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PubMed1985

Photoaffinity labeling of the progesterone receptor from human endometrial carcinoma.

A nude mouse model for the growth of human endometrial carcinoma and hormonal modulation of the progesterone receptor (PR) was established previously. This study describes the effect of 17 beta-estradiol and tamoxifen (TAM) on growth rate and PR concentration in a hormonally responsive human endometrial tumor (EnCa 101) grown in this experimental system and presents the first characterization of human endometrial carcinoma PR. EnCa 101 was transplanted subcutaneously into ovariectomized, BALB/c, nu/nu athymic mice and grown under 17 beta-estradiol-stimulated, TAM-stimulated, and control conditions. Both 17 beta-estradiol and TAM increased the growth rate of EnCa 101 in nude mice, and a parallel increase in the cytosol PR concentration was observed, from 130 +/- 55 (SD) fmol/mg protein to 1,311 +/- 598 fmol/mg protein and 710 +/- 310 fmol/mg protein, respectively. PR was partially purified by phosphocellulose and DEAE cellulose chromatography, and the DEAE eluate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and photoaffinity labeling with [17 alpha-methyl-3H]promegestone ([3H]R5020). Two PR-negative tumors (EnCa K and EnCa V) were also examined in parallel. Coomassie blue staining of gels revealed that the protein patterns of all of the partially purified preparations from EnCa 101, EnCa K, and EnCa V were essentially identical. In contrast, photolabeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of EnCa 101 grown in the presence of 17 beta-estradiol or TAM revealed incorporation of [3H]R5020 into proteins of molecular weight approximately 116,000 and 85,000. Labeled proteins of molecular weight 66,000, 45,000, and 35,000 were also observed. In each case, the labeling was competable with excess non-radioactive R5020. No incorporation of [3H]R5020 was observed in EnCa 101 grown in the absence of estrogen, nor was any observed in EnCa K or EnCa V.

Affinity Labels↗
PubMed1987

Photoaffinity labeling of the mitochondrial phosphate carrier by 4-azido-2-nitrophenyl phosphate.

The effect of 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive analogue of phosphate, on the phosphate carrier of pig-heart mitochondria has been investigated. In the dark, ANPP inhibits the transport of phosphate in a competitive manner with a Ki of 3.2 mM. Upon photoirradiation with visible light, [32P]ANPP binds covalently to the phosphate carrier and the inhibition becomes irreversible. Both the inhibition of phosphate transport and the incorporation of [32P]ANPP into the phosphate carrier depend on the concentration of the inhibitor and the pH of the medium. Incubation of the mitochondria with phosphate during illumination in the presence of ANPP protects the carrier against inactivation and decreases the amount of radioactivity which is found to be associated with the purified protein. By extrapolation it is calculated that at 100% inactivation of the phosphate carrier 0.35 mol of reagent are bound per mol of 33 kDa carrier protein. It is concluded that ANPP can be used for photoaffinity labeling of the mitochondrial phosphate carrier at the substrate-binding site.

Affinity Labels↗
PubMed1994

Stable expression of the cloned rat brain neurotensin receptor into fibroblasts: binding properties, photoaffinity labeling, transduction mechanisms, and internalization.

The study of the pharmacological, biochemical, and transduction properties of the cloned rat brain neurotensin receptor was carried out in thymidine kinase mutant fibroblasts stably transfected with the receptor cDNA. The interaction of neurotensin with transfected fibroblasts leads to a concentration-dependent stimulation of phosphatidylinositol hydrolysis and intracellular calcium. These effects are totally inhibited by the nonpeptide neurotensin antagonist SR48692. By contrast, this receptor remains unable to modulate intracellular levels of cyclic nucleotides. The transfected neurotensin receptor can be solubilized in an active form by digitonin with an identical pharmacological profile, whereas the detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonic acid is unable to solubilize the binding activity. The binding of iodinated neurotensin to transfected fibroblasts bearing the cloned receptor remains partly un-dissociated even after an acid washing step, indicating that the transfected neurotensin receptor retains the capacity to be internalized according to a temperature-dependent mechanism. Indeed, the sequestration of the neurotensin-receptor complex can be blocked by phenylarsine oxide. Finally, photoaffinity labeling experiments reveal that the cloned rat brain neurotensin receptor is expressed under two forms with molecular masses of 50 and 60 kDa. Labeling and internalization of these two proteins are totally blocked by the neurotensin antagonist SR48692.

Animals↗
PubMed1996

Photoaffinity labeling of the lutropin receptor with synthetic peptide for carboxyl terminus of the human choriogonadotropin alpha subunit.

Human choriogonadotropin (hCG) consists of an alpha subunit and a beta subunit. The existing evidence from various studies using truncation, substitution, synthetic hormone peptides, and hCG crystals suggests that the C-terminal region of the alpha subunit contacts the luteinizing hormone/choriogonoadotropin (LH/CG) receptor and is involved in receptor activation. Despite a deluge of the speculation and the important role of the alpha C-terminal region, direct evidence for its interaction with the receptor has been elusive. Because of the significant biological activity, it is imperative to prove the interaction of the alpha C-terminal region. For this purpose, decamer peptides corresponding to the alpha subunit sequence from His83 to Ser92 (alpha 83-92) were derivatized with the N-hydroxysuccinimide ester of 4-azidobenzoylglycine (ABG) and radioiodinated. The resulting ABG-125I-alpha 83-92 was capable of binding and activating the LH/CG receptor. Furthermore, UV-sensitive ABG-125I-alpha 83-92 exclusively photoaffinity-labeled an approximately of 86-kDa molecule. This labeled molecule was shown to be the LH/CG receptor by various methods including immunoprecipitation by anti-LH/CG receptor antiserum. In addition, evidence is presented that the amino group of alpha Lys91 of alpha 83-92 is in such close proximity to a carboxyl group of the receptor that this pair is cross-linked to form an amide, a zero length cross-link. This low affinity contact of alpha 83-92 and the receptor is sufficient for receptor activation and is crucial for the full understanding of the mechanistics of the receptor activation steps.

Affinity Labels↗
PubMed1987

Physicochemical characterization of photoaffinity-labeled angiotensin II receptors.

Specific receptor sites for angiotensin II (AII) were analyzed in the adrenal cortex and other target tissues including liver, anterior pituitary gland, and smooth muscle, after covalent labeling with the radioactive photoaffinity analog 125I-[Sar1,(4-N3)Phe8]-AII. The photoreactive AII derivative retained high affinity for adrenal receptors and full steroidogenic activity in adrenal glomerulosa cells. In bovine adrenal cortex, covalent labeling of AII receptors by the photoreactive analog was specifically inhibited by [Sar1]AII with an IC50 of about 5 nM. The Mr of the covalent AII-receptor complex during polyacrylamide gel electrophoresis of the labeled protein under reducing conditions was 58,000. Under non-reducing conditions, a minor band with Mr of 105,000 was also observed. Two labeled species were also found during gel permeation chromatography of the detergent-solubilized complex, with Mrs of 64,000 and 86,000. The pl of the solubilized photolabeled complex was absorbed to wheat germ lectin Sepharose 6MB and could be eluted by N-acetylglucosamine. The Mrs of specific AII-binding sites in several target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed significant differences within and between species. The most striking differences were between rat adrenal cortex (79,000) and both rat liver (60,000) and bovine adrenal cortex (58,000). After enzymatic deglycosylation, the Mr of the major component present in the bovine and rat adrenal cortex decreased by 40% and 55% to 35,000 and 34,000, respectively, suggesting that variations in carbohydrate content contribute to the physical heterogeneity of AII receptors in individual target tissues.

Adrenal Cortex↗
PubMed2001

A photoaffinity-labeled green leaf volatile compound 'tricks' highly selective and sensitive insect olfactory receptor neurons.

The sex pheromone of the scarab beetle, Phyllopertha diversa, is emitted by females and specifically detected by olfactory receptor neurons in the male and female antennae. Single sensillum recordings showed that, in contrast to the less sensitive pheromone sensilla in females, olfactory receptor neurons in the male antennae had a low threshold (1 ng), which rivals those of moths. The male and female antennae also possessed olfactory receptor neurons specific for the detection of floral and green leaf volatile compounds. Detectors for the green leaf volatile (Z)-3-hexenyl acetate had a threshold (10 pg) far below the sensitivity of the pheromone-detecting machinery. In addition, these neurons showed a remarkable selectivity even when challenged with related compounds at 10000-fold higher concentrations. Surprisingly, a diazo analog, (Z)-3-hexenyl diazoacetate, elicited slightly higher nervous activity than the natural ligand in the neurons specific and selective for (Z)-3-hexenyl acetate. The inability of the green leaf volatile-detecting machinery to discriminate the photoaffinity-labeled compound from the natural product indicates that the synthetic ligand interacts with odorant-binding protein, odorant receptor and odorant-degrading enzyme as does the cognate ligand.

Acetates↗
PubMed1991

Characterization and photoaffinity labeling of a calcitonin gene-related peptide receptor solubilized from human cerebellum.

Calcitonin gene-related peptide (CGRP) receptors were solubilized from human (h) cerebellum with use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of CGRP binding sites with apparent dissociation constants of 50 pM for the intact 125I-hCGRP-I(1-37) and 160 pM for the antagonist 125I-hCGRP-I(8-37). Unlabeled hCGRP-I and -II and hCGRP-I(8-37) displaced 125I-hCGRP-I from solubilized CGRP receptors with similar potencies (ID50 = 70-150 pM). Human CGRP-I(15-37), -(21-37), and -(28-37) were less potent (ID50 greater than or equal to 70 nM), suggesting that amino acid residues 8-14 may be important for maintaining high binding affinity. A novel photoreactive analogue of hCGRP-I, 125I-[C gamma-(4-azidoanilino)Asp3] hCGRP-I, was prepared by carbodiimide coupling of 4-azidoaniline to 125I-hCGRP-I. Photoaffinity labeling of soluble CGRP receptors with the photoreactive analogue and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed three specifically labeled binding proteins with apparent molecular weights (Mr) of 60,000, 54,000, and 17,000. Cross-linking of 125I-hCGRP-I and -II and 125I-hCGRP-I(8-37) to soluble CGRP binding sites using disuccinimidyl suberate revealed three specifically labeled binding proteins with the same Mr. The C-terminal fragment 125I-hCGRP-I(8-37), unlike the intact peptide, was, furthermore, cross-linked specifically to a 95,000 Mr protein. The CGRP receptor is N-glycosylated. Treatment with endoglycosidase F/N-glycosidase F converted the 60,000 and 54,000 to 46,000 and 41,000 Mr components.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1995

Photoaffinity labeling of the mitochondrial oxoglutarate carrier by azido-phthalonate.

The effect of azido-phthalonate, a photoreactive analogue of oxoglutarate, on the transport of oxoglutarate was investigated in proteoliposomes reconstituted with the purified oxoglutarate carrier. In the dark, azido-phthalonate inhibits the reconstituted oxoglutarate/oxoglutarate exchange in a competitive manner with a Ki of 0.38 mM. Upon photoirradiation, the inhibition of the oxoglutarate exchange by azido-phthalonate is not removed by passing the proteoliposomes through a Sephadex column. The light-induced inhibition of the oxoglutarate/oxoglutarate exchange activity by azido-phthalonate is time- and concentration-dependent. The kinetic analysis of transport inhibition by azido-phthalonate reveals that one molecule of this substrate analogue bound to the functional carrier molecule is responsible for complete inhibition of the carrier function. Azido-[3H]phthalonate binds to the oxoglutarate carrier covalently. Incubation of the proteoliposomes with oxoglutarate during photoirradiation in the presence of azido-phthalonate protects the carrier against inactivation and decreases the amount of radioactivity which is found to be associated with the carrier protein. It is concluded that azido-phthalonate can be used for photoaffinity labeling of the mitochondrial oxoglutarate carrier at the substrate-binding site.

Affinity Labels↗
PubMed1995

Purification, pharmacological characterization and photoaffinity labeling of sigma receptors from rat and bovine brain.

The sigma receptor/binding site, found in the brain and periphery, binds haloperidol, (+)-benzomorphans, N-propyl-3-(3-hydroxyphenyl)-piperidine (3-PPP) and certain atypical neuroleptics with high affinity. We have succeeded in ca. 6,000-fold purification of protein(s) from rat and bovine cerebellum which display pharmacology characteristic of the sigma receptor. This purification was achieved by affinity chromatography using a Sepharose gel linked to a new high-affinity ligand, (S)-3-(3-methoxyphenyl)-3'-oxo-3'-phenyl-N-propylpiperidine, an analog of (S)-3-PPP. Elution of the affinity column with haloperidol afforded material which, after reconstitution into bimolecular lipid vesicles, was pharmacologically characterized by specific radioligand binding assays using [3H]haloperidol combined with competitive displacement using appropriate selective ligands. Comparison of the relative rank orders of potency of the ligands in these selective sigma receptor assays corresponded well with values obtained with tissue homogenates. The observed enantioselectivity for the binding of SKF-10,047 and cyclazocine suggests that the material purified corresponds to the sigma 1 receptor subtype. SDS-PAGE indicated that the purified material consisted of two bands of approximate molecular masses 65 and 63 kilodaltons. Photoaffinity labeling of the affinity-purified receptor with [3H]azido-DTG led to incorporation of the label into material of molecular mass 50-70 kDa, by slicing of SDS gels, while similar photolabeling of crude cerebellar homogenates led to exclusive labeling of a 29 kDa polypeptide, as found previously using other tissues. Molecular sizing under non-denaturing conditions indicated the photolabeled species is a labile large receptor complex of mass ca. 300-500 kDa which gradually breaks down upon standing at -80 degrees C into the lower mass (50-70 kDa) material. The sigma receptor ligand binding subunit, which appears to be of the sigma 1 subtype, appears to be contained within the 29 kDa polypeptide, which may be a subunit of the 63-65 kDa protein, which in turn appears to be a component of a much larger receptor complex. It further appears that the 29 kDa polypeptide is readily dissociable from a larger photolabeled sigma receptor complex in tissue homogenates, but does not dissociate from the photolabeled affinity-purified CHAPS-solubilized sigma receptor.

Animals↗
PubMed1981

Biological activities of photoaffinity labeling analogues of kinins and their irreversible effects on kinin receptors.

Several analogues of bradykinin and [des-Arg9]bradykinin with potentially reactive groups have been tested for their biological activities. In these analogues, phenylalanine was replaced by the aromatic amino acid (4'-nitro)Phe, (4'-amino)Phe, (4'-azido)Phe, and (4'-diazonium)Phe, as well as with other residues. [Des-Arg9]bradykinin and the octapeptide analogues were tested on rabbit aorta strips, an assay organ containing the B1 receptor which is activated by the octapeptide [des-Arg9]bradykinin. Strips of rabbit jugular vein served as bioassays for bradykinin and the nonapeptide analogues, because the rabbit jugular vein bears the receptor B2 which is sensitive to bradykinin nonapeptides. The biological findings support the interpretation that kinins act on two different receptor types, since the potency orders of the analogues in the two bioassays are different. All potential photolabels retained reasonable affinities in the dark, except [(4'-azido)Phe8,des-Arg9]bradykinin, which, however, proved to be a weak and competitive antagonist of [des-Arg9]bradykinin on the rabbit aorta. Inactivation experiments with the unstable (4'-diazonium)Phe-containing peptides did not show any irreversible effect in the two bioassays. Photoaffinity labeling experiments with the azido and the nitro peptides gave irreversible and specific effects on both the rabbit aorta and the jugular vein. [(4'-Azido)Phe8,des-Arg9]bradykinin photolyzed at 365 nm on the rabbit aorta reduced the sensitivity of this tissue against [des-Arg9]bradykinin specifically to about one-third of its initial value. [(4'-Azido)Phe5]bradykinin reduced the sensitivity of the rabbit jugular vein to bradykinin by more than 50%. The observed irreversible effects were always loss of myotropic activity and never permanent contraction.

Affinity Labels↗
PubMed1995

Purification, photoaffinity labeling, and characterization of a single enzyme for 6-sulfation of both chondroitin sulfate and keratan sulfate.

A soluble sulfotransferase that could 6-sulfate both chondroitin sulfate and corneal keratan sulfate was purified 27,500-fold using a sequence of affinity chromatographic steps with heparin-Sepharose, wheat germ agglutinin-agarose, and 3',5'-ADP-agarose. The essentially pure enzyme had a specific activity 40 times greater than the most purified chondroitin 6-sulfotransferase previously reported and exhibited a single sharp Coomassie Blue-stained and a heavy silver-stained protein band of 75 kDa on SDS-polyacrylamide gel electrophoresis. Chromatography of the purified enzyme on Sephacryl demonstrated a size of 150 kDa, which indicated that the native enzyme exists as a dimer. In addition to 6-sulfation of nonsulfated GalNAc, the purified serum enzyme had the ability to sulfate GalNAc 4-sulfate residues to give GalNAc 4,6-disulfate residues. The purified enzyme exhibited a Km of 40 microM for adenosine 3'-phosphate 5'-phosphosulfate when either chondroitin sulfate or corneal keratan sulfate were used as the acceptors. Use of both chondroitin sulfate and keratan sulfate in the same experiment demonstrated mutual competition, establishing that the sulfation of these substrates is by the same enzyme. Photoaffinity labeling of the purified enzyme with 2-azidoadenosine 3',5'-di[5'-32P]phosphate occurred only with the 75-kDa protein, confirming that this is the chondroitin 6-sulfotransferase/keratan sulfotransferase.

Adenosine Triphosphate↗
PubMed2005

Photoaffinity labeling with 8-azidoadenosine and its derivatives: chemistry of closed and opened adenosine diazaquinodimethanes.

The reactive intermediate produced upon photolysis of 8-azidoadenosine was studied by chemical trapping studies, laser flash photolysis with UV-vis and IR detection, and modern computational chemistry. It is concluded that photolysis of 8-azidoadenosine in aqueous solution releases the corresponding singlet nitrene which rapidly tautomerizes to form a closed adenosine diazaquinodimethane in less than 400 fs. A perbenzoylated derivative of 8-azidoadenosine cannot undergo this tautomerization, and instead, it fragments upon photolysis to form an opened adenosine diazaquinodimethane. The singlet nitrene is too short-lived to be observed and, thus, to relax to the lowest triplet state or to become covalently attached to targeted biological macromolecules. The pivotal closed adenosine diazaquinodimethane, the product of nitrene tautomerization, has a lifetime of ca. 1 min or longer in water and in HEPES buffer at ambient temperature. However, this intermediate reacts rapidly with good nucleophiles such as amines, thiols, and phenolates, and significantly more slowly with weak nucleophiles such as alcohols and water. On the basis of these studies, it is clear that the closed adenosine diazaquinodimethane, and not the singlet or triplet nitrene, is the pivotal reactive intermediate involved in photolabeling and cross-linking studies using the 8-azidoadenosine family of photoaffinity labeling reagents.

Adenosine↗
PubMed1986

5-Azido-2'-deoxyuridine 5'-triphosphate: a photoaffinity-labeling reagent and tool for the enzymatic synthesis of photoactive DNA.

We have synthesized the photoactive deoxyuridine nucleotide 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP) and used it to synthesize light-sensitive DNA by enzymatic incorporation. In the absence of ultraviolet light, 5-N3dUTP is a substrate for Escherichia coli DNA polymerase I. In in vitro DNA synthesis reactions using bacteriophage M13 single-stranded DNA as the template and 5-N3dUTP in place of dTTP, a photoactive complementary strand was synthesized by DNA polymerase I. The complementary strand was not synthesized when the 5-N3dUTP was substituted for dCTP or when it was exposed to ultraviolet light prior to the addition of DNA polymerase I. Using a synthetic lac operator template of 26 bases and a 15-base primer, we generated a photoactive 26-base-pair lac operator by enzymatically incorporating 5-N3dUMP with DNA polymerase I. Crosslinking of this photoactive DNA fragment to lac repressor was totally dependent on the presence of UV light and was reduced 78% by 150 microM isopropyl beta-D-thiogalactoside. Under the same conditions no crosslinking to lac repressor was observed using a nonphotoactive 26-base-pair lac operator. Photoactivatable deoxyuridine analogs have potential application as reagents to crosslink DNA binding proteins to 5-azidouracil-containing DNA and as active-site-directed photoaffinity labelling reagents.

Affinity Labels↗
PubMed1989

Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. : Characterization, Solubilization, and Photoaffinity Labeling.

Using the novel radioligand, [(3)H]-9'-nor-fusicoccin-8'-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K(+), Mg(2+)-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [(3)H]-9'-nor-fusicoccin-8'-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (K(D)), K(D1) = 1.5 nanomolar and K(D2) = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 +/- 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin.

Journal Article↗
PubMed1985

Photoaffinity labeling of D-(-)-beta-hydroxybutyrate dehydrogenase by (arylazido)-beta-alanyl-substituted nicotinamide adenine dinucleotide.

(Arylazido)-beta-alanyl-substituted nicotinamide adenine dinucleotide (N3-NAD) is a photosensitive analogue of NAD capable of photoinduced nitrene generation and insertion into a nearby molecule. In the dark, N3-NAD can replace NAD as a cosubstrate for the mitochondrial D-(-)-beta-hydroxybutyrate dehydrogenase (BDH). With purified, phospholipid-reconstituted BDH and NAD as the variable substrate, the apparent Km and Vmax values were respectively 0.25 mM and 62.5 mumol min-1 (mg of protein)-1. With N3-NAD as the variable substrate, these values were respectively 0.59 mM and 5 mumol min-1 (mg of protein)-1. Photoirradiation of BDH in the presence of N3-NAD resulted in irreversible inhibition of the enzyme and incorporation into the protein of radioactivity from tritiated N3-NAD. Photoirradiation of BDH plus or minus NAD in the absence or presence of (arylazido)-beta-alanine caused little or no inhibition. The photoinhibition of BDH in the presence of N3-NAD was prevented nearly completely by addition of NADH, NAD plus beta-hydroxybutyrate, or NAD plus 2-methylmalonate and partially by addition of NAD. Moreover, the presence of NADH prevented, and prior partial modification of BDH at the NAD(H)-protectable site by N-ethylmaleimide decreased, the incorporation of radioactivity into BDH from photoirradiated [3H]N3-NAD. The above results suggest that N3-NAD can be used for photoaffinity labeling of BDH at the active site.

Affinity Labels↗
PubMed1985

Photoaffinity labeling of steroid 5 alpha-reductase of rat liver and prostate microsomes.

21-Diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione (Diazo-MAPD) inhibits steroid 5 alpha-reductase in liver microsomes of female rats with a Ki value of 8.7 +/- 1.7 nM, and the inhibition is competitive with testosterone. It also inhibits the binding of a 5 alpha-reductase inhibitor, [3H] 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA), to the enzyme in liver microsomes. The inhibition of 5 alpha-reductase activity and of inhibitor binding activity by diazo-MAPD becomes irreversible upon UV irradiation. [1,2-3H]Diazo-MAPD binds to a single high affinity site (Kd 8 nM, 125 pmol binding sites/mg of protein) in liver microsomes of female rats, and this binding requires NADPH. Without UV irradiation, this binding is reversible, and it becomes irreversible upon UV irradiation. Both the initial reversible binding and the subsequent irreversible conjugation after UV irradiation are inhibited by inhibitors (diazo-MAPD and 4-MA) and substrates (progesterone and testosterone) of 5 alpha-reductase, but they are not inhibited by 5 alpha-reduced steroids (5 alpha-dihydrotestosterone and 5 alpha-androstan-3 alpha, 17 beta-diol). NADPH stimulates the binding of [3H] diazo-MAPD to microsomes of male rat liver and prostate. UV irradiation also induces conjugation of [3H] diazo-MAPD to these microsomes. Photoaffinity labeled liver microsomes of female rats were solubilized and fractionated by high performance gel filtration. The radioactive conjugate eluted in one major peak at Mr 50,000.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMedDate not supplied

Comparative photoaffinity labeling study between azidophenyl, difluoroazidophenyl, and tetrafluoroazidophenyl derivatives for the GABA-gated chloride channels.

Syntheses of 2-aryl-substituted photoactivatable derivatives of 5-tert-butyl-1,3-dithiane and their oxidized bis-sulfone are described. The 4-azidoaryl and the 3,5-difluoro-4-azidoaryl groups were chosen as photosensitive moieties. Azidoaryl derivatives 3 and 13 were synthesized by diazotization and azidation of their corresponding arylamine precursors. The o-difluoroazidophenyl derivatives 2 and 9 were synthesized by transformation of the o-difluoro-substituted lithiophenyl into the corresponding azido derivative. The reversible binding properties of the photosensitive probes were established on bovine cortex P2 membranes by displacement of [3H]-1-phenyl-4-tert-butyl- 2,6,7-trioxabicyclo[2.2.2]-octane ([3H]TBOB), a specific ligand for the channel blocker binding site. The 2-(3',5'-difluro-4'-azidophenyl)-5-tert-butyl- 1,3-dithianebis-sulfone, compound 2, exhibited the best Ki of about 11 nM, compared to Kis of 180 and 570 nM, respectively, for probes 1 (azidotetrafluorophenyl analogue) and compared to Kis of 180 and 570 nM, respectively, for probes 1 (azidotetrafluorophenyl analogue) and 3 (azidphenyl analogue). On irradiation, probe 2 (0.6 microM) produced 18% irreversible loss of TBOB binding sites in brain membranes while probe 3 did not produce any photoinactivation. The loss observed with 2 was fully protectable by TBOB, demonstrating the specificity of the photochemical inactivation by compound 2 for the convulsant site of the GABAA receptor. These results, when compared to the photoaffinity labeling results obtained with the tetrafluorinated probe 1 (25% selective irreversible photochemical inactivation), establish a hierarchy between fluorinated and nonfluorinated arylazido probes and strengthen the potential of the newly described difluorinated probe 2, combining high affinity and good labeling efficacy.

Affinity Labels↗
PubMed1981

Photoaffinity labeling of the beta-adrenergic receptor.

A new photoactive beta-adrenergic antagonist, p-azidobenzylcarazolol (pABC) has been synthesized by combining a carbazole moiety with a p-azido-benzyl substituent. The compound has been labeled with tritium to a specific activity of 26 Ci/mmol. In frog erythrocyte membranes, [3H]p-azido-benzylcarazolol binds to the beta-adrenergic receptor with the expected beta 2 specificity and with high affinity (KD congruent to 100 +/- 10 pM). Unlabeled p-azido-benzylcarazolol can irreversibly inactivate the [3H]dihydroalprenolol-binding activity of frog erythrocyte membranes in a photodependent manner which can be prevented by beta-adrenergic agents. Incubation of frog erythrocyte membranes or digitonin-solubilized preparations of these membranes or digitonin-solubilized preparations of these membranes which had been enriched in beta-adrenergic receptors by a Sepharose-alprenolol chromatography step led to covalent incorporation of radioactivity into a Mr = 58,000 peptide. Specific incorporation of [3H]pABC into the Mr = 58,000 peptide could be prevented by both beta-adrenergic agonists and antagonists. This peptide has previously been purified and shown to contain the beta-adrenergic receptor-binding site (Shorr, R. G. L., Lefkowitz, R. J., and Caron, M. G. (1981) J. Biol. Chem. 256, 5820-5826). Thus, photoaffinity labeling of the beta-adrenergic receptor protein directly identifies the same hormone-binding subunit as has been isolated by conventional purification techniques.

Affinity Labels↗