Angiotensin-Induced Changes in the Apparent Size of Rat Liver Angiotensin Receptors

Abstract

Angiotensin receptors from rat liver were labeled using four different ligands : (Sar¹-(³H)Tyr⁴)-Angiotensin II ((³H)SarAII); (Sar¹-(³H)Tyr⁴-IIe⁸) -Angiotensin II ((³H)SarIIeAII); (Sar¹-(¹²⁵I)Tyr⁴-(4′-N₃)Phe⁸)-Anglotensin II (IN₃AII); (Sar¹-(¹²⁵I)Tyr⁴-(4′N₃D-Phe)⁸)-Angiotensin II (IN₃DPheAII) (³H) SarAII and IN₃AII behaved like agonists and (³H) SarlleAII and IN₃DPheAII like antagonists. All four ligands labeled the same population of sites. The azido derivatives allowed covalent labeling of receptors with a high yield (about 40%). Membranes were solubilized by Triton X-100 under experimental conditions which ensured complete solubilization of the liganded receptors in a stable form (less than 40% dissociation after 20 h). The apparent size of liganded angiotensin receptors was determined by gel filtration on Ultrogel ACA-34 columns and by SDS gel electrophoresis (in the case of covalent labeling). The apparent Stokes radius of solubilized angiotensin receptors was different wether the receptor was labeled with an agonist (Stokes radius = 6.2 ± 0. 1 nm (6) after labeling with (³H) SarAII) or with an antagonist (Stokes radii of 5. 5 ± 0. 1 (7), and 5.6 ± 0.1 nm (4) after labeling with (³H) SarIIeAII and IN₃DPheAII respectively). After covalent labeling with IN₃All anglotensin receptors were eluted as a mixture of light and heavy forms. SDS gel electrophoresis revealed only one molecular entity of M_r 64,000. It is concluded that binding of an agonist to liver angiotensin receptors triggers or stabilizes an interaction with another membrane component Involved in the coupling of the receptor to its primary effector.     

A high-affinity,radioiodinatable neuropeptide FF analogue incorporating a photolabile p-(4-hydroxybenzoyl)phenylalanine

A new radioiodinated photoaffinity compound, [¹²⁵I]YE(Bpa)WSLAAPQRFNH₂, derived from a peptide present in the rat neuropeptide FF (NPFF) precursor was synthesized, and its binding characteristics were investigated on a neuroblastoma clone, SH-SY5Y, stably expressing rat NPFF₂ receptors tagged with the T7 epitope. The binding of the probe was saturable and revealed a high-affinity interaction (K_D = 0.24 nM) with a single class of binding sites. It was also able to affinity label NPFF₂ receptor in a specific and efficient manner given that 38% of the bound radioligand at saturating concentration formed a wash-resistant binding after ultraviolet (UV) irradiation. Photoaffinity labeling with [¹²⁵I]YE(Bpa)WSLAAPQRFamide showed two molecular forms of NPFF₂ receptor with apparent molecular weights of 140 and 95 kDa in a 2:1 ratio. The comparison of the results between photoaffinity labeling and Western blot analysis suggests that all receptor forms bind the probe irreversibly with the same efficiency. On membranes of mouse olfactory bulb, only the high molecular weight form of NPFF₂ receptor is observed. [¹²⁵I]YE(Bpa)WSLAAPQRFamide is an excellent radioiodinated peptidic ligand for direct and selective labeling of NPFF₂ receptors in vitro.     

Solubilization and Sedimentation Analysis of Muscarinic Acetylcholine Receptors

Abstract

To investigate if G-protein-receptor interactions can be characterized using sucrose density gradients (SDG) we have determined the experimental conditions for muscarinic acetylcholine receptor (mAChR) solubilization and analysis on SDG. Solubilization of 65–80% of [³H]QNB bound mAChR was accomplished with 1% of detergent. Analysis of solubilized receptors on SDG containing 0.4M KCl and 0.1% detergent demonstrated that the physical properties of the receptor-detergent complexes are influenced by the solubilizing detergent as well as detergents included in the SDG. Neither GTPγS nor NaF and AlCl₃ altered the sedimentation properties of mAChR, suggesting that the solubilized mAChR is no longer associated with G-protein under these conditions. Receptors bound to [³H]oxotremorine and [³H]QNB had similar sedimentation properties, suggesting that, once solubilized, mAChRs do not remain associated with G-proteins. Covalent labeling with [³H]PrBCM followed by solubilization and analysis on SDS-gel electrophoresis demonstrated the presence of intact receptor molecule. These observations suggest that the changes in the sedimentation properties of detergent-receptor complexes are independent of G-protein interactions and are influenced by the nature of the detergent associated with the mAChR during analysis.     

Synthesis of the Nanomolar Photoaffinity GABAB Receptor Ligand CGP 71872 Reveals Diversity in the Tissue Distribution of GABAB Receptor Forms

A radioiodinated probe, [¹²⁵I]-CGP 71872, containing an azido group that can be photoactivated, was synthesized and used to characterize GABA_B receptors. Photoaffinity labeling experiments using crude membranes prepared from rat brain revealed two predominant ligand binding species at 130 and 100 kDa believed to represent the long (GABA_BR1a) and short (GABA_BR1b) forms of the receptor. Indeed, these ligand binding proteins were immunoprecipitated using a GABA_B receptor-specific antibody confirming the receptor specificity of the photoaffinity probe. Most convincingly, [¹²⁵I]-CGP 71872 binding was competitively inhibited in a dose-dependent manner by cold CGP 71872, GABA, saclofen, (−)-baclofen, (+)-baclofen and ( )-glutamic acid with a rank order and stereospecificity characteristic of the GABA_B receptor. Photoaffinity labeling experiments revealed that the recombinant GABA_BR2 receptor does not bind [¹²⁵I]-CGP 71872, providing surprising and direct evidence that CGP 71872 is a GABA_BR1 selective antagonist. Photoaffinity labeling experiments using rat tissues showed that both GABA_BR1a and GABA_BR1b are co-expressed in the brain, spinal cord, stomach and testis, but only the short GABA_BR1b receptor form was detected in kidney and liver whereas the long GABA_BR1a form was selectively expressed in the adrenal gland, pituitary, spleen and prostate. We report herein the synthesis and biochemical characterization of the nanomolar affinity [¹²⁵I]-CGP 71872 and CGP 71872 GABA_BR1 ligands, and differential tissue expression of the long GABA_BR1a and short GABA_BR1b receptor forms in rat and dog.     

Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor: A REGION CRITICAL FOR BIOLOGICAL ACTIVITY*

Understanding the molecular basis of natural ligand binding and activation of the glucagon-like peptide 1 (GLP1) receptor may facilitate the development of agonist drugs useful for the management of type 2 diabetes mellitus. We previously reported molecular approximations between carboxyl-terminal residues 24 and 35 within GLP1 and its receptor. In this work, we have focused on the amino-terminal region of GLP1, known to be critical for receptor activation. We developed two high-affinity, full agonist photolabile GLP1 probes having sites of covalent attachment in positions 6 and 12 of the 30-residue peptide (GLP1(7–36)). Both probes bound to the receptor specifically and covalently labeled single distinct sites. Chemical and protease cleavage of the labeled receptor identified the juxtamembrane region of its amino-terminal domain as the region of covalent attachment of the position 12 probe, whereas the region of labeling by the position 6 probe was localized to the first extracellular loop. Radiochemical sequencing identified receptor residue Tyr¹⁴⁵, adjacent to the first transmembrane segment, as the site of labeling by the position 12 probe, and receptor residue Tyr²⁰⁵, within the first extracellular loop, as the site of labeling by the position 6 probe. These data provide support for a common mechanism for natural ligand binding and activation of family B G protein-coupled receptors. This region of interaction of peptide amino-terminal domains with the receptor may provide a pocket that can be targeted by small molecule agonists.     

Comparison of the Surface Proteins and Glycoproteins On Erythrocytes of Calves Before and During Infection With Babesia Bovis

The surface proteins and glycoproteins on red cells from normal and Babesia bovis-infected calf blood have been compared. Several radiolabeling probes were used to label specifically external membrane molecules which were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by autoradiography or fluorography. No differences were observed among the Coomassie Blue-stained membrane proteins of erythrocytes from individual uninfected calves. Comparison of red cells from these animals also indicated no qualitative differences in the surface proteins with accessible tyrosyl residues labeled by lactoperoxidase-catalyzed radioiodnation, although some quantitative variation in the uptake of radioactivity into particular proteins was observed. the major radioiodinated bands on normal bovine erythrocytes had Mr of 165, 130, 90, and 45 kiloDaltons. However, labeling of surface glycoproteins by the periodate/[³H]NaBH₄ and galactose oxidase (± neuraminidase)/[³H]NaBH₄ methods showed significant differences in the surface proteins of red cells from individual uninfected calves. of 14 animals tested, 5 had major labeled glycoproteins of unique Mr. No changes were observed in radioiodinated surface proteins of total red cell samples from infected calves with 0.5-6% parasitemia. Radioiodination of concentrated infected red cells from the same samples (concentrated by selective hypotonic lysis of uninfected erythrocytes in KC1) resulted in the labeling of 3 new surface proteins, with Mr of 118, 115, and 60 kiloDaltons. the same new ¹²⁵I-labeled bands were identified on infected cells from 3 avirulent strains of B. bovis used in vaccine production. Furthermore, in concentrated infected cells there was very poor radiolabeling of major bands strongly labeled on uninfected cells (Mr 165, 130, and 90 kiloDaltons), suggesting parasite-induced loss of these proteins. Although there were some differences in ³H-labeled surface glycoproteins of red cells from normal and. B. bovis -infected blood, they were restricted to minor labeled bands and were not seen consistently. the labeled surface glycoproteins of concentrated infected cells were very similar to those of the uninfected red blood cells from infected blood.     

Identification of Mammalian Brain and Spinal Cord Opioid Receptor by Photoaffinity Labeling

Abstract

A radioiodinated photoreactive enkephal in derivative, ¹²⁵I(D-Ala² p-N₃-Phe⁴-Met⁵) enkephalin, was used to photoaffinity label the opioid receptor from the membranes of four mammalian brains (without cerebellum) and spinal cords. These included the cat, rabbit, guinea pig and mouse. The photolabeled membranes were analyzed by sodium dodecyl sulfate gel electrophoresis. A 43,000-daltons protein was specifically photolabeled in all the membranes tested, as the specific labeling of this protein was inhibited in the presence of 14.5 uM of (D-Ala² Met⁵) enkephalin. These data suggest that the 43,000-daltons protein is a binding protein of the opioid receptor in the different mammalian neural tissues.     

Differential docking of high-affinity peptide ligands to type A and B cholecystokinin receptors demonstrated by photoaffinity labeling

Type A and B cholecystokinin (CCK) receptors are highly homologous members of the class-I family of G protein-coupled receptors that bind CCK with high affinity. However, they have divergent structural specificities, with the type A receptor requiring seven carboxyl-terminal residues including a sulfated tyrosine and the type B receptor requiring only the carboxyl-terminal tetrapeptide. The aim of this work was to utilize affinity labeling to determine spatial approximations with photolabile p-benzoyl-l-phenylalanine (Bpa) residues sited at each end of CCK as docked at the type B CCK receptor, contrasting this with analogous work using similar probes docked at the type A receptor. Both probes were fully efficacious, potent agonists that stimulated intracellular calcium in receptor-bearing CHO-CCKBR cells (EC(50) values: Bpa(24) probe, 41 +/- 9 pM; Bpa(33) probe, 15 +/- 3.3 pM). They bound specifically, with high affinity (K(i) values: Bpa(24) probe, 0.60 +/- 0.17 nM; Bpa(33) probe, 0.58 +/- 0.11 nM). Cyanogen bromide cleavage of the covalently labeled receptor suggested the first extracellular loop as the region of labeling by each probe, distinct from the type A CCK receptor regions labeled using the same probes (third loop and amino-terminal tail, respectively). This was confirmed by subsequent enzymatic and chemical cleavage of labeled wild-type and mutant receptors. Sequential cycles of Edman degradation of labeled receptor fragments identified the specific residues within loop one labeled by each probe (Bpa(24) probe labeled Phe(122); Bpa(33) probe labeled Thr(119)). This provides a direct demonstration of distinct modes of docking the same high-affinity ligand to highly homologous receptors.     

5-Azido-8-ethynyl-NAADP: A bifunctional,clickable photoaffinity probe for the identification of NAADP receptors

Nicotinic acid adenine dinucleotide phosphate is an evolutionarily conserved second messenger, which mobilizes Ca²⁺ from acidic stores. The molecular identity of the NAADP receptor has yet to be defined. In pursuit of isolating and identifying NAADP-binding proteins, we synthesized and characterized a bifunctional probe that incorporates both a photoactivatable crosslinking azido moiety at the 5-position of the nicotinic ring and a ‘clickable’ ethynyl moiety to the 8-adenosyl position in NAADP. Microinjection of this 5N₃-8-ethynyl-NAADP into cultured U2OS cells induced robust Ca²⁺ responses. Higher concentrations of 5N₃-8-ethynyl were required to elicit Ca²⁺ release or displace ³²P-NAADP in radioligand binding experiments in sea urchin egg homogenates. In human cell extracts, incubation of ³²P-5N₃-8-ethynyl-NAADP followed by UV irradiation resulted in selective labeling of 23 kDa and 35 kDa proteins and photolabeling of these proteins was prevented when incubated in the presence of unlabeled NAADP. Compared to the monofunctional ³²P-5N₃-NAADP, the clickable ³²P-5N₃-8-ethynyl-NAADP demonstrated less labeling of the 23 kDa and 35 kDa proteins (~3-fold) but provided an opportunity for further enrichment through the ‘clickable’ ethynyl moiety. No proteins were specifically labeled by ³²P-5N₃-8-ethynyl-NAADP in sea urchin egg homogenate. These experiments demonstrate that 5N₃-8-ethynyl-NAADP is biologically active and selectively labels putative NAADP-binding proteins in mammalian systems, evidencing a ‘bifunctional’ probe with utility for isolating NAADP-binding proteins.     

Molecular Basis of Glucagon-like Peptide 1 Docking to Its Intact Receptor Studied with Carboxyl-terminal Photolabile Probes

The glucagon-like peptide 1 (GLP1) receptor is a member of Family B G protein-coupled receptors and represents an important drug target for type 2 diabetes. Despite recent solution of the structure of the amino-terminal domain of this receptor and that of several close family members, understanding of the molecular basis of natural ligand GLP1 binding to its intact receptor remains limited. The goal of this study was to explore spatial approximations between specific receptor residues within the carboxyl terminus of GLP1 and its receptor as normally docked. Therefore, we developed and characterized two high affinity, full-agonist photolabile GLP1 probes having sites for covalent attachment in positions 24 and 35. Both probes labeled the receptor specifically and saturably. Subsequent peptide mapping using chemical and proteinase cleavages of purified wild-type and mutant GLP1 receptor identified that the Arg¹³¹–Lys¹³⁶ segment at the juxtamembrane region of the receptor amino terminus contained the site of labeling for the position 24 probe, and the specific receptor residue labeled by this probe was identified as Glu¹³³ by radiochemical sequencing. Similarly, nearby residue Glu¹²⁵ within the same region of the receptor amino-terminal domain was identified as the site of labeling by the position 35 probe. These data represent the first direct demonstration of spatial approximation between GLP1 and its intact receptor as docked, providing two important constraints for the modeling of this interaction. This should expand our understanding of the molecular basis of natural agonist ligand binding to the GLP1 receptor and may be relevant to other family members.     

A carbene-generating photoaffinity probe for beta-adrenergic receptors

A new radioiodinated (2.2 Ci/μmol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [¹²⁵I]ICYP-diazirine binds with high affinity (K_d = 60 pM) to β-receptors from turkey erythrocyte membranes. Upon irradiation, [¹²⁵I]ICYP-diazirine is covalently incorporated in a M_r 40 000 protein. Stereoselective inhibition of photolabeling by the (?)enantiomers of alprenolol and isoproterenol indicated that the M_r 40 000 protein contains a β-adrenergic binding site. The yield of specific labeling was up to 8.2% of total β-receptor binding sites. The M_r 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [¹²⁵I]ICYP-diazirine resulted in specific labeling of two proteins with M_r 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximate M_r 67 000) was labeled specifically.     

Engineering streptokinase for generation of active site-labeled plasminogen analogs

We previously demonstrated that streptokinase (SK) can be used to generate active site-labeled fluorescent analogs of plasminogen (Pg) by virtue of its nonproteolytic activation of the zymogen. The method is versatile and allows stoichiometric and active site-specific incorporation of any one of many molecular probes. The limitation of the labeling approach is that it is both time-consuming and low yield. Here we demonstrate an improved method for the preparation of labeled Pg analogs by the use of an engineered SK mutant fusion protein with both COOH- and NH₂-terminal His₆ tags. The NH₂-terminal tag is followed by a tobacco etch virus proteinase cleavage site to ensure that the SK Ile¹ residue, essential for conformational activation of Pg, is preserved. The SK COOH-terminal Lys⁴¹⁴ residue and residues Arg253–Leu260 in the SK β-domain were deleted to prevent cleavage by plasmin (Pm) and to disable Pg substrate binding to the SK·Pg^∗/Pm catalytic complexes, respectively. Near elimination of Pm generation with the SKΔ(R253–L260)ΔK414–His₆ mutant increased the yield of labeled Pg 2.6-fold and reduced the time required more than 2-fold. The versatility of the labeling method was extended to the application of Pg labeled with a near-infrared probe to quantitate Pg receptors on immune cells by flow cytometry.     

SYNTHESIS OF NOVEL PEPTIDE NUCLEIC ACID-PEPTIDE CHIMERA FOR NON-INVASIVE IMAGING OF CANCER

A chelator-peptide-PNA-peptide chimera specific for KRAS has been prepared by continuous solid phase coupling with a C-terminal insulin-like growth factor 1 (IGF1) ligand, d(cys-ser-lys-cys), and N-terminal bis(s-benzoyl thioglycoloyl) diaminopropanoate chelator for radionuclide labeling. The probe was purified by RP-HPLC and characterized by MALDI-TOF mass spectroscopy. The probe was labeled with ^{99 m}Tc and ⁶⁴Cu. Both labeled probes accumulated in human pancreatic cancer xenografts in immunocompromised mice. Control experiments with mismatch chimeras and control xenografts will be necessary to determine the specificity of this molecular diagnostic strategy.     

Radioiodination of Parasite Antigens with 1,3,4,6,-Tetrachloro-3α,6α-diphenylglycoluril (IODOGEN): Studies with Zygotes of Plasmodium gallinaceum

The iodinating reagent 1,3,4,6,-tetrachloro-3α,6α-diphenylglycoluril (IODOGEN³) was used to label antigens on zygotes of Plasmodium gallinaceum with parallel studies using lactoperoxidase-catalyzed radioiodination for comparison. Proteins labeled by the IODOGEN method are most probably on the surface of the zygote, as the pattern of labeled proteins analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was very similar to the pattern of lactoperoxidase-labeled proteins. Furthermore, the labeled proteins represented only a subset of the total Coomassie Blue-stained proteins. The radioiodinated zygote proteins were immuno-reactive after IODOGEN or lactoperoxidase labeling. The IODOGEN method is technically much more simple than the lactoperoxidase method and does not require the addition of extraneous proteins or H₂O₂. The advantages of IODOGEN labeling, together with the essential equivalence of results obtained by these two, methods, make the IODOGEN method attractive for labeling parasite antigens in general.     

The Postnatal Development of the Gabaa/benzodiazepine Receptor in the Rat Red Nucleus

Abstract

The development of the GABA_A/Benzodiazepine receptor (GABA_AR) in the red nucleus was studied using ³H-flunitrazepam (FNZ) as the probe. Saturation binding assay showed that the ^Bmax of the ligand to the membranes of the nucleus increased from 0.50 ± 0.04 nmol/mg protein at postnatal day 4, to 0.71 ± 0.1 and 0.78 ± 0.08 at day 7 and day 10. At day 20 the ^Bmax decreased to a level near day 4 and persisted until day 40. However, the affinity of ³H-FNZ to the receptor remained quite constant. At least 4 proteins of 51kD, 53kD, 59kD and 62kD in the nucleus were labeled by ³H-FNZ, as revealed from photoaffinity binding and SDS-PAGE. The labeling of 53kD, 59kD and 62kD was high at earlier ages than day 10, whereas the 51kD was predominent from day 10 to day 40. Receptor binding autoradiography of the nucleus also showed that the most dense labeling was seen around day 10. The early transient increase in the GABA_AR of the red nucleus may indicate the plasticity of the nucleus in response to environmental changes after birth.     

Biodistribution of p-Iodobenzoyl (PIP) labeled antibodies in a murine lymphoma model

A monoclonal antibody against the murine T-cell antigen Thy 1.1 was radioiodinated using N-succin-imidyl p-iodobenzoate (PIP) in an attempt to decrease deiodination of the labeled antibody. The biodistribution of the PIP labeled antibody was compared to Iodogen labeled antibody in Thy 1.1⁺ lymphoma bearing AKR/Cum mice, where the antibody was tumor specific, and AKR/J mice where the antibody reacted with both tumor and normal T-cells. PIP labeling resulted in decreased iodine concentrations in stomach and salivary gland as compared to Iodogen labeling. There was little difference in radioiodine concentrations between the two preparations in tumor, lymphoid tissues or other organs. These results suggest deiodination of intact antibody plays little role in the clearance of radioiodinated anti-Thy 1.1 antibody from tissues.     

Biochemical Characterization and Solubilization of Human NK2 Receptor Expressed in Chinese Hamster Ovary Cells

Abstract

The human ileum neurokinin NK₂ receptor has been stably expressed in Chinese hamster ovary (CHO) cells using the dihydrofolate reductase (DHFR) expression system. Amplified cell populations expressing approximately 7×10⁵ NK₂ receptors/cell were selected in the presence of the DHFR inhibitor methotrexate. Cross-linking of [¹²⁵I]NKA to NK₂ receptor transfected cells revealed a specifically labeled protein of apparent molecular weight 64 kDa by SDS-polyacrylamide gel electrophoresis. This protein was deglycosylated by the enzymes N-glycosidase F and endoglycosydase F to a protein of apparent molecular weight of 39 kDa. The NK₂ receptor was solubilized in an active form from CHO cell membranes using the zwitterionic detergent CHAPS. This method represents a valuable approach for the production of significant amounts of NK₂ receptor protein from mammalian cells.     

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.     

Synthesis and structure determination of two new dinuclear end-to-end doubly bridged azido- and thiocyanato-copper(II) complexes derived from diethyldiethylenetriamine

Herein, we report the synthesis and characterization of two new dinuclear bridged azido and bridged thiocyanato complexes: [Cu₂(Et₂dien)₂(μ_1,3-N₃)₂](ClO₄)₂ (1) and [Cu₂(Et₂dien)₂(μ_N,S-NCS)₂]-(ClO₄)₂ (2) where Et₂dien = N,N-diethyldiethylenetriamine. In both complexes, the two copper centers are linked by two azide or two thiocyanate groups in end-to-end bonding fashion. The copper ions are penta-coordinated by three N-atoms of the Et₂dien ligand, one N atom from the bridging azido in 1 or from the thiocyanato group in 2. The fifth coordination site is occupied by N or S atom from the second bridging azide or thiocyanate ligand, respectively. The coordination geometry around the Cu(II) ions in the two complexes may be described as close to square pyramidal (SP) stereochemistry with severe distortion to trigonal bipyramidal (TBP) stereochemistry. The intradimer Cu?Cu distances are 5.264(1) and 5.571(1) Å for the azido and thiocyanato complex, respectively. The IR stretching frequencies of the azido, and the thiocyanato, ν(NCS⁻) groups in the 2030-2120 cm⁻¹ region are discussed in relation to other related species. The visible spectra of the complexes studied in different solvents reveal the assigned predominant SP stereochemistry in solution with the presence of a pronounced amount of TBP geometry in the thiocyanato complex. Moreover, the complexes undergo solvolysis through bond rupture and displacement of one of the bridged azido or thiocyanato ligands.     

Covalent Cross-Linking of Radiolabeled Human Chorionic Gonadotropin to Rat Ovarian Luteinizing Hormone Receptor with Glutaraldehyde

Abstract

Crude plasma membranes of pseudopregnant rat ovaries were incubated with ¹²⁵I-labeled human chorionic gonadotropin (¹²⁵I-hCG) and the formed luteinizing hormone (LH)/hCG receptor-¹²⁵I-hCG complex was solubilized, partially purified by Sepharose 6B gel filtration, cross-linked with glutaraldehyde and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. An apparent molecular weight (mol wt) of 130,000 was obtained for the non-reduced complex. A similar-sized complex was observed, when ³H-hCG (β-subunit labeled) instead of ¹²⁵I-hCG (α-subunit labeled) was used, indicating that the complex contains intact hCG. Upon reduction of the cross-linked receptor-¹²⁵I-hCG complex, a 105,000 mol wt complex in addition to the 130,000 one was observed. No smaller complexes appeared, however, upon reduction of the receptor-³H-hCG complex, suggesting that the α-subunit of hCG predominantly interacts with the receptor. The cross-linking efficiency was dependent on protein concentration, glutaraldehyde concentration, pH, reaction time and temperature. Under optimal conditions (2 mM glutaraldehyde, pH 7.0-8.0, 60 min, 20°C) no nonspecific complexes appeared and the efficiency of cross-linking of the partially purified detergent-solubilized receptor-¹²⁵I-hCG complex was approximately 30%. Glutaraldehyde thus provides a rapid and efficient cross-linking reagent to covalently attach ¹²⁵I-hCG to its receptor and is likely to be highly applicaple to other receptor-ligand systems as well.