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PubMed1982

Neurohormonal receptors and cyclic AMP-binding proteins in rabbit tracheal mucosa-submucosa.

Neurohormones and drugs that alter in vitro tracheal electrolyte transport and mucus glycoprotein secretion were examined for their ability to alter cyclic nucleotide accumulation in a smooth muscle-free preparation of rabbit tracheal mucosa-submucosa. cAMP levels were increased by beta-adrenergic agonists, histamine, 2-Cl-adenosine and prostaglandin E1. cGMP levels were increased by carbachol. The phosphodiesterase inhibitor isobutylmethylxanthine increased cAMP and cGMP levels and potentiated only the beta-adrenergic effects. The beta-adrenergic effects were blocked by (+/-)-propranolol and the effects of histamine by diphenhydramine, atropine and (+/-)-propranolol. Atropine blocked the carbachol effects. The isolated surface epithelium from rabbit trachea had higher basal cAMP levels and greater response to beta-adrenergic agonists and isobutylmethylxanthine than the mucosa-submucosa. Two major cAMP-binding proteins in the tracheal mucosa-submucosa were identified with the photoaffinity label 8-N3-[32P]cAMP. Agents that increased cAMP levels also decreased photoaffinity labelling, suggesting that these two cAMP-binding proteins were being occupied in the intact cell. The molecular weights of the proteins were 50 000 and 54 000 and correspond in electrophoretic mobility to the regulatory subunits of Type-I and Type-II cAMP-dependent protein kinases, respectively. The results are consistent with the hypothesis that epithelial functions in the airways are modulated by a number of agonists which increase cyclic nucleotide levels. The effects of beta-adrenergic agonists is apparently mediated by activation of adenylate cyclase and subsequent activation of cAMP-dependent protein kinases.

Adrenergic beta-Agonists↗
PubMed1991

Phorbol ester only partially mimics the effects of insulin on glucose transport and glucose-transporter distribution in 3T3-L1 adipocytes.

We examined the effects of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on the rate of hexose transport into 3T3-L1 adipocytes. Exposure of adipocytes to PMA (1 microM) for 60 min results in a 1.7-2.5-fold increase in the rate of hexose transport. This effect was mediated by translocation of two isoforms of glucose transporters to the plasma membrane, as determined by labelling in situ, photoaffinity labelling with a membrane-impermeant glucose analogue, and by immunoblotting of subcellular fractions. The PMA-induced stimulation of both transport and transporter translocation was substantially less than that induced by insulin in this cell line; the PMA-induced increase in plasma-membrane GLUT 1 and GLUT 4 transporter isoforms was only about 40% and 10% respectively of that induced by insulin. We suggest that the stimulation of transport by insulin and PMA occurs via different mechanisms, which is manifested by the ability of insulin to induce a much greater increase in the plasma-membrane content of GLUT 4 compared with the phorbol ester.

Adipose Tissue↗
PubMed1988

Protein kinase activity associated with the nuclear lamina.

A nuclear lamina-enriched fraction from Ehrlich ascites tumor cells contains a tightly bound protein kinase activity, which phosphorylates in vitro the nuclear lamins, a 52-kilodalton protein, and several unknown minor components. The enzyme(s) is thermolabile, independent of Ca2+ and cAMP, and inhibited by quercetin. After treatment with 4 M urea it remains bound to the nuclear lamina in an active state, but it is irreversibly inactivated in 6 M urea. The lamin proteins are phosphorylated on serine residues. Their two-dimensional phosphopeptide maps show multiple phosphorylation sites and a considerable similarity to the phosphopeptide maps of lamins labeled in vivo. Photoaffinity labeling experiments revealed several polypeptide fractions in the nuclear lamina fraction that are candidates for the protein kinase(s).

Animals↗
PubMed1997

Differential roles of T cell receptor alpha and beta chains in ligand binding among H-2Kd-restricted cytolytic T lymphocyte clones specific for a photoreactive Plasmodium berghei circumsporozoite peptide derivative.

To study the interaction of T cell receptor with its ligand, a complex of a major histocompatibility complex molecule and a peptide, we derived H-2Kd-restricted cytolytic T lymphocyte clones from mice immunized with a Plasmodium berghei circumsporozoite peptide (PbCS) 252-260 (SYIPSAEKI) derivative containing photoreactive Nepsilon-[4-azidobenzoyl] lysine in place of Pro-255. This residue and Lys-259 were essential parts of the epitope recognized by these clones. Most of the clones expressed BV1S1A1 encoded beta chains along with specific complementary determining region (CDR) 3beta regions but diverse alpha chain sequences. Surprisingly, all T cell receptors were preferentially photoaffinity labeled on the alpha chain. For a representative T cell receptor, the photoaffinity labeled site was located in the Valpha C-strand. Computer modeling suggested the presence of a hydrophobic pocket, which is formed by parts of the Valpha/Jalpha C-, F-, and G-strands and adjacent CDR3alpha residues and structured to be able to avidly bind the photoreactive ligand side chain. We previously found that a T cell receptor specific for a PbCS peptide derivative containing this photoreactive side chain in position 259 similarly used a hydrophobic pocket located between the junctional CDR3 loops. We propose that this nonpolar domain in these locations allow T cell receptors to avidly and specifically bind epitopes containing non-peptidic side chains.

Affinity Labels↗
PubMed1985

The role of extracellular calcium in corticotropin-stimulated steroidogenesis.

The role of extracellular Ca2+ in the binding of corticotropin (ACTH) to adrenocortical cell receptors as well as in the post-binding events involved in steroidogenesis were investigated. Binding studies using [125I-Tyr23,Phe2,Nle4]ACTH (1-38) peptide showed that extracellular Ca2+ is essential not only for the interaction of ACTH with its receptor, but also for continued occupancy of the receptor. In view of the requirement of Ca2+ for binding the hormone to the receptor, the role of Ca2+ in post-receptor events was investigated by covalently attaching the hormone to its receptor by photoaffinity labeling in the presence of Ca2+. Persistent activation of steroidogenesis induced by photoaffinity labeling in the presence of Ca2+ was depressed when cells were incubated in medium containing EGTA but was unaffected when the cells were merely washed and incubated in Ca2+-free medium. In the presence of EGTA, 8-Br-cAMP partially restored persistent activation of steroidogenesis. The concentration of extracellular Ca2+ required for restoring steroidogenesis was 10-fold lower than the concentration of Ca2+ needed for optimal binding of ACTH to its receptor. These results suggest that the primary role of extracellular Ca2+ in the action of ACTH is to facilitate the association of the hormone with its receptor.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Purification and characterization of Ca2+/calmodulin-dependent protein kinase I from bovine brain.

Ca2+/calmodulin-dependent protein kinase (Ca2+/CaM kinase I), which phosphorylates site I of synapsin I, has been highly purified from bovine brain. The physical properties and substrate specificity of Ca2+/CaM kinase I were distinct from those of all other known Ca2+/CaM kinases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified enzyme preparation consisted of two major polypeptides of Mr 37,000 and 39,000 and a minor polypeptide of Mr 42,000. In the presence of Ca2+ and calmodulin (CaM), all three polypeptides bound CaM, were autophosphorylated on threonine residues, and were labeled by the photoaffinity label 8-azido-ATP. Peptide maps of the three autophosphorylated polypeptides were very similar. The Stokes radius and the sedimentation coefficient of the enzyme were, respectively, 31.8 A and 3.25 s. A molecular weight of 42,400 and a frictional ratio of 1.38 were calculated from the above values, suggesting that Ca2+/CaM kinase I is a monomer. It is possible that the polypeptides of lower molecular weight are derived from the polypeptide of Mr 42,000 by proteolysis; alternatively, the polypeptides may represent isozymes of Ca2+/CaM kinase I. Synapsin I (site I) was the best substrate tested (Km, 2-4 microM) for Ca2+/CaM kinase I. Of many additional proteins tested, only protein III (a phosphoprotein related to synapsin I) and smooth muscle myosin light chain were phosphorylated. Ca2+/CaM kinase I was found in highest concentration in brain, where it showed widespread regional and subcellular distributions. In addition, the enzyme had a widespread and predominantly cytosolic tissue distribution. The widespread neuronal and tissue distribution of Ca2+/CaM kinase I suggests that other substrates might exist for this enzyme in both neuronal and non-neuronal tissues.

Adenosine Triphosphate↗
PubMed1985

The amine transporter from bovine chromaffin granules. Partial purification.

We have partially purified the amine transporter from bovine adrenal chromaffin granules in a single step utilizing affinity chromatography. A 5-hydroxytryptamine moiety has been coupled to a Sepharose 4B matrix in a position ortho to the hydroxyl group. When membranes solubilized with sodium cholate are chromatographed on the above matrix a 45,000 Mr polypeptide is highly enriched. The enrichment is dependent on the presence of the proper ligand on the matrix and is inhibited if the column is previously equilibrated with a soluble ligand. Enrichment of the above polypeptide is accompanied by an increase in the specific activity of the transporter as measured by its labeling by 4-azido-3-nitrophenylazo(5-hydroxytryptamine). The ability of reserpine, a competitive inhibitor of binding and transport, to inhibit labeling of the purified transporter correlates well with its known kinetic constants in the native membranes. The polypeptide purified is identical to the one previously identified as the putative transporter based on specific labeling by a photoaffinity label (Gabizon, R., Yetinzon, T., and Schuldiner, S. (1982) J. Biol. Chem. 257, 15145-15150). The results clearly support the contention that the 45,000 Mr peptide is the amine transporter or one of its subunits.

Amines↗
PubMed2004

Affinity labeling the dopamine transporter ligand binding site.

Photoaffinity labeling is a positive function approach that has been used in an effort to identify the cocaine-binding site on the dopamine transporter (DAT). Radioactive and non-radioactive analogs of cocaine and other dopamine uptake blockers are used to irreversibly label the DAT ligand-binding site and the protein is subjected to chemical or enzymatic treatments that cleave at specific amino acid residues. Analysis of cleavage products from radioactively photolabeled DAT using epitope-specific immunoprecipitation, gel electrophoresis, and autoradiography has identified the site of origin in the primary sequence of labeled fragments as small as 4 kDa. More precise localization of the site of labeling is done by subjecting photolabeled DAT to parallel or serial digestion with multiple cleavage methods, followed by analysis of radiolabeled peptides by reverse-phase HPLC. Fragment retention times are compared to calculated retention times of predicted digest peptides and to chemically or photochemically labeled synthetic peptides. The presence of authentic DAT sequence in HPLC fractions of digests from DAT labeled with non-radioactive ligands is further supported by MALDI and nanoelectrospray mass spectrometry. Using these methods we have identified two distinct regions of DAT that interact with multiple structurally related and diverse irreversible ligands, suggesting that these regions may be involved in the formation of ligand binding sites.

Affinity Labels↗
PubMed1998

Cellular distribution of secretin receptor expression in rat pancreas.

Secretin is an important regulator of pancreatic function, but the molecular basis of its actions is not well understood. We have, therefore, used in situ autoradiography, photoaffinity labeling, and RNase protection assays with healthy rat pancreas, dispersed acinar cells, and pancreas depleted of acinar cells to explore the cellular distribution and molecular identity of high-affinity secretin receptors in this complex organ. The autoradiographic examination of 125I-labeled [Tyr10]rat secretin-27 binding to normal pancreas demonstrated saturable and specific high-affinity binding sites on both acinar and duct cells, with a uniform lobular distribution, but with no binding above background over islets or vascular structures. Photoaffinity labeling demonstrated that the ductular binding site in acinar cell-depleted copper-deficient rat pancreas represented the same glycoprotein with a molecular weight of 50,000-62,000 that was present on acinar cells. RNase protection assays confirmed the molecular identity of the secretin receptors expressed on these distinct cells. The apparent absence or extreme low density of similar secretin receptors on islets and pancreatic vascular structures suggests that the pharmacological effects of secretin on those cells may either be indirect or mediated by another secretin family receptor that recognizes this hormone with lower affinity.

Animals↗
PubMed2004

P-glycoprotein substrate binding domains are located at the transmembrane domain/transmembrane domain interfaces: a combined photoaffinity labeling-protein homology modeling approach.

P-glycoprotein (P-gp) is an energy-dependent multidrug efflux pump conferring resistance to cancer chemotherapy. Characterization of the mechanism of drug transport at a molecular level represents an important prerequisite for the design of pump inhibitors, which resensitize cancer cells to standard chemotherapy. In addition, P-glycoprotein plays an important role for early absorption, distribution, metabolism, excretion, and toxicity profiling in drug development. A set of propafenonetype substrate photoaffinity ligands has been used in this study in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to define the substrate binding domain(s) of P-gp in more detail. The highest labeling was observed in transmembrane segments 3, 5, 8, and 11. A homology model for P-gp was generated on the basis of the dimeric crystal structure of Vibrio cholerae MsbA, an essential lipid transporter. Thereafter, the labeling pattern was projected onto the 3D atomic-detail model of P-gp to allow a visualization of the binding domain(s). Labeling is predicted by the model to occur at the two transmembrane domain/transmembrane domain interfaces formed between the amino- and carboxyl-terminal half of P-gp. These interfaces are formed by transmembrane (TM) segments 3 and 11 on one hand and TM segments 5 and 8 on the other hand. Available data on LmrA and AcrB, two bacterial multidrug efflux pumps, suggest that binding at domain interfaces may be a general feature of polyspecific drug efflux pumps.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Structure/function analysis of a dUTPase: catalytic mechanism of a potential chemotherapeutic target.

dUTP pyrophosphatase catalyses hydrolysis of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP) and inorganic pyrophosphate (PPi). Elimination of dUTP is vital since its misincorporation into DNA by DNA polymerases can initiate a damaging iterative repair and misincorporation cycle, resulting in DNA fragmentation and cell death. The anti-tumour activity of folate agonists and thymidylate synthase inhibitors is thought to rely on dUTP misincorporation. Furthermore, retroviral cDNA production may be particularly susceptible to the effects of dUTP misincorporation by virtue of the error-prone nature of reverse trans criptase. Consequently, dUTPase activity is an ideal point of intervention in both chemotherapy and anti-retroviral therapy. In particular, the dUTPase encoded by a human endogenous retrovirus (HERV-K) has been suggested to complement HIV infection and so is an attractive target for specific inhibition. Hence, we used site photoaffinity labelling, site-directed mutagenesis and molecular modelling to assign catalytic roles to the conserved amino acid residues in the active site of the HERV-K dUTPase and to identify structural differences with other dUTPase enzymes. We found that dUTP photoaffinity labelling was specific for a beta-hairpin motif in HERV-K dUTPase. Mutagenesis of aspartate residues Asp84 and 86 to asparagine within this beta-hairpin showed the carboxylate moiety of both residues was required for catalysis but not for dUTP binding. An increase in the pKa of both aspartate residues brought about by substitution of a serine residue with a glutamate residue adjacent to the aspartate residues increased activity by a factor of 1.67 at pH 8.0, implicating general base catalysis as the enzyme's catalytic mechanism. Conservative mutagenesis of Tyr87 to Phe resulted in a sevenfold reduction of dUTPase activity and a 3.3-fold reduction in binding activity, whilst substitution with an isoleucine residue totally abolished both catalytic activity and dUTP binding, suggesting that binding/activity is dependent on an aromatic side-chain at the base of the hairpin. Comparison of a homology-based three-dimensional model structure of HERV-K dUTPase with a crystallographic structure of the human dUTPase revealed displacement of a conserved alpha-helix in the HERV-K enzyme causing expansion of the HERV-K active site. This expansion may be responsible for the ability of the HERV-K enzyme to hydrolyse dTTP and bind the bulkier dNTPs in contrast to the majority of dUTPases which are highly specific for dUTP. Knowledge of the dUTPase catalytic mechanism and the distinctive topography of the HERV-K active site provides a molecular basis for the design of HERV-K dUTPase-specific inhibitors.

Amino Acid Sequence↗
PubMed1999

Molecular characterization of the human neuropeptide Y Y2-receptor.

Five neuropeptide Y receptors, the Y1-, Y2-, Y4-, Y5- and y6-subtypes, have been cloned, which belong to the rhodopsin-like G-protein-coupled, 7-transmembrane helix-spanning receptors and bind the 36-mer neuromodulator NPY (neuropeptide Y) with nanomolar affinity. In this study, the Y2-receptor subtype expressed in a human neuroblastoma cell line (SMS-KAN) and in transfected Chinese hamster ovary cells (CHO-hY2) was characterized on the protein level by using photoaffinity labeling and antireceptor antibodies. Two photoactivatable analogues of NPY were synthesized, in which a Tyr residue was substituted by the photoreactive amino acid 4-(3-trifluoromethyl)-3H-diazirin-3-ylphenylalanine ((Tmd)Phe), [Nalpha-biotinyl-Ahx2,(Tmd)Phe36]NPY (Tmd36), and the Y2-receptor subtype selective [Nalpha-biotinyl-Ahx2,Ahx5-24,(Tmd)Phe27]N PY (Tmd27). Both analogues were labeled with [3H]succinimidyl-propionate at Lys4 and bind to the Y2-receptor with affinity similar to that of the native ligand. A synthetic fragment of the second (E2) extracellular loop was used to generate subtype selective antireceptor antibodies against the Y2-receptor. Photoaffinity labeling of the receptor followed by SDS-PAGE and detection of bound radioactivity and SDS-PAGE of solubilized receptors and subsequent Western blotting revealed the same molecular masses. Two proteins correspondingly have been detected for each cell line with molecular masses of 58 +/- 4 and 50 +/- 4 kDa, respectively.

Amino Acid Sequence↗
PubMed1997

Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane of suspension-cultured rice cells by affinity labeling.

A high-affinity binding protein for the N-acetylchito-oligosaccharide elicitor of phytoalexin biosynthesis was identified by photoaffinity labeling and affinity cross-linking in the plasma membrane of suspension-cultured rice cells. Both a [125I]-labeled photolabile 2-(4-azidophenyl)ethylamino conjugate ([125I]-GN8-AzPEA) and a [125I]-labeled 2-(4-aminophenyl)ethylamino conjugate ([125]-GN8-APEA) of N-acetylchito-octaose were synthesized. The two conjugates were separately incubated with the plasma membrane prepared by aqueous two-phase partitioning, and covalently cross-linked to the elicitor binding site by irradiation with UV light or treatment with the cross-linking agent glutaraldehyde, respectively. Autoradiography of the SDS-PAGE gel of the solubilized membrane proteins revealed the labeling of a single 75 kDa band in both cases. The incorporation of the radiolabeled ligands into the 75 kDa protein showed a saturable mode of binding, with half-maximal incorporation at 45 and 52 nM for photoaffinity labeling and affinity cross-linking, respectively. The labeling of the 75 kDa protein was inhibited by N-acetylchito-oligosaccharides in a size-dependent manner, and N-acetylchito-octaose (GlcNAc)8 showed a half-maximal inhibition at concentrations of the order of 10 nM. However, neither chito-octaose (GlcN)8, cellopentaose nor alpha-1,4 linked N-acetylgalactosamine octamer (GalNAc)8 at concentrations as high as 25 microM inhibited the labeling of the 75 kDa protein. These results are in good agreement with the sensitivity and the specificity of the 'high-affinity binding site' previously identified by binding assays, as well as with the activities of these oligosaccharides in the induction of phytoalexin biosynthesis and other cellular responses. These results suggest that the 75 kDa protein identified by the affinity labeling represents a functional receptor for this elicitor.

Affinity Labels↗
PubMed1987

Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA.

A photoactive nucleotide analogue of dUTP, 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP), was synthesized from dUMP in five steps. The key reaction in the synthesis of 5-N3dUTP is the nitration of dUMP in 98% yield in 5 min at 25 degrees C using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. Reduction of the resulting 5-nitro compound with zinc and 20 mM HCl gave 5-aminodeoxyuridine monophosphate (5-NH2dUMP). Diazotization of 5-NH2dUMP with HNO2 followed by the addition of NaN3 to the acidic diazonium salt solution gave a photoactive nucleotide derivative in 80-90% yield. The monophosphate product was identified as 5-N3dUMP by proton NMR, UV, IR, and chromatographic analysis as well as by the mode of synthesis and its photosensitivity. After formation of 5-N3dUTP through a chemical coupling of pyrophosphate to 5-N3dUMP, the triphosphate form of the nucleotide was found to support DNA synthesis by Escherichia coli DNA polymerase I at a rate indistinguishable from that supported by dTTP. When UMP was used as the starting compound, 5-N3UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E. coli RNA polymerase. To prepare [gamma-32P]-5-N3dUTP for use as an active-site-directed photoaffinity labeling reagent, a simple method of preparing gamma-32P-labeled pyrimidine nucleotides was developed. [gamma-32P]-5-N3dUTP is an effective photoaffinity labeling reagent for DNA polymerase I and was found to bind to the active site with a 2-fold higher affinity than dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1994

Characterization of two membrane-associated beta-glucosidases from maize (Zea mays L.) coleoptiles.

We isolated membrane vesicles from maize (Zea mays L.) coleoptiles and identified in these vesicles a 58 kDa (pm58) and a 60 kDa (pm60) protein by photoaffinity labelling with 5-azido-[7-3H]indole-3-acetic acid ([3H]N3IAA). Photoaffinity labelling was effectively competed for by auxins as well as by flavonoids. The labelled proteins were solubilized by Triton X-114 from the vesicles and partially purified. Microsequence analysis revealed that pm60 is a beta-glucosidase. This was confirmed by biochemical and immunological analysis. We show that pm60 has a beta-D-glucoside glucohydrolase (EC 3.2.1.21) activity. It uses p-nitro-phenyl beta-D-glucopyranoside (PNPG) as a substrate, with a pH optimum of 5.0. The Km for PNPG is 0.652 mM and the Vmax. 6.24 mumol.min-1.mg-1. The beta-glucosidase activity of pm60 was competitively inhibited by IAA and 1-naphthylacetic acid as well as by gluconolactam and glucose. N-terminal amino-acid-sequence analysis of pm58 revealed similarity to pm60, suggesting that both proteins are encoded by different members of a gene family.

Affinity Labels↗
PubMed1996

An inhalational anesthetic binding domain in the nicotinic acetylcholine receptor.

To determine inhalational anesthetic binding domains on a ligand-gated ion channel, I used halothane direct photoaffinity labeling of the nicotinic acetylcholine receptor (nAChR) in native Torpedo membranes. [14C]Halothane photoaffinity labeling of both the native Torpedo membranes and the isolated nAChR was saturable, with Kd values within the clinically relevant range. All phospholipids were labeled, with greater than 95% of the label in the acyl chain region. Electrophoresis of labeled nAChR demonstrated no significant subunit selectivity for halothane incorporation. Within the alpha-subunit, greater than 90% of label was found in the endoprotease Glu-C digestion fragments which contain the four transmembrane regions, and the pattern was different from that reported for photoactivatable phospholipid binding to the nAChR. Unlabeled halothane reduced labeling more than did isoflurane, suggesting differences in the binding domains for inhalational anesthetics in the nAChR. These data suggest multiple similar binding domains for halothane in the transmembrane region of the nAChR.

Affinity Labels↗
PubMed1996

Purification and properties of two rat liver phenobarbital-inducible UDP-glucuronosyltransferases that catalyze the glucuronidation of opioids.

Glucuronidation of xenobiotics and endobiotics is catalyzed by a group of intrinsic membrane proteins of the endoplasmic reticulum of cells: the UDP-glucuronosyltransferases. Two isoforms with glucuronidation activity toward opioids have been purified and characterized from liver microsomes obtained from phenobarbital-treated Wistar rats. The proteins have been identified as the gene products of UGT2B1 and UGT1.1r. The purified proteins exhibited the same apparent KM values for morphine glucuronidation (2-3 mM). However, the purified UGT1.1r enzyme exhibited glucuronidation activity toward buprenorphine and bilirubin with high efficiency, but the UGT2B1 protein did not react with these compounds. Both purified enzymes glucuronidated chloramphenicol, 4-hydroxybiphenyl, chrysin, and ibuprofen. Flunitrazepam photoaffinity labeling was demonstrated for both enzymes, and naloxone, the opioid antagonist, antagonized the photoaffinity labeling reactions.

Amino Acid Sequence↗
PubMed1999

NEM modification prevents high-affinity ATP binding to the first nucleotide binding fold of the sulphonylurea receptor, SUR1.

Pancreatic beta-cell ATP-sensitive potassium channels, composed of SUR1 and Kir6.2 subunits, serve as a sensor for intracellular nucleotides and regulate glucose-induced insulin secretion. To learn more about the interaction of SUR1 with nucleotides, we examined the effect of N-ethylmaleimide (NEM) modification. Photoaffinity labeling of SUR1 with 5 microM 8-azido-[alpha-32P]ATP or 8-azido-[gamma-32P]ATP was inhibited by NEM with Ki of 1.8 microM and 2.4 microM, and Hill coefficients of 0.94 and 1.1, respectively. However, when the cysteine residue in the Walker A motif of the first nucleotide binding fold (NBF1) of SUR1 was replaced with serine (C717S), photoaffinity labeling was not inhibited by 100 microM NEM. These results suggest that NBF1 of SUR1 has a NEM-sensitive structure similar to that of NBF1 of MDR1, a multidrug transporter, and confirm NBF1 as the high-affinity ATP binding site on SUR1.

ATP Binding Cassette Transporter, Subfamily B, Mem↗