephalin. Preparation, purification by high performance liquid chromatography and binding characteristics

125I[D-Ala2, Met5] enkephalin with high specific activity (122-185 Ci/mmol) was prepared and purified by Sep-Pak C18 reverse phase cartridge followed by high performance liquid chromatography (HPLC). HPLC at pH 3.0 resolved 125I[D-Ala2, Met5] enkephalin into two fractions, which ran as a single spot in thin-layer chromatography with the same Rf values. Alkaline hydrolysates of the HPLC-purified fractions showed a single spot corresponding to monoiodotyrosine standard when analysed by thin-layer chromatography. Binding kinetics of the tracer was found to approach equilibrium after 30 min at 24 degrees. Scatchard analysis of the saturation equilibrium binding studies gave an equilibrium dissociation constant of 3.58 nM and the number of binding site of 30 fmol/mg protein. Enkephalin analogs were capable of displacing 125I[D-Ala2, Met5] enkephalin binding from the rat brain plasma membrane. The effective concentration of [D-Ala2, Met5] enkephalin and [D-Ala2, Leu5] enkephalin for 50% inhibition of 125I[D-Ala2, Met5] enkephalin binding was estimated to be 79 nM and 23 nM, respectively. Both substance P and gastrin tetrapeptide failed to displace the 125I[D-Ala2, Met5] enkephalin binding to any significant extent. The 125I[D-Ala2, Met5] enkephalin prepared by the present procedure is therefore a useful tracer. This method of preparing radioiodinated peptide may be applicable to other enkephalin analogs or neuropeptides in general.     

A unique hydrophobic domain of rat brain globular acetylcholinesterase for binding to cell membranes

Both salt-soluble and detergent-soluble rat brain globular acetylcholinesterases (SS- and DS- AChE EC 3.1.1.7) are amphiphiles, as shown by detergent dependency of enzymatic activity and binding to liposomes. Proteinase K and papain treatment transformed SS-AChE and DS-AChE into forms that, in absence of detergent, no longer aggregated nor bound to liposomes. In contrast, phosphatidylinositol-specific phospholipase C had no effect on these properties. Labeling DS-AChE with 3-(trifluoromethyl)-3-(m-(¹²⁵I)-iodophenyl) diazirine ([¹²⁵I]TID) revealed, by polyacrylamide gel electrophoresis under reducing conditions, one single band of 69 kD apparent molecular mass. The same pattern was previously obtained with Bolton and Hunter reagent-labeled enzyme (1). Proteinase K treatment transformed the 11 S [¹²⁵I]TID labeled AChE into a 4 S form which no longer showed¹²⁵I-radioactivity and was unable to bind to liposomes. These results are compatible with the existence of a hydrophobic segment present both on salt-soluble and detergent-soluble 11 S AChE as well as on the minor forms 4 S and 7 S. This segment is not linked to the catalytic subunits by disulfide bounds in contrast to the 20 kD non-catalytic subunit described by Inestrosa et al. (2).Abbreviations used AChE acetylcholinesterase - SS-AChE salt-soluble AChE - DS-AChE detergent-soluble AChE - BSA bovine serum albumin - ChE serum (butyryl) cholinesterase - ConA-Sepharose concanavalin A-Sepharose 4B - DMAEBA-Sepharose dimethylaminoethylbenzoic acid-Sepharose 4B - PC-Chol-SA liposomes phosphatidylcholine-cholesterol-stearylamine liposomes - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - ¹²⁵I-TID 3-(trifluoromethyl)-3-(m-(¹²⁵I)-iodophenyl) diazirine     

Iodotyrosylation of peptides using tertiary-butyloxycarbonyl-l-[125I]iodotyrosine N-hydroxysuccinimide ester

A rapid method for the iodotyrosylation of peptides using tertiary-butyloxycarbonyl-l-[¹²⁵I]-iodotyrosine N-hydroxysuccinimide ester is described. The method, like that of Bolton and Hunter (1973, Biochem. J.133, 529–539), uses nonoxidizing conditions, is applicable to tyrosine-free peptides, and results in an easily isolated product with decreased cationic charge. The present method has the additional advantage that the derivatized peptide is readily deblocked to form a radiolabeled product with the same net charge as the starting material.     

A comparison of two procedures for labelling the surface of the hydatid disease organism, Echinococcus granulosus, with 125I

Living, intact protoscoleces of the British horse and sheep strains of Echinococcus granulosus were subjected to surface radioiodination procedures using 125I and Iodogen and 125I-Bolton Hunter reagent. Subsequent combined electron microscopy and autoradiography revealed specific surface membrane labelling with the Iodogen procedure, but significant tegumental labelling with the Bolton-Hunter reagent. The two parasite strains yielded different profiles of electrophoretically separated labelled proteins; the Iodogen method, not surprisingly, resulted in a less complex pattern of labelled polypeptides than the Bolton and Hunter reagent.