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PubMed1998

Identification of two uridine binding domain peptides of the UDP-glucose-binding site of rabbit muscle glycogenin.

Glycogenin, the autoglucosyltransferase that initiates the de novo biosynthesis of glycogen, photoaffinity labeled with [beta32P]5-azido-UDP-glucose. The photoinsertion of the azidouridine derivative showed activating ultraviolet light dependency, saturation effects, and inhibition by UDP-glucose, thus demonstrating the specificity of the interaction. In the absence of Mn2+, the requirement for the catalytic activity of glycogenin, the photolabeling decreased by 70%. Competitive binding experiments indicated that the pyrophosphate or a phosphate was the moiety of UDP-glucose implicated in the strongest interaction at the binding site. Proteolytic digestion of photolabeled glycogenin resulted in the identification of two labeled fragments, 89-143 and 168-233, that carried the uridine binding sites. This is the first report of the region of glycogenin that harbors the UDP-glucose-binding domain.

Amino Acid Sequence↗
PubMed1998

Amino acid sequence, post-translational modifications, binding and labelling of porcine odorant-binding protein.

An odorant-binding protein, migrating in SDS-PAGE with an apparent molecular weight of 22 kDa and an isoelectric point of 4.2, has been purified from pig nasal mucosa. Its complete amino acid sequence was determined by a combination of mass spectrometry and Edman degradation procedures. The protein consists of a single polypeptide chain of 157 amino acids, presenting at the N-terminus a pyroglutamic acid residue. The two cysteine residues, occurring in the primary structure at positions 63 and 155, are involved in an intramolecular disulphide bridge. Sequence comparison with other lipocalins revealed a good similarity with bovine odorant-binding protein, the only member of this class which does not contain disulphide bonds and of which the three-dimensional structure recently has been resolved. Nine out of the 1 6 residues lining the binding pocket in bovine OBP are conserved in the porcine protein, suggesting structural similarities in this region of the molecule. The synthesis of a fluorescent photoaffinity labelling agent and of two tin-containing thymol analogues is also described. These compounds together with other ligands were able to bind the protein as revealed by competitive binding experiments.

Amino Acid Sequence↗
PubMed2006

ATP binding site in the plant ADP-glucose pyrophosphorylase large subunit.

The ATP binding region in the catalytically inactive large subunit (LS) of the potato tuber ADP-glucose pyrophosphorylase was identified and investigated. Mutations at the ATP binding significantly affected not only the apparent affinities for ATP and Glc-1-P, and catalytic rate but also in many instances, sensitivity to 3-phosphoglycerate. The catalytic rates of the LS mutant enzymes correlated most strongly with changes in the affinity toward ATP, a relationship substantiated by photoaffinity labeling studies with azido-ATP analog. These results indicate that the LS, although catalytically defective, interacts cooperatively with the catalytic small subunit in binding substrates and effectors and, in turn, influencing net catalysis.

Adenosine Triphosphate↗
PubMedDate not supplied

Photoaffinity analogue for the anti-inflammatory drug alpha-trinositol: synthesis and identification of putative molecular targets.

alpha-Trinositol (alpha T), or Ins(1,2,6)P3, is a semisynthetic inositol trisphosphate produced commercially by the partial degradation of phytic acid with phytase. The molecular targets mediating the mechanism of action of this novel anti-inflammatory, analgesic, and antivasoconstrictive drug are unknown. A new photoaffinity analogue, 4-[3H]BZDC-alpha T, has been prepared in which the [3H]-p-benzoyldihydrocinnamoyl ([3H]BZDC) photophore is tethered through an O-(5-aminopentanoyl) linkage to the 4-OH of alpha T. Photoaffinity labeling experiments with two human tissues, umbilical cord vascular smooth muscle cells and platelet membranes, revealed proteins that were selectively labeled by 4-[3H]BZDC-alpha T. Thus, co-incubation with alpha T but not with Ins(1,3,4,5)P4 during photolysis competitively displaced labeling of a 55 kDa platelet protein. In vascular epithelial cells, alpha T and Ins(1,3,4,5)P4 both displaced labeling of a 55 and 43 kDa proteins. The identification of putative protein targets for alpha T in smooth vascular tissue may have important implications in elucidation of the mechanism of action of this unusual drug.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2000

Cloning and characterization of mammalian UDP-glucose glycoprotein: glucosyltransferase and the development of a specific substrate for this enzyme.

The endoplasmic reticulum enzyme UDP-glucose glycoprotein:glucosyltransferase (UGGT) has the unique property of recognizing incompletely folded glycoproteins and, if they carry an N -linked Man(9)GlcNAc(2)oligosaccharide, of catalyzing the addition of a glucose residue from UDP-glucose. Using peptide sequence information, we have isolated the complete cDNA of rat liver UGGT and expressed it in insect cells. The cDNA specifies an open reading frame which codes for a protein of 1527 residues including an 18 amino acid signal peptide. The protein has a C-terminal tetrapeptide (HEEL) characteristic of endoplasmic reticulum luminal proteins. The purified recombinant enzyme shows the same preference for unfolded polypeptides with N -linked Man(9)GlcNAc(2)glycans as the enzyme purified from rat liver. A genetically engineered Saccharomyces cerevisiae strain capable of producing glyco-proteins with Man(9)GlcNAc(2)core oligosaccharides was constructed and secreted acid phosphatase (G0-AcP) was purified. G0-AcP was used as an acceptor glycoprotein for UGGT and found to be a better substrate than the previously used soybean agglutinin and thyroglobulin. Recombinant rat UGGT has a K (m) of 44 microM for UDP-glucose. A proteolytic fragment of UGGT was found to retain enzymatic activity thus localizing the catalytic site of the enzyme to the C-terminal 37 kDa of the protein. Using site-directed mutagenesis and photoaffinity labeling, we have identified residues D1334, D1336, Q1429, and N1433 to be necessary for the catalytic activity of the enzyme.

Acid Phosphatase↗
PubMed1998

Photochemical localization of the semotiadil binding region within the cardiac Ca2+ channel alpha1 subunit. Comparison with the skeletal muscle counterpart.

We have previously identified the binding region of a new Ca2+ antagonist semotiadil in the skeletal muscle Ca2+ channel. To the same semotiadil derivatives, the cardiac counterpart showed distinct and different binding characteristics: semotiadil and its photoaffinity analog D51-4700 inhibited [3H]PN200-110 binding to cardiac membrane preparations with IC50 values of 13-20 microM, which are 10 times higher than those in skeletal muscle. Hill slopes of the binding inhibition were 0.94-1.0 for the cardiac channels compared to 0.63-0.67 for the skeletal muscle channels. A possible explanation for the difference is that the semotiadil binding site is differently conferred in cardiac and skeletal muscle Ca2+ channels. To reveal this within the primary structure, photoaffinity labeling of cardiac membranes was employed. [3H]D51-4700 was photo-incorporated in several polypeptides but only the alpha1 subunit of the Ca2+ channel was photolabeled in a specific manner. Antibody mapping of the [3H]D51-4700-labeled alpha1 subunit with several anti-peptide antibodies revealed that the labeled site was located solely in a peptide fragment between Cys1461 and Lys1529. This region encompasses the labeled site of skeletal muscle, but contains several non-identical amino acid residues, which may participate in expressing different binding characteristics between the two muscle type Ca2+ channels.

Amino Acid Sequence↗
PubMed2000

Preparation and use of a photoactivatable glucose-6-phosphate analogue.

A benzophenone-containing derivative of glucose-6-phosphate, 6-[(3-([2,3-3H2]-p-benzoyl dihydrocinnamidoylpropyl-1-oxy)phosphoryl]-D-glucopyranose ([3H]BZDC-Glc-6-P) was synthesized and employed to photoaffinity label proteins on intact rat liver microsomes. The use of a non-photoactivatable, UV-transparent desoxy analogue of BZDC, named p-benzyldihydrocinnamoyl (BnDC), is introduced as a general method to achieve competition when hydrophilic ligands are modified with hydrophobic photophores.

Animals↗
PubMed2003

Synthesis of a biotin-tagged photoaffinity probe of 2-azetidinone cholesterol absorption inhibitors.

The design and synthesis of a biotin-tagged photoreactive analogue C-4 of the cholesterol absorption inhibitor Ezetimibe is described. Photoaffinity labeling of intestinal brush border membrane vesicles with C-4 and subsequent streptavidin-biotin chromatography leads to selective extraction of a 145 kDa integral membrane protein as the molecular target for cholesterol absorption inhibitors.

Animals↗
PubMed2003

Identification and purification of sulfotransferases for 20-hydroxysteroid from the larval fat body of a fleshfly, Sarcophaga peregrina.

Sulfotransferase (ST) activity for 20-hydroxyecdysone (20E) was identified in a larval fat body lysate of the fleshfly, Sarcophaga peregrina, but not in the hemolymph. The activity was highly sensitive to 2,6-dichloro-4-nitrophenol (DCNP) (IC50=0.61 microM), a specific inhibitor of phenol ST (P-ST), but insensitive to triethylamine, a hydroxysteroid ST inhibitor. These results suggest that 20E-specific ST enzymes belong to the P-ST family, despite the fact that 20E is a hydroxysteroid. In addition to 20E ST activity, a relatively high level of 2-naphthol ST activity was detected in the fat body lysate. The ST activity for both substrates transiently decreased to the 50% of maximal levels, 6 hrs after induction of pupation. The ST enzymes were separated on a DEAE-cellulose column. The 20E-ST enzymes were eluted around 50 mM KCl as two separate peaks of close proximity and the P-ST was eluted at 0.1 M KCl. The 20E ST enzymes were further purified using 3'-phosphoadenosine 5'-phosphate (PAP)-agarose affinity column chromatography. Both of the eluted active fractions demonstrated 43-kDa proteins on SDS-polyacrylamide gel. Photoaffinity labeling with [35S]-3'-phosphoadenosine 5'-phosphosulfate (PAPS) showed 43-kDa bands in the fat body lysate, as well as in the purified fractions. These results suggest that the 43-kDa proteins catalyze 20E sulfation within the fat body of S. peregrina.

Adenosine Diphosphate↗
PubMed2003

Use of synthetic isoprenoid analogues for understanding protein prenyltransferase mechanism and structure.

Protein prenylation involves the post-translational modification of specific protein-derived cysteine residues with farnesyl or geranylgeranyl groups through thioether linkages. Because a large number of proteins that participate in signal transduction processes require this modification, there has been intense interest in developing inhibitors of these enzymes and in clarifying the biological function of prenylation. Isoprenoid analogues have proven to be versatile tools for probing the mechanism and structure of prenyltransferases. Mechanistic probes have been created to investigate the stereochemical course and substituent effects in prenyltransferase catalyzed reactions. They have also been used to measure kinetic isotope effects and search for possible cationic intermediates. Photoaffinity labeling analogues containing either diazotrifluoropropionate or benzophenone units have been used to identify the location of isoprenoid binding sites in these enzymes. Biophysical probes incorporating fluorescent moieties or isotopic labels have been used to measure isoprenoid dissociation constants or prenyl group conformation when bound to the enzyme. Analogues containing noncognate alkene isomers or bulky substituents have also contributed to an understanding of isoprenoid recognition. Most recently, photoactive and isomeric isoprenylated analogues are providing insights into the function of protein prenylation.

Benzophenones↗
PubMed2002

Development of a biologically active secretin analogue incorporating a radioiodinatable photolabile p-(4-hydroxybenzoyl)phenylalanine in position 10.

Photoaffinity labeling is a powerful approach for direct elucidation of residue-residue approximations as a ligand is bound to its receptor, providing important constraints for molecular modeling. Probes utilized for this need to incorporate photolabile sites of covalent attachment and an indicator, such as a radiolabel. Radioiodine provides a particularly useful high specific radioactivity label, but due to its size, can only be accommodated in limited positions within a peptide ligand. In this work, we attempted to develop a probe for the secretin receptor that would directly provide spatial approximation data for position 10 of secretin, its site of radiolabeling. This was achieved by incorporation into a secretin analogue of the radioiodinatable and photolabile benzophenone moiety, p-(4-hydroxybenzoyl)phenylalanine (OH-Bpa). An unintended additional modification of secretin in synthesizing this probe was the elimination of Gly(4). This probe was shown to bind to the secretin receptor specifically and saturably (K(i)=25.3+/-6.0 nM). It represented a full agonist, stimulating intracellular cAMP in a concentration-dependent manner (EC(50)=4.2+/-0.7 nM). It was also able to affinity label the secretin receptor in a specific and efficient manner. This probe should provide the opportunity to identify the region of the secretin receptor in spatial approximation with position 10, within the pharmacophore of secretin, leading to refinement of molecular conformational models of this agonist-bound receptor.

Amino Acid Sequence↗
PubMed2003

Synthesis, characterization, and biological properties of 8-azido- and 8-amino-substituted 2',5'-oligoadenylates.

A series of 8-azido- and 8-amino-substituted 2',5'-oligoadenylatyes was prepared by a uranyl-ion catalyzed polymerization of the corresponding 8-substituted adenosine phosphorimidazolide. Subsequent 5'-dephosphorylation of the resulting 5'-phosphoryl 2',5'-linked oligomers with alkaline phosphatase gave the corresponding core oligomers. The CD spectra indicated that the 8-aminoadenosine analogue of the 2',5'-linked trimer has an anti-orientation as in naturally occurring 2',5'-oligoadenylates, while 8-azido-substituted 2',5'-oligoadenylates have a syn-orientation. The 8-substituted oligomers showed enhanced resistance against digestion by snake venom phosphodiesterase. The 2',5'-linked 8-azidoadenylate trimer and tetramer displayed strong RNase L binding and activating ability, although the corresponding dimer is devoid of such activities. In contrast, very low or no RNase L binding and activating ability were observed in the 8-aminoadenosine analogue of 2',5'-oligoadenylates. Results indicate that the bulkiness and ionic character of the 8-substituting group have significant effects on the ability of these analogues to bind and activate RNase L. Furthermore, the orientation of the glycosidic base in the 2-5A analogues may change from syn to anti during binding to RNase L. The 8-azidoadenosine analogues of 2-5A will be useful tools in the photoaffinity labeling of RNase L, due to their strong RNase L binding ability. In addition, these 8-azidoadenosine compounds may be considered as candidates for experimental therapeutic agents because they have enhanced stability to enzyme degradation while retaining the ability to activate RNase L.

Adenine Nucleotides↗
PubMed2001

Characterization of the type A cholecystokinin receptor hormone-binding domain: use of contrasting and complementary methodologies.

Insights into the molecular basis of binding of the peptide hormone, cholecystokinin, to its G protein-coupled receptor is of substantial interest and may contribute to the successful production and refinement of receptor-active drugs. A number of methodological approaches provide complementary data to contribute to these insights. These include receptor mutagenesis, ligand structure-activity data, conformational analysis of ligand and receptor fragments, and photoaffinity labeling. In this work, we compare and contrast each of these methods and provide our current view of the cumulative impact of the current data on molecular conformational models of the agonist-occupied type A cholecystokinin receptor. These support the key roles played by extracellular loop and tail regions of this receptor for binding its natural peptide ligand.

Amino Acid Sequence↗
PubMed2002

Characterization of anthracenediones and their photoaffinity analogs.

In an attempt to overcome the cardiotoxicity and cross-resistance problems caused by the anticancer drugs anthracyclines and anthracenediones during chemotherapy, we have developed a series of aza-anthracenedione compounds by modifying the chromophore and the side arms of anthracyclines and anthracenediones. One of these aza-anthracenediones, 6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione (BBR 2778), which is currently under phase II clinical trials, showed remarkable antitumor activity and appeared to lack a cardiotoxic effect in preclinical studies. However, it was still cross-resistant against multidrug resistance (MDR) cells expressing P-glycoprotein (P-gp). In contrast, another aza-anthracenedione, 6,9-bis[[2-(dimethylamino)ethyl]amino]benzo[g]isoquinoline-5,10-dione, which has side arm structures different from those of BBR 2778, was highly active against MDR cells. In this study, BBR 2778, BBR 2378, and an anthracenedione compound, 1,4-bis[(2-aminoethyl)amino]-5,8-dimethyl-9,10-anthracenedione, were used to assess the relationship between the chemical structures of these drugs and their interactions with DNA and P-gp. In addition, the biological and pharmacological influences of photoaffinity labeling were also studied for BBR 2778 and DEH. As the results indicate, the photolabeled analogs of BBR 2778 and DEH were less DNA-reactive and less cytotoxic. The more lipophilic compound, BBR 2378, and the photolabeled analogs of BBR 2778 and DEH inhibited P-gp labeling by azidopine better than did the more hydrophilic parental compounds. These studies suggested that the DNA binding affinity of BBR 2778 and DEH could be important in determining their cytotoxicity, and that the chemical structure of the side arms and the lipophilicity of these drugs are critical in determining their cross-resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Expression and coupling characteristics of the CRH and orexin type 2 receptors in human fetal adrenals.

Hormones produced by the fetal adrenal regulate fetal growth, steroidogenic activity, and intrauterine homeostasis, which are essential for the maintenance of pregnancy and the preparation of the fetus for extrauterine life. There is a functional interaction between CRH and the fetal adrenal, as CRH increases dehydroepiandrosterone sulfate production in cultured fetal adrenal cells. Moreover, in a rodent model administration of orexin A induced corticosterone production. To examine this relationship in more detail we measured the expression of the different subtypes of CRH and orexin receptors and their specific coupling to G protein alpha-subunits upon activation with CRH and orexin A, respectively. Using RT-PCR and fluorescent in situ hybridization analysis, we demonstrated the presence of CRH receptors 1alpha and 2alpha, and orexin type 2 receptor mRNA. None of the other CRH receptor variants or orexin type 1 receptor were detected. Immunofluorescent analysis and Western blotting confirmed the protein expression of both receptors, which also bind fluo-CRH and fluo-orexin with high affinity. Immunoblotting analysis confirmed the expression of prepro-orexin and orexin A in fetal adrenals. Using photoaffinity labeling, we determined which G proteins are coupled to the CRH and orexin receptors in fetal adrenals when challenged with CRH or orexin. Treatment of fetal adrenal membranes with CRH (100 nM) increased the labeling of G(o) and, to a lesser extent, G(s), but not G(i) and G(q), whereas treatment with orexin A (100 nM) increased the labeling of G(s) and G(i), but not G(o) and G(q). These findings provide new insights into the components of the signal transduction machinery in human fetal adrenals and demonstrate for the first time the presence of functional orexin receptors outside of the CNS in humans.

Adrenal Glands↗
PubMed2002

Moleular models for cholecystokinin-A receptor.

Numerous techniques have been used to elucidate the structural basis for interaction of cholecystokinin (CCK)-related peptides with their hormone-binding receptor, the CCK-A receptor (CCK-AR), including structure-activity relationship studies, site-directed mutagenesis, photoaffinity-labeling, and solution NMR analysis of both CCK peptide ligands and peptide fragments derived from the CCK-A receptor. Different structural models have been developed for the peptide-receptor complexes using various subsets of the available experimental data (Giragossian & Mierke 2001; Ding et al. 2002; Escrieut et al. 2002). Here, we review details of the various models and evaluate the impact of selected experimental data sets on model development.

Models, Molecular↗
PubMed1998

Association of estramustine resistance in human prostatic carcinoma cells with modified patterns of tubulin expression.

Estramustine (EM) is an antimicrotubule drug used in the treatment of hormone refractory advanced prostate cancer. To investigate the mechanism of resistance to EM, we compared its effects on human prostate cancer cells (DU145) and an estramustine-resistant derived cell line (E4). Immunofluorescence demonstrated that EM caused depolymerization of microtubules and blocked cells in mitosis in DU145 cells, with less effect in E4 cells. Using tubulin isotype-specific antibodies, a threefold increase in betaIII and approximately twofold increase in betaI + II isotype in E4 cells compared to DU145 cells were observed. A most interesting observation concerned an increase in the posttranslational modification of alpha-tubulin of both polyglutamylation and acetylation in the E4 cells. Significant to this observation, using direct EM photoaffinity labeling of tubulin, drug binding to the most acidic posttranslationally modified forms of alpha-tubulin was shown to be minimal. Taken together, these results indicate that the modification of the tubulin expression pattern may be responsible for estramustine resistance by both lowering the amount of drug bound to microtubules and inducing more stable microtubules.

Animals↗
PubMed2003

Is the V3 loop involved in HIV binding to CD4?

The entry of the human immunodeficiency virus into cells requires the interaction of the viral envelope glycoprotein gp120 with CD4 and a chemokine receptor. The gp120 binding site has been previously mapped to the Ig-CDR2-like region of CD4 first domain. A second area of this domain (Ig-CDR3-like region) is involved in gp120-CD4 interactions, but its gp120 counterpart remained so far unknown. Using a photoaffinity labeling experiment, we demonstrate that a peptide, mapping the (307-330)m region of HIV-MN-gp120 V3 loop, binds a sequence including a part of the Ig-CDR3-like region. These results may contribute to explain the complex mechanism of human immunodeficiency virus penetration, helping the development of new therapeutic agents.

Amino Acid Sequence↗

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