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PubMed1984

Rapid laser flash photoaffinity labeling of binding sites for a noncompetitive inhibitor of the acetylcholine receptor.

Photoaffinity labeling of the nicotinic acetylcholine receptor from Torpedo marmorata electric tissue was performed in the presence of cholinergic effectors in the millisecond to second time range by a combination of a stopped-flow apparatus and a high-energy pulse laser. The label applied was [3H]triphenylmethylphosphonium, a lipophilic cation previously shown to be a specific blocker of the acetylcholine receptor ion channel. With the receptor in the resting state most of the label was incorporated into the alpha polypeptide chains. In the presence of agonists and antagonists increasing incorporation into the delta- and (less pronounced) the beta-chain was observed. The time course of this increase had a half-life of about 0.4 s, being slower than receptor activation and channel opening. in the resting, active, and even rapidly desensitized state, the alpha polypeptide chains appear to be the primary targets of the photoaffinity reaction. The action spectrum of the photolabeling has a sharp maximum at lambda = 270 nm and a small-side maximum at lambda = 290 nm. It does not resemble the absorption spectrum of the label and may hint at amino acid side chains as the moieties activated by UV light causing the photolabeling. The effector specificity of the observed slow increase of label incorporation into the delta polypeptide chain was investigated. It does not prove that slow desensitization is the underlying event. The agonists acetylcholine and carbamoylcholine as well as treatment of receptor-rich membranes with phospholipase A2 (but not phospholipase D) triggered labeling of delta, but antagonists such as D-tubocurarine and most conspicuously flaxedil had a similar effect.

Affinity Labels↗
PubMed1989

Analysis of structural features of dihydropyridine analogs needed to reverse multidrug resistance and to inhibit photoaffinity labeling of P-glycoprotein.

Synthetic dihydropyridine analogs were screened to determine whether they would reverse multidrug resistance of a multidrug-resistant human KB carcinoma cell line, KB-C1. Among twenty-four dihydropyridine analogs examined, thirteen almost completely overcame drug resistance (group A), nine partially overcame resistance (group B) and two did not reverse resistance (group C). The twenty-two compounds that reversed drug-resistance (groups A and B) were hydrophobic dihydropyridine derivatives. Three compounds that reversed resistance, NK-113, NK-138 and NK-194, increased the accumulation of [3H]vincristine in the resistant KB-C1 cells, but not in the parental KB cells, nor in a revertant cell line, KB-C1-R2. NK-101 (group C), which did not reverse resistance, had no effect on drug accumulation. Enhanced efflux of vincristine from the resistant cells was inhibited completely by NK-194, but NK-194 did not affect vincristine influx. Nine of the twenty-four compounds were screened to determine whether they inhibited photoaffinity labeling of the cell surface protein gp170 (P-glycoprotein) in KB-C1 cells by N-(p-azido-[3-125I]-salicyl)-N'-beta-aminoethylvindesine [( 125I]NASV). All five compounds of group A, NK-138, NK-194, NK-200, NK-203 and NK-220, inhibited the photoaffinity labeling of gp170 at less than 10-100 microM, whereas NK-113 and NK-196 of group B inhibited the labeling at 100-200 microM. By contrast, NK-101 and NK-102 of group C did not inhibit labeling even at 2000 microM. These studies confirm the relationship among reversal of multidrug resistance, decreased efflux of vincristine, and inhibition of [125I]NASV labeling of P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Identification of a UDP-glucose-binding site of human UDP-glucose dehydrogenase by photoaffinity labeling and cassette mutagenesis.

We have identified a UDP-glucose-binding site within human UDP-glucose dehydrogenase (hUGDH) by photoaffinity labeling with a specific probe, [(32)P]5N(3)UDP-glucose, and cassette mutagenesis using a synthetic hUGDH gene. Photolabel-containing peptides were generated by photolysis followed by tryptic digestion and isolated using the phosphopeptide isolation kit. Photolabeling of these peptides was effectively prevented by the presence of UDP-glucose during photolysis, demonstrating a selectivity of the photoprobe for the UDP-glucose-binding site. Amino acid sequencing and compositional analysis identified the UDP-glucose-binding site of hUGDH as the region containing the sequence, ASVGFGGSXFQK, corresponding to A268-K279 of the amino acid sequence of hUGDH. The unidentified residue, X, can be designated as a photolabeled C276 because the sequences including the cysteine residue in question have a complete identity with those of other UGDH species known. The importance of the C276 residue in the binding of UDP-glucose was further examined with mutant proteins at the C276 site. The mutagenesis at C276 has no effect on the expression of the mutants (C276G, C276K, C276E, C276L, and C276Y). Enzyme activities of the C276 mutants were not measurable under normal assay conditions, suggesting an important role for the C276 residue. No incorporation of [(32)P]5N(3)UDP-glucose was also observed for the mutants. These results indicate that C276 plays an important role for efficient binding of UDP-glucose to hUGDH.

Amino Acid Sequence↗
PubMed1996

Identification of the ATP transporter of rat liver rough endoplasmic reticulum via photoaffinity labeling and partial purification.

In order to identify the ATP transporter in rat liver rough endoplasmic reticulum (RER), a photoreactive azido derivative of ATP, 3'-O-(p-azidobenzoyl)-ATP (AB-ATP), was synthesized by the reaction of ATP with N-hydroxysuccinimido 4-azidobenzoate (NHS-AB). The activity of the ATP transporter was determined by measuring the influx of [8-14C]ATP. The ATP transport had an apparent Km value of 6.5 microM and a Vmax of 1 nmol min-1 (mg of protein)-1. The transport of ATP was specifically inhibited by AB-ATP and 4, 4'-diisothiocyanatostilbene-2', 2'-disulfonic acid (DIDS). Under a dim light, AB-ATP was a competitive inhibitor of the ATP transport with Ki value of 0.19 microM, which indicates that AB-ATP has a high affinity for the ATP transporter, so it can be utilized as a photoaffinity probe for the identification of the ATP transporter in rat liver RER. An SDS--PAGE analysis of RER vesicles photolabeled with [gamma-32P]AB-ATP indicates the presence of a 56-kDa protein. The 56-kDa protein was completely protected from photoaffinity labeling by 10 microM ATP but not by 30 microM GTP. The specific labeling of the 56-kDa protein was sensitive to the anion transport inhibitor DIDS. In order to confirm whether the apparent uptake of ATP was due to the 56-kDa protein, the ATP transporter was partially purified through two successive ion-exchange chromatography steps (DEAE and Mono-S). The fraction showing the high activity of the ATP transporter also contained the 56-kDa protein photolabeled with [gamma-32P]AB-ATP. On the basis of the photoaffinity labeling and reconstitution experiment, we conclude that the 56-kDa protein represents the ATP transporter in rat liver RER.

ATP-Binding Cassette Transporters↗
PubMed1984

Photoaffinity labeling of insulin-sensitive hexose transporters in intact rat adipocytes. Direct evidence that latent transporters become exposed to the extracellular space in response to insulin.

Irradiation of intact rat adipocytes with high intensity ultraviolet light in the presence of 0.5 microM [3H] cytochalasin B results in the labeling of Mr 43,000 and 46,000 proteins that reside in the plasma membrane fraction. In contrast to the Mr 46,000 protein, the Mr 43,000 component is not observed in the microsome fraction and exhibits lower affinity for [3H]cytochalasin B. Photolabeling of the Mr 43,000 protein is inhibited by cytochalasin D, indicating it is not a hexose transporter component. The Mr 46,000 protein exhibits characteristics expected for the glucose transporter such that D-glucose or 3-O-methylglucose but not cytochalasin D inhibits its photolabeling with [3H] cytochalasin B. Furthermore, insulin addition to intact cells either prior to or after photoaffinity labeling of the Mr 46,000 protein causes a redistribution of this component from the low density microsomes to the plasma membrane fraction, as expected for the hexose transporter. Photolabeling of transporters in both the low density microsome and plasma membrane fractions is inhibited when intact cells are equilibrated with 50 mM ethylidene glucose prior to irradiation with [3H]cytochalasin B. Incubation of intact cells with 50 mM ethylidene glucose for 1 min at 15 degrees C leads to an intracellular concentration of only 2 mM. Under these conditions, the photoaffinity labeling in intact cells of hexose transporters that fractionate with the low density microsomes is unaffected, indicating these transporters are not exposed to the extracellular medium. In contrast, photolabeling in intact insulin-treated cells of hexose transporters that fractionate with the plasma membrane is inhibited under these incubation conditions. The results demonstrate that insulin action results in the exposure to the extracellular medium of previously sequestered hexose transporters.

3-O-Methylglucose↗
PubMed1990

Peripheral-type benzodiazepine receptors in human glioblastomas: pharmacologic characterization and photoaffinity labeling of ligand recognition site.

Peripheral-type benzodiazepine receptors (PBR), unlike central-type benzodiazepine receptors, are found in low concentrations in normal brain. Because PBR have been described in neoplastic cells of neuroglial origin, they have been suggested for imaging human glial tumors and for directing cytotoxic therapy at these tumors. Little information exists, however, on the presence or pharmacology of PBR in human glial tumors. Using radioligand binding techniques, we have demonstrated that 6 out of 6 glioblastoma (GBM) specimens had high concentrations of PBR [( 3H]PK 11195 binding sites) which were significantly greater than in 5 normal human frontal cortex samples. The pharmacologic specificity of these sites differed significantly from that of PBR in human and rat kidney specimens. Saturation binding experiments revealed a small number of high affinity sites and a substantial number of sites of intermediate affinity. Under in vitro binding conditions the more numerous lower affinity site is the major contributor to specific binding measurements. The ligand recognition site of the PBR in human GBM tissue was photoaffinity labeled using [3H]PK 14105, a nitrophenyl analogue of PK 11195. Subsequent SDS-polyacrylamide gel electrophoresis revealed specific incorporation of label into a 17,300 molecular weight component. There was no specific incorporation into normal human frontal cortex, but a component of very similar molecular weight was demonstrated in human kidney. We conclude that human glioblastomas consistently express PBR sites that are present in greater density than in normal human brain. Imaging of human glial tumors with analogues of PK 11195 thus appears feasible. Further molecular characterization of the photoaffinity-labeled PBR may also provide new information on the biology of these tumors.

Affinity Labels↗
PubMed1999

Demonstration of a direct interaction between residue 22 in the carboxyl-terminal half of secretin and the amino-terminal tail of the secretin receptor using photoaffinity labeling.

An understanding of the molecular basis of hormonal activation of receptors provides important insights for drug design. Toward this end, intrinsic photoaffinity labeling is a powerful tool to directly identify the ligand-binding domain. We have developed a new radioiodinatable agonist ligand of the secretin receptor that incorporates a photolabile p-benzoyl-L-phenylalanine (Bpa) into the position of Leu22 and have utilized this to identify the adjacent receptor domain. The rat [Tyr10,Bpa22]secretin-27 probe was a fully efficacious agonist, with a potency to stimulate cAMP accumulation by Chinese hamster ovary SecR cells similar to that of natural secretin (EC50 = 68 +/- 22 pM analogue and 95 +/- 25 pM secretin). It bound specifically and with high affinity (Ki = 5.0 +/- 1.1 nM) and covalently labeled the Mr = 57,000-62,000 secretin receptor. Cyanogen bromide cleavage of the receptor yielded a major labeled fragment of apparent Mr = 19,000 that shifted to Mr = 9,000 after deglycosylation. This was most consistent with either of two glycosylated domains within the amino-terminal tail of the receptor. Immunoprecipitation with antibody directed to epitope tags incorporated into each of the candidate domains established that the fragment at the amino terminus of the receptor was the site of labeling. This was further localized to the amino-terminal 30 residues of the receptor by additional proteolysis of this fragment with endoproteinase Lys-C. This provides the first direct demonstration of a contact between a secretin-like agonist and its receptor and will contribute a useful constraint to the modeling of this interaction.

Amino Acid Sequence↗
PubMed1995

Photoaffinity labeling with 2(-)[2-(4-azido-3(-)[125I]- iodophenyl)ethylamino]adenosine and autoradiography with 2(-)[2-(4-amino-3(-)[125I]iodophenyl)ethylamino]adenosine of A2a adenosine receptors in rat brain.

The A2a adenosine receptor agonist 2(-)[2-(4-amino-3- iodophenyl)ethylamino]adenosine is a potent coronary vasodilator. The corresponding radioiodinated ligand, [125I]APE, discriminates between high- and low-affinity conformations of A2a adenosine receptors. In this study, [125I]APE was used for rapid (24-h) autoradiography in rat brain sections. The pattern of [125I]APE binding is consistent with that expected of an A2a-selective radioligand. It is highest in striatum, nucleus accumbens, and olfactory tubercle, with little binding to cortex and septal nuclei. Specific [125I]APE binding to these brain regions is abolished by 1 microM 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680) but is little affected by 100 nM 8-cyclopentyl-1,3-dipropylxanthine. Conversion of [125I]APE to the corresponding arylazide results in [125I]AzPE. The rank-order potency of compounds to compete for [125I]AzPE binding in the dark is CGS-21680 > D-(R)-N6-phenylisopropyladenosine > N6- cyclopentyladenosine, indicating that it also is an A2a-selective ligand. Specific photoaffinity labeling by [125I]AzPE of a single polypeptide (42 kDa) corresponding to A2a adenosine receptors is reduced 55 +/- 4% by 100 microM guanosine 5'-O-(3-thiotriphosphate) and 91 +/- 1.3% by 100 nM CGS-21680. [125I]APE and [125I]AzPE are valuable new tools for characterizing A2a adenosine receptors and their coupling to GTP-binding proteins by autoradiography and photoaffinity labeling.

Adenosine↗
PubMed1996

125I-APE binding to adenosine receptors in coronary artery: photoaffinity labeling with 125I-azidoAPE.

Coronary arteries are known to contain adenosine receptors that elicit vasodilation. Past attempts to characterize these receptors by radioligand binding have been unsuccessful. In the present study, a newly synthesized iodinated adenosine analogue, [125I]2-[2-(4-amino-3-iodophenhyl)ethylamino]adenosine (125I-APE), was found to bind to adenosine receptors in porcine coronary artery smooth muscle membranes. Specific 125I-APE binding is temperature sensitive with maximal binding detected at 4 degrees C. 125I-APE binds to a high affinity low density site with a KD of 0.59 +/- 0.11 nM and a Bmax 7 +/- 0.8 fmoles/mg protein. A high abundance lower affinity site is suggested by the fact that APE competes for 125I-APE binding with a concentration that inhibits 50% (IC50) of 0.96 microM. Competition with various other adenosine receptor agonists results in a potency order of (IC50, microM): 2-phenylaminoadenosine (CV 1808, 0.34) > APE (0.96) > CGS 22988 (5.2) > 2-chloroadenosine (30) > CGS 21680 and NECA (> 100). Agonist binding is not affected by GppNHp (10(-7)-10(-3) M). Among antagonists the potency order is (microM): CGS 15943 (1.1) > 8-(3-chlorostyryl)-caffeine (CSC, 5.3) > 8-sulfophenyltheophylline (SPT, 86) > theophylline (> 100). These binding characteristics are similar to the properties of a putative A4 binding site characteristic of A2a receptors assayed at a low temperature. Photoaffinity labeling of porcine coronary artery membrane proteins with the azide derivative of 125I-APE revealed a 45,000-Da binding site. Photolabeling is prevented by coincubation of membranes at 4 degrees C with various adenosine receptor antagonists (1 microM CSC, 1 microM CGS 15943 or 100 microM theophylline). In conclusion, adenosine receptors of coronary arteries have been detected for the first time by radioligand binding and photoaffinity labeling. This ligand appears to label porcine A4 binding sites that may correspond to A2a receptors assayed at 4 degrees C.

Adenosine↗
PubMed1985

Photoaffinity labelling of hepatic plasma membranes suggests two classes of hepatic insulin receptor.

Photoaffinity labelling of hepatic insulin receptors revealed specifically-labelled bands of 130, 90 and 40 kDa. Endogenous protease activity in hepatic plasma membranes, as well as contaminating proteases present in preparations of clostridial collagenase, degraded some of the 130-kDa insulin-binding subunit to a 115-kDa form. However, a large proportion of the 130-kDa subunits were resistant to degradation, suggesting the presence of two classes of insulin receptor in hepatic plasma membranes. In one class the 130-kDa subunit was sensitive to proteolysis, while in the other it was not. In contrast, the 130-kDa receptor subunits of adipose tissue were all resistant to such degradation. Scatchard analysis of control and collagenase-treated plasma membranes demonstrated that conversion of the 130-kDa subunit to a 115-kDa form did not affect the insulin-binding characteristics of the receptor. It was also apparent that insulin binds to a single class of high-affinity sites in hepatic plasma membranes.

Affinity Labels↗
PubMed1978

Photoaffinity labeling of the ribosomal peptidyl transferase site with synthetic puromycin analogues.

A photoaffinity labeling puromycin analogue, Nepsilon-(2-nitro-4-azidophenyl)-L-lysinyl puromycin aminonucleoside (NAP-Lys-Pan), was synthesized and used for investigation of the peptidyl transferase center of 70S riobsomes. Visible light irradiation of NAP-Lys-Pan led to covalent linkage of the analogue with Escherichia coli ribosomes. In a subsequent step, poly(uridylic acid) was employed to direct Ac[14C]Phe-tRNA to the P sites of the photolabeled ribosomes. Transpeptidation of Ac[14C]phenylalanine to the bound NAP-Lys-Pan resulted in selective incorporation of radioactive label into the peptidyl transferase A site. Dissociation of the ribosomes into subunits, and digestion of the RNA components, indicated that the radioactive label was incorporated into a protein fraction of the 50S subunit.

Acyltransferases↗
PubMed1989

Photoaffinity labeling of indole-3-acetic acid-binding proteins in maize.

The photoaffinity labeling agent 5-azidoindole-3-acetic acid, an analog of the endogenous plant hormone indole-3-acetic acid (an auxin), was used to identify indole-3-acetic acid-binding proteins in maize. Two peptides with subunit molecular masses of 24 and 22 kilodaltons are specifically labeled in a saturable manner. Both peptides are slightly acidic and behave as dimers under nondenaturing conditions. The possibility that one of these peptides is the auxin receptor that mediates cell elongation in maize is discussed.

Journal Article↗
PubMed1985

Photoaffinity labeling of the V1 vasopressin receptor in plasma membranes from rat liver.

Photoaffinity labeling experiments were performed with membranes from rat liver containing V1 vasopressin receptors. Photoreactive analogues of [1-beta-mercaptopropionic acid]vasopressin [( Mpa1], vasopressin, or deamino-vasopressin) retaining a high binding affinity (apparent dissociation constants: 5 X 10(-9) M-3 X 10(-8) M) and agonistic properties were used. The tritium-labeled analogue [Mpa1,Lys(N epsilon-4-azidobenzoyl)8]vasopressin preferentially and specifically labels a 30-kDa polypeptide and with lower efficiency a 38-kDa polypeptide. The analogue [Mpa1,Dab4(N gamma-(N-4-azido-2-nitrophenyl-beta-Ala4]arginine-vasopressin specifically labels the 38-kDa polypeptide. The labeling of these two membrane proteins is completely suppressed by an excess of arginine-vasopressin; bradykinin or angiotensin II do not inhibit the incorporation of the reactive vasopressin analogues into these proteins. The results suggest that the rat hepatic V1 receptor exists in the plasma membrane in an oligomeric form composed of two subunits with a molecular mass of 30 and 38 kDa.

Affinity Labels↗
PubMed2006

Differential spatial approximation between secretin and its receptor residues in active and inactive conformations demonstrated by photoaffinity labeling.

Understanding of the conformational changes in G protein-coupled receptors associated with activation and inactivation is of great interest. We previously used photoaffinity labeling to elucidate spatial approximations between photolabile residues situated throughout the pharmacophore of secretin agonist probes and this receptor. The aim of the current work was to develop analogous photolabile secretin antagonist probes and to explore their spatial approximations. The most potent secretin antagonist reported is a pseudopeptide ([psi(4, 5)]secretin) in which the peptide bond between residues 4 and 5 was replaced by a psi(CH(2)-NH) peptide bond isostere. We have developed a series of [psi(4, 5)]secretin analogs incorporating photolabile benzoyl phenylalanine residues in positions 6, 22, and 26. Each bound to the secretin receptor saturably and specifically, with affinity similar to their parental peptide. At concentrations with no measurable agonist activity, each probe covalently labeled the secretin receptor. Peptide mapping using proteolytic cleavage, immunoprecipitation, and radiochemical sequencing identified that each of these three probes labeled the amino terminus of the secretin receptor. Whereas the position 22 probe labeled the same residue as its analogous agonist probe and the position 6 probe labeled a residue within two residues of that labeled by its analogous agonist probe, the position 26 probe labeled a site 16 residues away from that labeled by its analogous agonist probe. Thus, whereas structurally related agonist and antagonist probes dock in the same general region of this receptor, conformational differences in active and inactive states result in substantial differences in spatial approximation at the carboxyl-terminal end of secretin analogs.

Amino Acid Sequence↗
PubMed1987

Photoaffinity labelling of the phenylalkylamine receptor of the skeletal muscle transverse-tubule calcium channel.

The tritiated arylazido phenylalkylamine (-)-5-[(3-azidophenethyl)[N-methyl-3H]methylamino]-2-(3,4, 5-trimethoxyphenyl)-2-isopropylvaleronitrile was synthesized and used to photoaffinity label the phenylalkylamine receptor of the membrane-bound and purified calcium channel from guinea-pig skeletal muscle transverse-tubule membranes. The photoaffinity ligand binds reversibly to partially purified membranes with a Kd of 2.0 +/- 0.5 nM and a Bmax of 17.0 +/- 0.9 pmol/mg protein. Binding is stereospecifically regulated by all three classes of organic calcium channel drugs. A 155 kDa band was specifically photolabelled in transverse-tubule particulate and purified calcium channel preparations after ultraviolet irradiation. Additional minor labelled polypeptides (92, 60 and 33 kDa) were only observed in membranes. The heterogeneous 155 kDa region of the purified channel was resolved into two distinct silver-stained polypeptides after reduction (i.e. 155 and 135 kDa). Only the 155 kDa polypeptide carries the photoaffinity label and it is concluded that the 135 kDa polypeptide (which migrates as a 165 kDa band under alkylating conditions) is not a high-affinity drug receptor carrying subunit of the skeletal muscle transverse-tubule L-type calcium channel.

Affinity Labels↗
PubMed2005

Design of dantrolene-derived probes for radioisotope-free photoaffinity labeling of proteins involved in the physiological Ca2+ release from sarcoplasmic reticulum of skeletal muscle.

Bifunctional dantrolene derivatives have been synthesized as probes for radioisotope-free photoaffinity labeling with the aim of elucidating the molecular mechanism of skeletal muscle contraction. GIF-0430 and GIF-0665 are aromatic azido-functionalized derivatives that were designed to selectively inhibit physiological Ca2+ release (PCR) from sarcoplasmic reticulum (SR) in mouse skeletal muscle without a strong effect on Ca2+-induced Ca2+ release (CICR). These photoaffinity probes consist of either an azidomethyl or an ethynyl group, respectively, which could function as a tag for introduction of an optional detectable marker unit by an appropriate chemoselective ligation method after the photo-cross-linking operation. Actually, the former probe worked to photolabel its target proteins specifically as confirmed by subsequent fluorescent visualization.

Animals↗
PubMed1998

Synthesis of photoaffinity label analogues of alpha-tocopherol.

Photoaffinity analogues of alpha-tocopherol have been synthesized that incorporate the photosensitive 4-azido-2,3,5,6-tetrafluorobenzyloxy group at the terminus of unbranched analogues of the naturally occurring phytyl side chain. An intermediate from these syntheses has also been used to generate a supported ligand for bioaffinity chromatography of alpha-tocopherol binding proteins.

Chromatography, Affinity↗
PubMed1982

Photoaffinity-labeling and fluorescence-distribution studies of gonadotropin-releasing hormone receptors in ovarian granulosa cells.

Photoaffinity labeling of rat ovarian granulosa cells and membrane preparations with a bioactive photoaffinity derivative of gonadotropin-releasing hormone resulted in identification of two specific components with apparent molecular weights of 60,000 and 54,000. Fluorescent visualization of gonadotropin-releasing hormone receptors in these cells, by using a bioactive rhodamine derivative of the hormone, indicated that the fluorescently labeled receptors were initially distributed uniformly on the cell surface and then formed patches that subsequently internalized (at 37 degrees C) into endocytic vesicles. These processes were dependent on specific binding sites for the rhodamine-labeled peptide on the granulosa cells. These studies may provide an experimental basis for understanding the molecular events involved in the action of the hormone in the ovary.

Affinity Labels↗