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PubMed1995

Functional significance of interaction of H-FABP with sulfated and nonsulfated taurine-conjugated bile salts in rat liver.

To identify the cytosolic proteins of rat hepatocytes involved in transcellular transport of sulfated and taurine-conjugated bile salts in comparison with only taurine-conjugated bile salts, photoaffinity labeling studies were performed with [3H]-7,7-ASLCT (7,7-azi-3 alpha-sulfatolithocholyl[2'-3H(N)]taurine), [3H]-7,7-ACT ((7,7-azi-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)- 2'-[2'-3H(N)]aminoethanesulfonate), and [3H]-7,7-ALCT (7,7-azilithocholyl[2'-3H(N)]taurine) using isolated hepatocytes and intact liver tissue. Photoaffinity labeling of isolated hepatocytes with [3H]-7,7-ASLCT on the one hand and with [3H]-7,7-ACT and [3H]-7,7-ALCT on the other resulted in a different labeling pattern of cytosolic polypeptides, without a relevant incorporation of radioactivity into subunits of glutathione transferases. This suggests that glutathione transferases play no role in the transport of dianionic or of monoanionic bile salts. With [3H]-7,7-ACT and [3H]-7,7-ALCT a moderate incorporation of radioactivity was found in polypeptides with apparent M(r)s of 33,000, 38,000, and 54,000, whereas with [3H]-7,7-ASLCT, a polypeptide with an apparent M(r) of 14,000, identified as H-FABP, was markedly and almost exclusively labeled. Photoaffinity labeling of specimens of intact liver tissue resulted in a labeling pattern of cytosolic polypeptides comparable to that obtained from photolabeled isolated hepatocytes. All results suggest that transcellular transport of dianionic sulfated as well as taurine-conjugated bile salts and of monoanionic taurine-conjugated bile salts follows different pathways. In intracellular transport of taurine-conjugated bile salts, several cytosolic polypeptides may have a function, whereas, in transport of taurine-conjugated 3 alpha-sulfato bile salts, only H-FABP appears to be involved.

Affinity Labels↗
PubMedDate not supplied

Study of the interaction between actin and antitumor substance aplyronine A with a novel fluorescent photoaffinity probe.

The interaction between actin and aplyronine A, a potent antitumor and actin-depolymerizing substance of marine origin, was investigated by photoaffinity labeling experiments. Photoaffinity probes consisting of a side-chain portion of aplyronine A as a ligand, a diazirine moiety as a photoaffinity group, and a fluorophore as a detecting group were synthesized. Photolabeling experiments between actin and the probe were carried out. Actin was successfully photolabeled by the fluorescent probe and visualized clearly. The present results provide the first chemical evidence for the direct interaction between actin and the side-chain portion of aplyronine A.

Actins↗
PubMed1987

The surface cyclic AMP receptor in Dictyostelium. Levels of ligand-induced phosphorylation, solubilization, identification of primary transcript, and developmental regulation of expression.

A monospecific polyclonal antiserum to the surface cAMP receptor of Dictyostelium has been developed by immunization with purified receptor immobilized on particles of polyacrylamide and on nitrocellulose paper. In Western blots, the antiserum displays high affinity and specificity for both the R (Mr 40,000) and D (Mr 43,000) forms of the receptor previously identified by photoaffinity labeling with 8-azido-[32P] cAMP. These bands, labeled with the photoaffinity label or with 32 Pi, were quantitatively and specifically immunoprecipitated, supporting co-purification data that all represent the same polypeptide. The R form, found in unstimulated cells, contained at least 0.2 mol of phosphate/mol of receptor. The D form, generated by cAMP stimulation of intact cells, contained at least 4 mol of phosphate/mol of receptor. In the absence of detergents, the receptor was exclusively located on membranes. The receptor was solubilized effectively in Triton X-100 and sedimented as a broad peak of 5-7 S on sucrose velocity gradients. Western blots of membranes isolated at different times after starvation indicate that the appearance of cell surface cAMP binding sites during the aggregation stage of development (5-6 h) is due to de novo synthesis of receptor protein. Pulse labeling with [35S]methionine indicated that the receptor is most rapidly synthesized during the preaggregation stage of development (1-3 h), prior to its maximal accumulation in membranes. The serum specifically immunoprecipitates a polypeptide of Mr 37,000 from an in vitro translation reaction using RNA isolated from preaggregation stage cells. The time course of expression of the mRNA coding for the Mr 37,000 polypeptide parallels the rate of receptor synthesis in vivo.

Affinity Labels↗
PubMed1991

Peptide subunits of gamma-aminobutyric acidA/benzodiazepine receptors from bovine cerebral cortex.

The gamma-aminobutyric acidA/benzodiazepine receptor complexes from bovine cerebral cortex were purified by immunoaffinity chromatography, and the main component peptide subunits were characterized. The peptide band originally thought to be a single beta subunit [57,000 Mr band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] is composed of at least four different peptides of 54,000-57,000 Mr. Two peptides of 55,000 and 57,000 Mr were recognized by the beta subunit-specific monoclonal antibody 62-3G1. Peptides in the range of 54,000-57,000 Mr were photoaffinity-labeled with [3H]muscimol. A different 57,000 Mr peptide was photoaffinity-labeled by [3H]flunitrazepam, but neither was recognized by the monoclonal antibody 62-3G1 nor photoaffinity-labeled with [3H]muscimol. Some peptides could be identified by their differential mobility shift in SDS-PAGE after treatment with endoglycosidase H. Two additional subunit peptides of 51,000 and 53,000 Mr were also photoaffinity-labeled by [3H]flunitrazepam and reacted with antiserum A. However, the 57,000 Mr peptide that also was photoaffinity-labeled by [3H]flunitrazepam did not react with antiserum A.

Affinity Labels↗
PubMed1985

Mechanism of interferon action. The interferon-induced phosphoprotein P1 possesses a double-stranded RNA-dependent ATP-binding site.

Protein P1, the interferon-induced protein phosphorylated in the presence of dsRNA in human amnion U-cells, was covalently labeled with [alpha-32P]ATP following ultraviolet irradiation. The photoaffinity labeling of protein P1 was dependent upon double-stranded RNA. Antibody prepared against phosphorylated protein P1 immunoprecipitated the double-stranded RNA-dependent photoaffinity-labeled product. The extent of photoaffinity labeling was significantly decreased by the addition of unlabeled ATP, GTP, or AMP; adenosine had little effect on the photoaffinity labeling of protein P1. These results suggest that protein P1 possesses a site capable of binding an adenine nucleotide in a double-stranded RNA-dependent manner.

Adenosine Triphosphate↗
PubMed2000

Localization of the 1,4-dihydropyridine drug acceptor of P-glycoprotein to a cytoplasmic domain using a permanently charged derivative N-methyl dexniguldipine.

INTRODUCTION: P-glycoprotein (P-gp) is a 170 kDa ATPase which can transport a wide range of natural product cytotoxic drugs out of cells, thus conferring the multidrug resistance (MDR) phenotype. METHODS: In this paper we used the 1,4-dihydropyridine (1,4-DHP) MDR-reversing agent dexniguldipine (DN), and a derivative with a quaternary nitrogen which is permanently charged, N-methyl-DN, to explore the sidedness of block of [3H]-vinblastine transport by P-gp. RESULTS: In cytotoxicity assays, 1 microM DN sensitized MCF7 ADR cells, causing a 13-fold decrease in the EC50 of vinblastine from 400 +/- 80 nM to 30 +/- 25 nM. In marked contrast, N-methyl-DN was without effect. In intact MCF7 ADR cells, DN reversed the [3H]vinblastine uptake deficit with an EC50 of 445 +/- 100 nM, again, N-methyl-DN was inactive. In photoaffinity labelling studies using the arylazide [3H]-B9209-005 in whole cells, DN potently inhibited incorporation of the photoaffinity label into P-gp whilst N-methyl-DN was without effect. However, in photoaffinity labelling studies in membrane fragments, both DN and N-methyl-DN potently inhibited [3H]-B9209-005 photoaffinity labelling of P-gp. Furthermore, in membrane fragments [3H]-vinblastine binding to P-glycoprotein was potently inhibited by both N-methyl-DN (Ki 10.7 +/- 4.9 nM) and DN (Ki 11.2 +/- 3.8 nM), and both N-methyl-DN and DN blocked ATP-dependent [3H]-vinblastine transport into inside-out vesicles. Thus, in intact cells the permanently charged 1,4-dihydropyridine, N-methyl-DN is unable to reverse the MDR phenotype or photoaffinity labelling of P-gp. However, in cell fragments and inside-out vesicles, N-methyl-DN binds avidly to P-gp and this binding blocks [3H]-vinblastine transport. CONCLUSION: These data are consistent with the hypothesis that 1,4-DHPs block [3H]-vinblastine binding, and thereby transport by P-gp, by acting at a domain accessible only from the cytoplasm.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Characterization of rhodamine 123 binding to P-glycoprotein in human multidrug-resistant cells.

The overexpression of P-glycoprotein is currently believed to be responsible for the enhanced efflux or decreased influx of cytotoxic drugs across the cell membrane in drug-resistant cells. P-glycoprotein has been proposed to mediate the efflux of a large number of structurally and functionally unrelated drugs. Although it has been suggested that P-glycoprotein binds directly to many lipophilic cations, it remains unclear whether one or more sites in P-glycoprotein mediate its broad substrate specificity. In this report, a photoactive derivative of rhodamine 123 (Rh123) [125I-azidosalicylic acid (ASA)-Rh123] was synthesized and used in a photoaffinity labeling assay to demonstrate, for the first time, direct and specific binding to P-glycoprotein. The photoaffinity labeling of P-glycoprotein by ASA-Rh123 was specifically inhibited in the presence of vinblastine and verapamil but not in the presence of colchicine. Surprisingly, ASA-Rh123 photoaffinity labeled a 6-kDa V8 peptide in P-glycoprotein that was previously shown to be photoaffinity labeled by another multidrug resistance-associated drug, [125I]iodoarylazidoprazosin. Photoaffinity labeling of mitochondria from drug-sensitive or -resistant cells with 125I-ASA-Rh123 did not reveal significant differences in the mitochondrial proteins from sensitive or resistant cells. Interestingly, however, 125I-ASA-Rh123 did photolabel a 66-kDa protein in mitochondria that was not detected in plasma membrane preparations with this assay. Taken together, our results demonstrate for the first time that Rh123 binds specifically to P-glycoprotein and that its binding site may be shared by other multidrug resistance-associated drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

[Analysis of GTP-binding protein function with a photoaffinity GTP analog].

The mechanism whereby hormones or neurotransmitters activate G proteins and their intracellular effectors can be studied in reconstituted systems using purified components. However, the regulation of receptor-G protein signaling appears to be substantially more complex in the cell and several additional components participate in this event. To study the relationship among G proteins receptors, and effectors in complex systems, such as membranes of permeable cells, it is necessary to employ methods that selectively allow the examination of G protein activation. One such method is photoaffinity labeling using a hydrolysis-resistant, photoaffinity GTP analog, P3 (4-azidoanilido)-P1-guanosine 5'-triphosphate (AAGTP). Here we describe the synthesis and purification of [32P]AAGTP as well as a procedure suitable studying the G protein function in membrane preparations. Photoaffinity labeling in rat cerebral cortex membranes showed that at least four G proteins (GsH, GsL, Gi, and Go) were labeled by [32P]AAGTP. [32P]AAGTP labeling on Gs and Gi was altered in concert with the activation states of those G proteins. An agonist-specific increase in [32P]AAGTP labeling of the G protein alpha-subunit in a membrane preparation has also been demonstrated. Thus, the photoaffinity labeling method with [32P]AAGTP makes it possible to investigate the behavior of individual G proteins in complex systems such as membrane preparations.

Affinity Labels↗
PubMed1988

cAMP-binding proteins in medullary tubules from rat kidney: effect of ADH.

Little is known of the regulatory steps in the cellular action of vasopressin (AVP) on the renal epithelium, subsequent to the cAMP generation. We studied cAMP-binding proteins in the medullary collecting tubule (MCT) and the thick ascending limb of Henle's loop (MTAL) microdissected from the rat kidney by use of photoaffinity labeling. Microdissected tubules were homogenized and photoaffinity labeled by incubation with 1 microM 32P-labeled 8-azido-adenosine 3',5'-cyclic monophosphate (N3-8-[32P]-cAMP); the incorporated 32P was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Both in MCT and MTAL preparations, the analyses showed incorporation of N3-8-[32P]cAMP into two bands (Mr = 49,000 and Mr = 55,000) that comigrated with standards of the cAMP-dependent protein kinase regulatory subunits RI and RII. In MCT, most of the 32P (80%) was incorporated into RI, whereas in MTAL the 32P incorporated into RI and RII was equivalent. When freshly dissected MCT segments were incubated with 10(-12)-10(-6) M AVP, the subsequent photoaffinity labeling of RI with N3-8-[32P]cAMP was markedly diminished in a dose-dependent manner compared with controls. Our results suggest that cAMP binds in MCT and MTAL to regulatory subunits RI and RII of cAMP-dependent protein kinase. However, in MCT the dominant type of cAMP-dependent protein kinase appears to be type I. The outlined procedure is suitable to indirectly measure the occupancy of RI by endogenous cAMP generated in MCT cells in response to physiological levels (10(-12) M) of AVP.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1990

Identification and structural characterization of alpha 1-adrenergic receptor subtypes.

Rat liver and brain membrane alpha 1-adrenergic receptors were purified greater than 500-fold by successive chromatographic steps using heparin-agarose, an affinity matrix constructed by coupling a novel derivative of the alpha 1-selective antagonist prazosin to Affigel-102 and wheat germ agglutinin-agarose. Several lines of evidence were obtained for the existence in brain of an alpha 1-adrenergic receptor subtype that is structurally distinct from that previously characterized in liver and other tissues using photoaffinity labeling, protein purification, and DNA cloning techniques. The alpha 1-selective ligand chlorethylclonidine (CEC) (an alkylating agent) irreversibly inactivates 100% of [3H]prazosin binding sites in partially purified preparations of rat liver. Under identical conditions, only 50% of brain receptors are irreversibly inactivated. Computer modeling of data obtained from the competition by the alpha antagonists WB4101 and phentolamine for [3H]prazosin binding to partially purified preparations of rat liver is best fit by assuming a single class of low affinity sites for both ligands. However, analysis of partially purified brain preparations indicates the presence of two binding sites with different affinities for these antagonists. Additionally, prior alkylation of brain receptors with CEC results in the loss of low affinity phentolamine and WB4101 binding sites. The CEC-insensitive site in brain, which displays high affinity for phentolamine and WB4101, is resistant to photoaffinity labeling by [125I]azidoprazosin. This is not due to a markedly lower affinity of the CEC-insensitive sites for the photoaffinity label, because competition studies with [127I]azidoprazosin revealed a single class of high affinity sites in partially purified brain samples. Photoaffinity labeling of partially purified liver and brain samples not treated with CEC results in the specific labeling of a single protein of Mr 80,000. No specifically labeled protein is observed for partially purified brain samples that had previously been incubated with CEC. Treatment of photoaffinity-labeled liver and brain receptors with N-glycanase to cleave N-linked oligosaccharides results in a single Mr 55,000 protein. Taken together, these data provide evidence for the existence of a single receptor subtype (alpha 1b) in rat liver and for two subtypes (alpha 1a and alpha 1b) in rat brain. Furthermore, the insensitivity of the alpha 1a subtype to CEC and the resistance of the alpha 1a subtype to covalent labeling by an alpha 1b-selective photoaffinity probe suggest that the primary structures of the two receptor subtypes differ, such that an amino acid(s) in the alpha 1b subtype that incorporates CEC and the photoaffinity label is lacking in the alpha 1a subtype.

Affinity Labels↗
PubMed1999

NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron-sulfur cluster N2 to quinone.

The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron-sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[3H]pyridaben ([3H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron-sulfur cluster N2 to quinone.

Azirines↗
PubMed1988

Dopamine D2 receptor binding subunits of Mr congruent to 140,000 and 94,000 in brain: deglycosylation yields a common unit of Mr congruent to 44,000.

The ligand-binding subunit of the porcine striatal dopamine D2 receptor was identified by photoaffinity labeling with [125I]N-azidophenethylspiperone ([125I]NAPS). Upon photolysis, [125I]NAPS covalently incorporated into a broad band of apparent Mr congruent 140,000 with an appropriate pharmacological profile for D2 receptors as assessed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Smaller subunits of apparent Mr congruent 94,000 and 34,000 were specifically labeled by [125I]NAPS with an appropriate D2 receptor profile and were similar to the major ligand-binding subunits of photoaffinity-labeled canine striatal D2 receptors. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of the Mr congruent to 140,000/94,000 subunits upon denaturing electrophoresis in either the absence or presence of thiol-reducing/alkylating reagents. In order to investigate the possible basis for the existence of these high molecular weight forms of the D2 receptor, we assessed the carbohydrate nature of photolabeled D2 ligand-binding subunits by the use of lectin affinity chromatography and specific exo- and endoglycosidase treatments. Both photoaffinity-labeled D2 receptor proteins from porcine striatum (Mr congruent to 140,000 and 94,000) were glycoproteins as indexed by their absorption and specific elution from wheat germ agglutinin lectin resins. The exoglycosidase neuraminidase altered the electrophoretic mobility of both the Mr congruent to 140,000 and 94,000 labeled subunits to a single band of apparent Mr congruent to 51,000. Prior removal of sialic acid residues did not alter the reversible binding characteristics of [3H]spiperone to D2 receptors. Complete removal of receptor-associated N-linked carbohydrate by the endoglycosidase glycopeptidase F (peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase) produced a further increase in the mobility of the Mr congruent to 51,000 subunit to apparent Mr congruent to 44,000. The porcine Mr congruent to 34,000 photolabeled peptide is an N-linked glycoprotein as assessed by lectin affinity chromatography and susceptibility to digestion by glycopeptidase F to a peptide of apparent Mr congruent to 23,000.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity Labels↗
PubMed1990

Defined geometry of binding between triantennary glycopeptide and the asialoglycoprotein receptor of rat heptocytes.

Three derivatives of a triantennary glycopeptide, each containing a single uniquely located 6-amino-galactose residue at either position 6', 6, or 8, were modified at the 6-amino group by attachment of a photolyzable reagent and radiolabeled by iodination of tyrosine. These were allowed to bind to the asialoglycoprotein receptor of isolated rat hepatocytes and photolyzed for affinity labeling. (formula; see text) Each probe specifically labeled either the major (RHL1) or minor (RHL2/3) subunits which comprise the receptor. A photolyzable group attached to galactose residue 6 6' specifically radiolabeled RHL1, whereas a photolyzable group attached to galactose 8 specifically labeled RHL2/3. Photoaffinity labeling of a soluble rat hepatic lectin preparation demonstrated that the minor subunits (RHL2/3) were no longer labeled by the triantennary probe with a photolyzable group at galactose 8. The inhibitory potency of a variety of complex glycopeptides against radiolabeled ligand binding to both rat hepatocytes and soluble lectin are in agreement with photoaffinity results that galactose 8 of triantennary glycopeptide is of unique importance by binding solely to the minor subunits (RHL2/3) of the asialoglycoprotein receptor on hepatocytes. Conversely, galactose residues 6 and 6' bind specifically to the major subunit (RHL1), indicating a precise binding geometry between the trivalent ligand and lectin.

Affinity Labels↗
PubMed2002

ATP binding/hydrolysis by and phosphorylation of peroxisomal ATP-binding cassette proteins PMP70 (ABCD3) and adrenoleukodystrophy protein (ABCD1).

The 70-kDa peroxisomal membrane protein (PMP70) and adrenoleukodystrophy protein (ALDP), half-size ATP-binding cassette transporters, are involved in metabolic transport of long and very long chain fatty acids into peroxisomes. We examined the interaction of peroxisomal ATP-binding cassette transporters with ATP using rat liver peroxisomes. PMP70 was photoaffinity-labeled at similar efficiencies with 8-azido-[alpha-32P]ATP and 8-azido-[gamma-32P]ATP when peroxisomes were incubated with these nucleotides at 37 degrees C in the absence Mg2+ and exposed to UV light without removing unbound nucleotides. The photoaffinity-labeled PMP70 and ALDP were co-immunoprecipitated together with other peroxisomal proteins, which also showed tight ATP binding properties. Addition of Mg2+ reduced the photoaffinity labeling of PMP70 with 8-azido-[gamma-32P]ATP by 70%, whereas it reduced photoaffinity labeling with 8-azido-[alpha-32P]ATP by only 20%. However, two-thirds of nucleotide (probably ADP) was dissociated during removal of unbound nucleotides. These results suggest that ATP binds to PMP70 tightly in the absence of Mg2+, the bound ATP is hydrolyzed to ADP in the presence of Mg2+, and the produced ADP is dissociated from PMP70, which allows ATP hydrolysis turnover. Properties of photoaffinity labeling of ALDP were essentially similar to those of PMP70. Vanadate-induced nucleotide trapping in PMP70 and ALDP was not observed. PMP70 and ALDP were also phosphorylated at a tyrosine residue(s). ATP binding/hydrolysis by and phosphorylation of PMP70 and ALDP are involved in the regulation of fatty acid transport into peroxisomes.

ATP Binding Cassette Transporter, Subfamily D, Mem↗
PubMed1989

N-linked oligosaccharides are responsible for rat striatal dopamine D2 receptor heterogeneity.

The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-(p-azido-m-[125I]iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass (Mr). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 Mr; after digestion with peptide-N4-[N-acetyl-beta-glucosaminyl] asparagine amidase, all fractions yielded a single peptide approximately 40,000 Mr. Treatment of photoaffinity labeled membranes with alpha-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000-54,000 Mr. Treatment of the receptor with neuraminidase followed by endo-alpha-N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of approximately 31,000 Mr is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide. Complete removal of N-linked oligosaccharide from the dopamine D2 receptor did not change the rank order potency of agonist and antagonist compounds to compete for [3H]spiperone binding to crude membrane fractions. The dopamine D2 receptor represents a highly glycosylated neural receptor.

Affinity Labels↗
PubMedDate not supplied

Solubilization and partial purification of S-adenosyl-L-homocysteine binding proteins from the rat brain.

Rat brain proteins presenting high-affinity binding of S-adenosyl-L-homocysteine were solubilized and purified. Extraction of binding protein was carried out in the presence of Triton X-100 and 1 M NaCl; this solubilized fraction exhibits similar kinetic properties than the membrane proteins. Purification was performed using affinity chromatography on S-adenosyl-L-homocysteine carboxyhexyl Sepharose 48 conjugate. The analysis of the affinity gel eluate by SDS-PAGE showed high purification ratios for two proteins exhibiting 54 and 68 kDa. Three activity peaks were separated when solubilized membrane proteins were submitted to isoelectric focusing; the activity peaks corresponded to proteins of pH1 6.0, 6.5, and 7.2. SDS-PAGE separation of proteins contained in each peak showed protein aggregation; a 54-kDa subunit was present in each aggregate. Solubilized membrane proteins were labeled by photoaffinity labeling with tritiated S-adenosyl-L-homocysteine; the 54- and 68-kDa proteins were found among the specifically labeled proteins. Finally, according to the previous data from the literature, the purified S-adenosyl-L-homocysteine binding proteins do not seem to be the same as adenosine receptors or phosphatidylethanolamine-N-methyltransferase.

Affinity Labels↗
PubMed2001

A binary system of photoreagents for high-efficiency labeling of DNA polymerases.

To increase the efficiency of photoaffinity labeling of DNA polymerases, a binary system of photoaffinity reagents was applied. Photoreactive radioactive primers were synthesized by DNA polymerases beta (pol beta) or DNA polymerase from Thermus thermophilus (pol Tte) using a template-primer duplex in the presence of a dTTP analogue containing 4-azidotetrafluorobenzoyl group linked via spacers of varying length to 5-position of uridine ring- 5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-4-dUTP) or 5-[N-[[(2,3,5,6-tetrafluoro-4-azidobenzoyl)-butanoyl]-amino]-trans-3-aminopropenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-9-dUTP). The reaction mixtures were UV irradiated (lambda = 365-450 nm) in the absence or presence of a dTTP analog, containing a pyrene moiety-5-[N-(4-(1-pyrenyl)-butylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr- 8-dUTP) or 5-[N-(4-(1-pyrenyl)-ethylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr-6-dUTP). The most efficient crosslinking of both DNA polymerases was observed in the case of photoreactive DNA primer, carrying the FAB-4-dUMP moiety at the 3'-end, and Pyr-6-dUTP as a sensitizer. The binary system of photoaffinity reagents allows increasing photoaffinity labeling of the both DNA polymerases in comparison to the primer crosslinking without photosensitizer.

DNA Primers↗
PubMed1990

Identity of the calmodulin-binding proteins in bovine lens plasma membranes.

Two bovine lens plasma membrane proteins have been identified that bind calmodulin in a Ca2(+)-dependent fashion. Electrophoretic analysis of lens membrane proteins photoaffinity-labeled with benzophenone[125I]calmodulin confirmed our previous observation [Louis, Johnson and Turnquist (1985) Eur. J. Biochem. 150, 271-8] that two major products are formed with Mr = 46 kDa and 36 kDa. Limited proteolysis of lens membrane proteins with chymotrypsin resulted in the formation of a 21-kDa-fragment that was derived from the hydrolysis of the major lens membrane protein MP26; this correlated with the loss of the 46-kDa complex, and the formation of a 41-kDa photoaffinity-labeled complex. Use of the [125I]calmodulin gel overlay procedure confirmed that the 46-kDa and 41-kDa photoaffinity-labeled complexes reflect the interaction of calmodulin with both MP26 and its 21-kDa chymotryptic fragment, respectively. Proteolysis of lens membranes with higher concentrations of chymotrypsin resulted in the hydrolysis of the 18-kDa protein which correlated with the generation of a 12-kDa fragment; this paralleled the loss of the 36-kDa photoaffinity labeled complex and the formation of a 28-kDa-complex. The [125I]calmodulin gel overlay procedure demonstrated that the 36-kDa and 28-kDa photoaffinity-labeled complexes reflect the interaction of calmodulin with the 18-kDa lens membrane protein and its likely 12-kDa chymotryptic fragment, respectively. Identification of these calmodulin-binding proteins suggests that MP26 and the 18-kDa membrane protein are likely candidates for the proposed calcium sensitive, calmodulin-dependent gating of lens fiber cell junctions.

Affinity Labels↗