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PubMed1989

Interaction of a nuclear location signal with isolated nuclear envelopes and identification of signal-binding proteins by photoaffinity labeling.

The nuclear envelope (NE) separates the two major compartments of eukaryotic cells, the nucleus and the cytoplasm. Recent studies suggest that the uptake of nuclear proteins into the nucleus is initiated by binding of nuclear location signals (NLSs) contained within these proteins to receptors in the NE, followed by translocation through the nuclear pore complex. To examine the binding step without interference from intranuclear events, we have used a system consisting of (i) purified rat liver NEs fixed onto glass slides and (ii) the prototype simian virus 40 large T antigen (SV40 T) NLS conjugated to nonnuclear carrier proteins, and we have visualized the receptor-ligand interaction by indirect immunofluorescence. In this system, incubation of isolated NEs with the wild-type SV40 T NLS conjugate with carrier proteins resulted in binding that was signal sequence-dependent, could be competitively blocked with excess conjugated and unconjugated wild-type peptide, did not require ATP, and was not affected by the transport-inhibiting lectin wheat germ agglutinin. In contrast, only minimal binding was observed with a mutant SV40 T NLS conjugate. These results are consistent with those obtained in other, more complex in vitro systems and suggest that binding of the SV40 T NLS is receptor-mediated. Binding is largely abolished by extraction of the NE with the nonionic detergent Triton X-100, suggesting that the receptor is soluble in detergent. We find in the Triton X-100 supernatant four major NLS-binding proteins with apparent molecular masses of 76, 67, 59, and 58 kDa by photoaffinity labeling with a highly specific crosslinker, azido-NLS. The reduced complexity of the system described here should be useful for the functional study of other potential NLSs for the identification and isolation of their binding sites and for the screening of antibodies raised against these binding sites.

Affinity Labels↗
PubMed1992

Characterization of human striatal A2-adenosine receptors using radioligand binding and photoaffinity labeling.

The adenosine agonist [3H]CGS21680 (2-[4-[[2-carboxyethyl]phenyl]ethylamino]-5'- N-ethylcarboxamidoadenosine) bound to A2 receptors in human striatal membranes with a Kd of 17.8 +/- 1.1 nM and a Bmax of 313 +/- 10 fmol/mg protein. The addition of 100 microM GTP diminished both the affinity of agonist radioligand for A2 adenosine binding sites and the total binding, resulting in Kd and Bmax values of 28.6 +/- 1.0 nM and 185 +/- 22 fmol/mg of protein. Adenosine ligands competed for [3H]CGS21680 with the expected potency order. The adenosine antagonist [3H]XAC (8-[4-[[[[(2-aminoethyl)-amino]carbonyl]methyl] oxy]phenyl]-1,3-dipropylxanthine), although A1-selective in the rat, binds to human striatal A2 receptors with high affinity. 25 nM CPX (8-cyclopentyl-1,3-dipropylxanthine), an A1-selective antagonist, was added to the incubation medium and effectively eliminated 91% of [3H]XAC (1 nM) binding to human A1 receptors, yet preserved 90% of binding to A2 receptors. [3H]XAC exhibited saturable, specific binding (50% of total) to A2 sites with a Kd of 2.98 +/- 0.54 nM and a Bmax of 0.71 +/- 0.23 pmol/mg protein (25 degrees C, non-specific binding defined with 100 microM NECA). The potency order for antagonists against 1 nM [3H]XAC was CGS15943A greater than XAC approximately PD115,119 greater than PAPA-XAC greater than CPX greater than HTQZ approximately XCC approximately CP-66,713 greater than theophylline approximately caffeine, indicative of an A2-type binding site. A2a-receptors were found to be present in the human cortex, albeit at a much lower density than in the striatum. Photoaffinity labeling using 125I-PAPA-APEC revealed a molecular weight of 45K, but proteolytic cleavage was observed, resulting in fragments of MW 43K and 37K. In the absence of proteolytic inhibitors the 37K fragment, which still bound 125I-PAPA-APEC, was predominant.

Adenosine↗
PubMed1982

4-Azidophlorizin, a high affinity probe and photoaffinity label for the glucose transporter in brush border membranes.

A new phlorizin derivative (2'-O-(beta-D-glucopyranosyl)-4-azidophloretin, 4-azidophlorizin) has been synthesized and its affinity for the D-glucose, Na+ co-transport system in brush border vesicles from intestinal and renal membranes has been compared with that of phlorizin. The extent of the reversible interaction of the ligand with the transporter in dim light has been evaluated from three separate measurements: (1) Ki', the constant for fully-competitive inhibition of (Na+, delta psi)-dependent D-glucose uptake, (2) Kd', the dissociation constant of 4-azido[3H]phlorizin binding in the presence of an NaSCN inward gradient, and (3) Ki", the constant for fully-competitive inhibition of the specific ((Na+, delta psi)-dependent, D-glucose protectable) high-affinity [3H]phlorizin binding. In experiments with vesicles derived from rat kidney, all three constants (Ki', Kd' and Ki") were essentially equal and ranged between 3.2 and 5.2 microM, that is, the azide derivative has almost the same affinity for this transporter as phlorizin itself. On the other hand, compared to phlorizin, the 4-azidophlorizin has a lower affinity for the transporter in vesicles prepared from rabbit; its Ki' values are some 15-20-times larger than those determined with rat membranes. However, the affinity of the azide for the sugar transporter in membranes from either the intestine or kidney of the same animal species (rabbit or rat) was essentially the same. In spite of the lower affinity for the transporter in either membrane system from the rabbit, results described elsewhere (Hosang, M., Gibbs, E.M., Diedrich, D.F. and Semenza, G. (1981) FEBS Lett., 130, 244-248) indicate that 4-azidophlorizin is an effective photoaffinity label in this species also. Photolysis of the azide yields a reactive intermediate which reacts with a 72 kDa protein in rabbit intestine brush borders. Covalent labeling of this protein occurred under conditions which suggests that it is (a component of) the glucose transporter.

Affinity Labels↗
PubMed1991

The beta-glucan synthase from Lolium multiflorum. Detergent solubilization, purification using monoclonal antibodies, and photoaffinity labeling with a novel photoreactive pyrimidine analogue of uridine 5'-diphosphoglucose.

The membrane-bound beta-glucan synthase from Italian ryegrass (Lolium multiflorum L.) endosperm cells has been solubilized by both non-ionic and zwitterionic detergents. A complex relationship exists between the ratio of (1----3)-, (1----4)-, and (1----3, 1----4)-beta-glucan products of the solubilized enzyme, the cations present, and the concentration of the uridine 5'-diphosphoglucose substrate. Monoclonal antibodies directed against the beta-glucan synthase complex were generated by immunization of mice with an unfractionated microsomal reparation. Hybridoma cell lines were screened using a combination of indirect enzyme-linked immunosorbent assay followed by an enzyme-capture assay. The purified monoclonal antibodies were used with Pan-sorbin (stablized protein A-bearing staphylococcal cells) to immunoprecipitate an active beta-glucan synthase complex which had been solubilized from a microsomal preparation with 0.6% CHAPS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitated synthase complex revealed four major polypeptides of apparent molecular mass 30, 31, 54, and 58 kDa together with several minor components. The immunoprecipitated beta-glucan synthase complex was capable of synthesizing both (1----3)- and (1----4)-beta-glucans. A new photoreactive pyrimidine analogue of uridine 5'-diphosphoglucose, 5-[3-(p-azidosalicylamide]allyl-uridine 5'-diphosphoglucose was synthesized in a three-step reaction sequence involving mercuration of UDP-Glc, alkylation of 5-Hg-UDP-Glc, and acylation of 5-(3-amino)allyl-UDP-Glc and characterized by chemical and spectroscopic analysis. The analogue inhibits (Kiapp 16 microM) and, upon UV irradiation, irreversibly inactivates the beta-glucan synthase. The analogue was iodinated with Na125I to give a radiolabeled, photoreactive compound, and was used in photoaffinity labeling of UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, and several putative UDP-Glc-binding proteins from L. multiforum. The radiolabeled analogue specifically labeled the 31-kDa polypeptide in the immunoprecipitated synthase complex. The photolabeling of this polypeptide is strictly dependent on UV irradiation, is blocked by uridine 5'-diphosphoglucose and uridine 5'-diphosphate, and reaches saturation at analogue concentrations above 300 microM. These results indicate that the 31-kDa polypeptide in the beta-glucan synthase complex bears a uridine 5'-diphosphoglucose-binding site and is involved in the catalysis of beta-glucan synthesis.

Affinity Labels↗
PubMed1985

Photoaffinity labeling of the follitropin receptor.

A photoactivatable derivative of human follitropin was used to identify the follitropin receptor on porcine granulosa cells. The hormone was condensed with a heterobifunctional reagent, the N-hydroxysuccinimide ester of 4-azidobenzoylglycine, and radioiodinated. The 125I-labeled hormone (125I-hormone) derivative associated with the same number of receptors as 125I-hormone itself, but with a slightly lower Ka, 1.12 X 10(10) M-1 compared with 1.4 X 10(10) M-1 for the 125I-hormone. The binding could be blocked with untreated hormone. Its alpha and beta subunits could be cross-linked to produce alpha beta dimer by photolysis. When the 125I-hormone derivative bound to the cells was photolyzed for crosslinking and the products resolved by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, two new bands (106 and 61 kDa) of lower electrophoretic mobility appeared in addition to the alpha, beta, and alpha beta bands. Formation of these crosslinked complexes required photolysis, and the 125I-hormone derivative specifically bound to cells bearing the receptor. Binding could be blocked by excess untreated follitropin but not with human choriogonadotropin and thyrotropin. Under nonreducing conditions, one major band (104 kDa) of cross-linked complexes appeared. Upon reduction with dithiothreitol and second-dimensional electrophoresis, the 104-kDa band produced two smaller complexes of 75 and 61 kDa, indicating the loss of two components and the existence of intercomponent disulfides. Successful production of the 104-kDa complex requires blocking of free sulfhydryl groups with N-ethylmaleimide. It is, however, independent of various protease inhibitors or the temperature and the time period of hormone incubation with cells or the plasma membrane fraction. The mass estimates and the interaction with the hormone of the photoaffinity-labeled components are discussed.

Affinity Labels↗
PubMed1992

Use of azidobestatin as a photoaffinity label to identify the active site peptide of leucine aminopeptidase.

Aminopeptidases catalyze the hydrolysis of amino acid residues from the amino terminus of peptide substrates. They are found in most cells and tissues, and their activity has been implicated in myriad fundamental biochemical and physiological processes. Nevertheless, little is known about the structure of the aminopeptidase active sites. Beef lens leucine aminopeptidase (blLAP) can be considered prototypical of many enzymes in this family of peptidases. Bestatin, [(2S,3R)-(3-amino-2-hydroxy-4-phenyl-butanoyl)-L-leucine] is a nonhydrolyzable substrate analogue of a peptide, PheLeu, which is rapidly cleaved by blLAP. Bestatin incorporates elements of the putative tetrahedral intermediate, and this results in a greater than 10(5)-fold enhancement of binding relative to analogous peptides. Bestatin is the most tightly bound inhibitor of many aminopeptidases. Bestatin was successively converted to nitrobestatin, p-aminobestatin, [3H]-p-aminobestatin, and finally [3H]-p-azidobestatin (pAB). Like bestatin, pAB is a slow binding inhibitor of LAP (Ki*, the dissociation constant for the final complex, = approximately 4 x 10(-9); Ki, the dissociation constant for the initial collision complex, = approximately 10(-8). The t1/2 for binding of 2 x 10(-8) M and 8 x 10(-8) M bestatin are approximately 60 min and approximately 38 min, respectively. pAB, nitrobestatin, bestatin, and physiological peptides appear to bind in the same site, the first three with similar avidity. In the dark, pAB and bestatin protect low concentrations of the enzyme against inactivation upon extensive dialysis. The t1/2 for photoactivation of pAB is approximately 3 s. Irradiation of blLAP for such short periods of time resulted in insignificant change in activity. blLAP which was placed in 254-nm light in the presence of pAB was inactivated significantly. Treatment of photolabeled blLAP with trypsin produces only two peptides. Autoradiography and scintillation counting indicate that the active site is in the peptide which includes residues 138-487. Treatment of the same blLAP with hydroxylamine produces two different peptides, with the active site in the peptide 323-487. This indicates that the active site is in the carboxyl-terminal one-third of the protomer. It is likely that this photoaffinity label will be useful in identifying active sites in other aminopeptidases as well.

Affinity Labels↗
PubMed1994

An 125I-labeled N6-substituted azido analog of NAD+ for the photoaffinity labeling of NAD(+)-linked enzymes.

125I-N6-(N-[6-N-(5-iodo-4-azidosalicyl)-aminohexyl]- aminocarbamoylmethyl)-nicotinamide adenine dinucleotide (125I-N6-I-ASA-AH-NAD+) was synthesized by coupling N6-([6-aminohexyl]-carbamoylmethyl)-NAD+ with 4-azidosalicylic acid N-hydroxysuccinimide ester followed by radioiodination. The utility of 125I-N6-I-ASA-AH-NAD+ as an effective site-directed photoprobe was demonstrated by the photolabeling of both glutamate dehydrogenase and 15-hydroxyprostaglandin dehydrogenase. Both enzymes can be saturated with labeled probe with apparent dissociation constants comparable to those reported for NAD+. Photoincorporation of the probe into both enzymes was found to be protected specifically by NAD+. These results indicate that 125I-N6-I-ASA-AH-NAD+ can be a specific photoprobe for NAD(+)-linked enzymes.

Affinity Labels↗
PubMed1986

Structural and pharmacological differences between codfish and rat brain alpha 1-adrenergic receptors revealed by photoaffinity labeling with 125I-APDQ.

The photoaffinity probe 125I-APDQ has been used to characterize alpha 1-receptor peptides in the cod and rat brains. In the cod brain a major specific peptide of Mr = 68,000 could be covalently labeled by 125I-APDQ as revealed by SDS-PAGE. In the rat brain a specific peptide with Mr = 77,000 was instead labeled. When a number of adrenergic agonists and antagonists were tested for their ability to protect the labeling by 125I-APDQ their potencies were those expected for alpha 1-receptors in both species. The ligand binding peptide in the cod brain also distinguished between stereoisomers of epinephrine as expected for a physiological receptor. However, there was a distinct difference between the cod and rat alpha 1-receptor in that the beta-agonist 1-isoprenaline was equipotent to 1-norepinephrine in the cod whereas it was less potent in the rat. The protecting ability of the tested agents were also matched by their ability to displace the alpha 1-adrenergic ligand 3H-prazosin from alpha 1-receptor binding sites in brain membranes from both species. Thus, the codfish alpha 1-receptor seems to be different from mammalian alpha 1-receptors both structurally and pharmacologically.

Affinity Labels↗
PubMed1982

The beta 1-adrenergic receptor of the turkey erythrocyte. Molecular heterogeneity revealed by purification and photoaffinity labeling.

The beta 1-adrenergic receptor of turkey erythrocytes has been purified by a combination of affinity and high performance steric exclusion chromatography. These procedures provide preparations with specific activities of greater than 15,000 pmol/mg of protein with an overall recovery of approximately 30% of the receptor activity solubilized from membrane preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated purified receptor reveals two bands of labeled protein with apparent Mr = 40,000 +/- 2,000 and 45,000 +/- 3,000 in a 3-4:1 ratio. These same two peptides can also be labeled specifically and in approximately the same ration in both membranes and purified preparations using the photoaffinity probe 125I-labeled p-azidobenzylcarazolol. When the two purified polypeptides are completely separated by high performance liquid chromatography and subjected to detailed ligand binding studies, identical beta 1-adrenergic specificities are found for the two receptor forms. Preliminary characterization of these two proteins by partial protease digestion suggests a large degree of similarity between them, albeit with some significant differences. These results demonstrate that both purification and photoaffinity labeling identify two polypeptides in turkey erythrocyte membranes as containing a beta 1-adrenergic receptor binding site. The functional and structural relationships of these two forms of the receptor remain to be elucidated.

Affinity Labels↗
PubMed1983

Conformational and allosteric changes in fructose 1,6-bisphosphatase upon photoaffinity labeling with 2-azidoadenosine monophosphate.

The photoaffinity analog 2-azido-AMP was found to be a potent allosteric inhibitor of pig kidney fructose 1,6-bisphosphatase. UV-induced covalent incorporation of 2-azido-[8-3H]AMP fully inactivated the enzyme at a level stoichiometric with its subunit composition (4 mol of analog/mol of tetramer). The photoincorporation and inactivation were prevented by the presence of AMP but not by the substrate, IMP, or adenosine. Enzyme fully modified with 2-azido-AMP was capable of binding fructose 1,6-bisphosphate but not AMP. The analog thus specifically modified the enzyme's allosteric sites. Titrations of the native enzyme's fast reacting cysteines with 5,5'-dithiobis-(2-nitrobenzoic acid) distinguished four conformational states of the enzyme: E(+/- Mg2+), E(AMP), E(AMP)Mg2+, E(Fru-1,6-P2)x. AMP had a biphasic effect with a low affinity phase that could be mimicked by IMP, a competitive inhibitor of fructose 1,6-bisphosphate binding. Enzyme fully modified with 2-azido-AMP was isolated and observed to have a similar conformation and to undergo similar ligand-induced conformational changes as the native enzyme-AMP complex. Random modification of 50% of the subunits with 2-azido-AMP shifted the AMP inhibition curve from sigmoidal to hyperbolic without changing the inhibition constant. AMP-induced cooperativity in the pig kidney fructose 1,6-bisphosphatase appears to be restricted to 2 subunits without alterations in AMP affinity.

Adenosine Monophosphate↗
PubMed1997

Biosynthesis of chondroitin sulfate. Purification of glucuronosyl transferase II and use of photoaffinity labeling for characterization of the enzyme as an 80-kDa protein.

A photoaffinity analogue, [beta-32P]5-azido-UDP-GlcA, was used to photolabel the enzymes that utilize UDP-GlcA in cartilage microsomes and rat liver microsomes. SDS-polyacrylamide gel electrophoresis analysis of photolabeled cartilage microsomes, which are specialized in chondroitin sulfate synthesis, showed a major radiolabeled band at 80 kDa and other minor radiolabeled bands near 40 and 60 kDa. Rat liver microsomes, which are enriched for enzymes of detoxification by glucuronidation, had a different pattern with multiple major labeled bands near 50-60 and 35 kDa. To determine that the photolabeled 80-kDa protein is the GlcA transferase II, we have purified the enzyme from cartilage microsomes. This membrane-bound enzyme, involved in the transfer of GlcA residues to non-reducing terminal GalNAc residues of the chondroitin polymer, has now been solubilized, stabilized, and then purified greater than 1350-fold by sequential chromatography on Q-Sepharose, heparin-Sepharose, and WGA-agarose. The purified enzyme exhibited a conspicuous silver-stained protein band on SDS-polyacrylamide gel electrophoresis that coincided with the major radiolabeled band of 80 kDa. SDS-polyacrylamide gel analysis of photoaffinity-labeled active fractions from the Q-Sepharose, heparin-Sepharose, and WGA-agarose also indicated only the single radiolabeled band at 80 kDa. Intensity of photolabeling in each of the fractions examined coincided with enzyme activity. The photolabeling of this 80-kDa protein was saturable with the photoprobe and could be inhibited by the addition of UDP-GlcA prior to the addition of the photoprobe. Thus, the photolabeling with [beta-32P]5-azido-UDP-GlcA has identified the GlcA transferase II as an 80-kDa protein. The purified enzyme was capable of transferring good amounts of GlcA residues to chondroitin-derived pentasaccharide with negligible transfer to pentasaccharides derived from hyaluronan or heparan.

Affinity Labels↗
PubMed2006

Positively coded photoaffinity label for altering isoelectric points of proteins.

Novel diazirinyl photoaffinity ligand, which contains (3-trifluoromethyl) phenyldiazirine and penta(epsilon-Boc-Lys) as a photoreactive code, allows the introduction of a positive cascade to alter the pI value of labeled components, facilitating the isolation of photolabeled biocomponents with isoelectric focusing techniques.

Isoelectric Point↗
PubMed1985

Photoaffinity labeling of atrial natriuretic factor receptors of rat kidney cortex plasma membranes.

Synthetic rat atrial natriuretic factor (ANF) was derivatized with the N-hydroxysuccinimide ester of [125I]iodoazidosalicylic acid to yield a radioactive photoaffinity probe. Incubation of purified plasma membranes from rat kidney cortex with this photoaffinity probe resulted in the specific labeling of a 140-kDa glycoprotein. The photoaffinity labeling of this protein was inhibited by ANF but not by reduced and alkylated ANF nor by other unrelated peptides. A 140-kDa band was also specifically labeled in liver plasma membranes but not in adipocyte plasma membranes. These observations suggest strongly that the 140-kDa glycoprotein is the ANF receptor.

Adipose Tissue↗
PubMed1984

Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase.

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5'-[beta gamma-imido]triphosphate or 20 mM-guanosine 5'-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP-binding site of the EGF-receptor kinase with apparent high affinity and that this analogue is an effective photoaffinity label for the kinase. Furthermore, these studies demonstrate that the EGF receptor, identified by using monoclonal antibodies, contains an ATP-binding site, providing further confirmation that the EGF receptor and EGF-dependent protein kinase are domains of the Mr 170000 protein.

Adenosine Triphosphate↗
PubMed1986

Purification of bile acid-binding proteins from rat hepatic cytosol. Use of a photoaffinity label to detect novel Y' binders.

A 125I-labelled photolabile derivative of cholic acid has been used to investigate the organic anion-binding Y' fraction from rat liver, prepared by the method of Sugiyama, Y., Yamada, T. and Kaplowitz, N. (1982) Biochimica Biophysica Acta, 709, 342-352. The use of this photoaffinity probe led to the discovery of previously undescribed bile acid-binding proteins. A comprehensive purification scheme for the Y' proteins which allows the isolation of these novel binding species is described. Electrophoretic analysis shows that the Y' binders can be divided into two groups. The proteins in group 1 are dimeric and the 5B, 6E and 7F binding species consist of subunits with approximate molecular masses of 19.6, 15.6 and 14.9 kDa, respectively. The group 2 binding proteins, 5C, 5D and 8C, are monomeric and have molecular masses of approximately 36.2, 36.2 and 33 kDa, respectively. Calculation of the incorporation of 125I by these proteins showed that the group 1 proteins displayed a significantly greater specific incorporation of radioactivity than group 2. The specificity of 125I-labelled 3 beta-azidocholylhistamine is further demonstrated by analysis of tryptic digests of photoaffinity labelled Y' binders and glutathione S-transferases AA, A, D and F by reverse-phase high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). The majority of the radioactivity was shown to be incorporated into a single component, which was not coincident with the free photoaffinity label.

Affinity Labels↗
PubMed1992

Photoaffinity labeling of the somatostatin receptor: identification of molecular subtypes.

Pharmacological studies have suggested that the somatostatin (SS) receptor is heterogeneous and may exhibit subtypes selective for SS-14 and SS-28. Whether this heterogeneity can be explained by separate molecular forms of the receptor protein is unclear. In the present study, we have developed a novel photosensitive azido derivative of the octapeptide SS analog Tyr3 SMS (EE 581) and used it as a photoaffinity probe to characterize the molecular components of the SS receptor in five receptor positive tissues (normal rat brain, pituitary, pancreas, and adrenal cortex, and mouse AtT-20 pituitary tumor cells). [125I]EE-581 labeled specific high affinity binding sites in all these tissues (Kd range 1.3-1.67 nM). Photoaffinity labeled membrane SS receptors were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. Three specifically labeled SS receptor proteins of 80 kilodaltons (kDa), 58 kDa, and 32 kDa were identified and exhibited a tissue-specific distribution. The 58 kDa species was the exclusive form in pancreas, adrenal cortex, and AtT-20 cells and the dominant form in brain. The 32 kDa receptor protein was expressed as a minor form (ratio of 58 kDa:32 kDa 3:1), exclusively in brain. The 80 kDa receptor was found only in the pituitary where it occurred as the sole SS receptor species. Competition experiments showed that the 58 kDa and 32 kDa receptor proteins in brain reacted with SS-14 greater than SS-28; in contrast, the 58 kDa protein in AtT-20 cells bound SS-28 greater than SS-14 suggesting the existence of distinct subtypes of the 58 kDa receptor in these two tissues. These data represent the first systematic evaluation of the molecular forms of SS receptor proteins by photoaffinity labeling in different target tissues and provide direct evidence for molecular heterogeneity and SS-14/SS-28 selectivity; a major 58 kDa protein present in most tissues, an additional 32 kDa protein uniquely expressed in brain, and an 80 kDa protein exclusive to the normal pituitary.

Adrenal Glands↗
PubMed1994

Photoaffinity labeling of chloroquine-binding proteins in Plasmodium falciparum.

A photoreactive analog of chloroquine, N-(4-(4-diethylamino-1-methylbutylamino)quinolin-6-yl)-4- azi do-2- hydroxybenzamide (referred to as ASA-Q), has been synthesized and shown to mimic the action of chloroquine in possessing substantial antimalarial activity against a chloroquine-sensitive strain of Plasmodium falciparum. As for chloroquine, ASA-Q is less effective at killing drug-resistant strains of malaria, and the resistance can be modulated using the reagent verapamil. ASA-Q has been radiolabeled with Na125I and used as a photoaffinity probe for labeling chloroquine-binding proteins in malaria-infected erythrocytes. Two proteins have been identified with apparent molecular masses of 42 and 33 kDa in both chloroquine-sensitive and chloroquine-resistant strains of malaria. Photoaffinity labeling of the two proteins by iodo-ASA-Q was competitively inhibited by an excess of unlabeled chloroquine. The structurally related antimalarials amodiaquine and quinine also inhibited labeling of the two proteins, while verapamil and doxycycline had no effect. We suggest that the two labeled proteins are the macromolecular targets of chloroquine action in malaria parasites.

Affinity Labels↗