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PubMed1992

Photoaffinity labeling of the organic cation/H+ exchanger in renal brush border membrane vesicles.

The brush border membrane of the proximal tubule contains two efflux pathways for organic cations from the cell to the tubular fluid: a P-glycoprotein and an organic cation/H+ exchanger. There is evidence that they transport many of the same substrates. Their structural relatedness is unknown and is the subject of this report. The experimental approach was to identify the exchanger with photoaffinity labeling reagents. The rationale was that if the P-glycoprotein and the organic cation/H+ exchanger transport many of the same substrates, then they might be photoaffinity labeled by the same reagents. [125I]Iodoarylazidoprazosin and [3H]azidopine are two reagents, which have been used, to photoaffinity label the P-glycoprotein. We found that several polypeptides were photolabeled in a time- and concentration-dependent manner. The photoincorporation into only two of these polypeptides (41 and 28 kDa) was blocked extensively by the presence of known substrates for the exchanger. The photoaffinity labeling of only the 41-kDa polypeptide was affected by treatment with the chemical reagents, N-ethylmaleimide and dithiothreitol, which are known to affect the exchanger reaction. The findings are consistent with the interpretation that a 41-kDa polypeptide is, or is a component of, the exchanger.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

The gamma 2 subunit is an integral component of the gamma-aminobutyric acidA receptor but the alpha 1 polypeptide is the principal site of the agonist benzodiazepine photoaffinity labeling reaction.

Polyclonal antibodies were raised to a synthetic peptide whose amino acid sequence was derived from the novel gamma-aminobutyric acidA (GABAA) receptor subunit, gamma 2. These anti-gamma 2 1-15 Cys antibodies reacted specifically with the GABAA receptor purified from adult bovine cerebral cortex in an enzyme-linked immunosorbent assay. Anti-gamma 2 1-15 Cys antibodies specifically immunoprecipitated [3H]flunitrazepam photoaffinity-labeled native receptor in parallel with anti-alpha 1 324-341 antibodies. Immunoprecipitation of sodium dodecyl sulphate (SDS) denatured photoaffinity-labeled receptor by anti-gamma 2 1-15 Cys antibodies, however, resulted in a significant decrease in the maximum percentage of radioactivity immunoprecipitated compared to that by anti-alpha 1 324-341 antibodies. In immunoblots, anti-gamma 2 1-15 Cys antibodies reacted with a broad band in the molecular weight range Mr 43,000-49,000 which was distinct from that recognized by anti-alpha 1 324-341 antibodies. The anti-alpha 1 324-341 immunoreactive band was the main subunit irreversibly photoaffinity labeled by [3H]flunitrazepam, i.e. Mr 53,000. These results demonstrate for the first time that the gamma 2 subunit is an integral component of the GABAA receptor but it is the alpha 1 subunit that is the principal site of the agonist benzodiazepine photoaffinity labeling reaction. It supports a role of both the alpha 1 and gamma 2 polypeptides in the formation of the central benzodiazepine binding site within a GABAA receptor oligomer.

Affinity Labels↗
PubMed1983

Photoaffinity labeling: theory and practice.

The potential for generating a reactive reagent in situ by photoexcitation and for labeling sites not containing reactive nucleophilic residues provides photoaffinity labeling with a special advantage in the study of ligand-binding sites. There are, however, problems in the application of the photoaffinity technique. A number of concerns about this approach are examined by using as a background studies with arylazido-beta-alanyl nucleotide probes developed in this laboratory. It is concluded that photoaffinity labeling is likely to emerge as one of the major techniques for elucidating structure-function relationships within biological membranes. In the future the design of photoprobes with shorter activation half-lives may make it possible to investigate time-dependent (i.e., conformational) changes occurring at the active site of enzymes and within complex ligand-binding sites.

Adenosine Triphosphate↗
PubMed1995

Photoaffinity labeling of retinoic acid-binding proteins.

Retinoid-binding proteins are essential mediators of vitamin A function in vertebrate organisms. They solubilize and stabilize retinoids, and they direct the intercellular and intracellular trafficking, transport, and metabolic function of vitamin A compounds in vision and in growth and development. Although many soluble retinoid-binding proteins and receptors have been purified and extensively characterized, relatively few membrane-associated enzymes and other proteins that interact with retinoids have been isolated and studied, due primarily to their inherent instabilities during purification. In an effort to identify and purify previously uncharacterized retinoid-binding proteins, it is shown that radioactively labeled all-trans-retinoic acid can be used as a photoaffinity labeling reagent to specifically tag two known retinoic acid-binding proteins, cellular retinoic acid-binding protein and albumin, in complex mixtures of cytosolic proteins. Additionally, a number of other soluble and membrane-associated proteins that bind all-trans-[11,12-3H]retinoic acid with high specificity are labeled utilizing the same photoaffinity techniques. Most of these labeled proteins have molecular weights that do not correspond to any known retinoid-binding proteins. Thus, photoaffinity labeling with all-trans-retinoic acid and related photoactivatable retinoids is a method that should prove extremely useful in the identification and purification of novel soluble and membrane-associated retinoid-binding proteins from ocular and nonocular tissues.

Affinity Labels↗
PubMed1983

Photoaffinity-labelling of the glycine receptor of rat spinal cord.

The irreversible incorporation upon ultraviolet illumination of the glycine receptor antagonist, [3H]strychnine, into synaptic membrane fractions of rat spinal cord has been investigated. The specificity of this photoaffinity-labelling reaction for the glycine receptor was demonstrated by the following results: (a) the Kd value (9.7 nM) of the glycine-displaceable irreversible incorporation of [3H]strychnine was similar to the previously reported Kd of [3H]strychnine binding to the glycine receptor; (b) pre-illumination of the membranes with unlabelled strychnine led to a corresponding reduction in the number, but not the affinity, of reversible glycine-displaceable [3H]strychnine binding sites; (c) the ultraviolet light-induced incorporation into the membranes of [3H]strychnine was inhibited by different glycine receptor agonists; other neurotransmitter substances had little or no effect. Also, [3H]strychnine alone was shown to be stable upon illumination with ultraviolet light; this suggests that photocrosslinking of [3H]strychnine may require energy transfer from specific groups of its high-affinity receptor binding site. Upon sodium dodecyl sulphate/polyacrylamide gel electrophoresis a single labelled polypeptide with a relative molecular mass of 48000 was revealed from spinal cord membranes photoaffinity-labelled with [3H]strychnine. Spinal cord membranes photoaffinity-labelled with the gamma-aminobutyric acid receptor ligand [3H]flunitrazepam, however, gave a single polypeptide with a relative molecular mass of 5- 0000. Treatment of membranes, labelled with [3H]strychnine, by endoglycosidase H did not alter the relative molecular mass of the 48000-Mr labelled polypeptide. Trypsin treatment, on the other hand, successively produced major fragments of relative molecular masses of 42000 and 37000. Also, even after extensive treatment with trypsin or chymotrypsin, greater than or equal to 90% of the radioactivity incorporated into the labelled membranes remained membrane-associated. It is concluded that the strychnine binding site of the glycine receptor is located on a protease-inaccessible, i.e. probably hydrophobic domain of the 48000-Mr subunit.

Affinity Labels↗
PubMed1985

Photoaffinity labelling of androgen receptors with 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one.

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in calf uterus and rat prostate. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of 98 kD. In rat prostate cytosol an androgen receptor with a molecular mass of 46 kD could be photoaffinity labelled with R1881. The photoaffinity labelling procedure described here provides a method for studying the hormone binding domain of androgen receptors in partial purified preparations.

Affinity Labels↗
PubMed1986

Photoaffinity labeling of the gonadotropin receptor with native, asialo, and deglycosylated choriogonadotropin.

Human choriogonadotropin (hCG) is a heterodimeric hormone composed of an alpha and a beta subunit. hCG and its asialo (ashCG) and deglycosylated (dghCG) forms vary in their ability to stimulate hormone responsive adenylate cyclase. ashCG is a partial agonist, and dghCG is an antagonist. Photoactivatable moieties were coupled to hCG, ashCG, and dghCG, and the derivatives were radioiodinated. Competitive binding studies indicate that all of the derivatives had a similar affinity for the gonadotropin receptor on porcine granulosa cell membranes. Radiolabeled derivatives were used to photoaffinity label the gonadotropin receptor. Radiolabeled complexes were separated by NaDodSO4/PAGE. All of the derivatives produced similar autoradiographic patterns, except that dghCG produced an additional 48-kDa complex. To investigate the structure of the complexes further, peptide mapping of proteolytic digests was used. All, except for the 48-kDa complex, generated similar peptide maps indicating a relationship between those complexes in which the smaller components are part of the larger. The 48-kDa complex contained both subunits of 40-kDa dghCG. Therefore, this complex is expected to contain an additional component of 8 kDa. The complex was generated whether the hormone-receptor complex was photoaffinity labeled on cells, on isolated membranes, or after solubilizing in detergent. Formation was blocked by excess hCG and did not occur in the absence of UV irradiation. We conclude that the hCG derivatives are able to photoaffinity label the hCG receptor but that the dghCG derivative can photoaffinity label an additional component that was not observed when derivatives of hCG or ashCG were used to label the receptor.

Adenylyl Cyclases↗
PubMed1992

Effects of GTP analogues and activation of endogenous protein kinases on photoaffinity labeling with [3H](+)PN200-110 of crude membranes from rat heart and brain.

The effects of GTP analogues and conditions in which various endogenous protein kinases were activated on photoaffinity labeling with [3H](+)PN200-110 (PN) of crude membranes from rat cardiac muscle and whole brain were investigated. Photoaffinity labeling with 20 nM [3H](+)PN of these crude membranes was decreased by 100 microM GTP-gamma-S, but not by 100 microM GTP or 100 microM GDP-beta-S. Similar results were obtained on the effects of GTP and its analogues on the specific binding of 20 nM [3H](+)PN to these crude membranes under the same conditions. Activation of endogenous protein kinases in these crude membranes did not influence the photoaffinity labeling with [3H](+)PN. These results suggested the binding sites, or DPH-sensitive, or L-type, calcium channels in curde membranes from rat cardiac muscle and whole brain are directly or indirectly modulated by endogenous GTP-binding protein, but not by various endogenous protein kinases in these crude membranes.

Affinity Labels↗
PubMed1993

Catechol O-methyltransferase pharmacogenetics: photoaffinity labelling and western blot analysis of human liver samples.

The level of catechol O-methyltransferase (COMT) activity and COMT thermal stability in human tissue are controlled by a common genetic polymorphism. We studied individual hepatic biopsy samples shown previously to have phenotypically high, low or intermediate COMT activities and thermal stabilities to test the hypothesis that the molecular mass (M(r)) and/or isoelectric point (pI) of the enzyme might differ in tissue from subjects with different presumed genotypes for the COMT genetic polymorphism. COMT was partially purified from each hepatic tissue sample by sequential ion exchange and gel filtration chromatography, and photoaffinity labelling was performed with [3H-methyl]-S-adenosyl-L-methionine ([3H-methyl]-Ado-Met), the methyl donor for the COMT enzymatic reaction. Two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) analysis of individual samples consistently showed the presence of three [3H-methyl]-Ado-Met photoaffinity labelled proteins with pI values of 5.4, 5.5 and 5.7, all three of which had M(r) values of approximately 27.1 kDa. The same pattern was observed in all samples irrespective of COMT phenotype. Western blot analysis of 2-D SDS-PAGE gels performed with rabbit polyclonal antibodies to partially purified human kidney COMT showed a pattern similar to that found during photoaffinity labelling. Once again, the same pattern was found in all samples irrespective of COMT phenotype. Therefore, neither photoaffinity labelling nor Western blot analysis revealed differences in either M(r) or pI of cytoplasmic COMT in hepatic tissue from subjects selected on the basis of different phenotypic expression of the COMT genetic polymorphism.

Affinity Labels↗
PubMed1985

8-Azidoflavins as photoaffinity labels for flavoproteins.

8-Azidoflavins have been synthesized and their potential as photoaffinity labels for flavoproteins has been explored. They are very photolabile, and in aqueous media they react with solvent to yield 8-aminoflavins and 8-hydroxlaminoflavins as the main products. They fulfill the criteria expected of a good photoaffinity label, since they bind stoichiometrically at the flavin-binding site of flavoproteins, thus minimizing problems of nonspecific labeling. Second, they absorb strongly in the visible, so that the reactive nitrene can be generated without short wavelength light, minimizing the possibility of light-induced damage of the protein. Third, in the absence of light, 8-N3-flavins are stable, permitting a study of their binding to apoproteins. 8-Azidoflavins have been bound to hen egg white riboflavin-binding protein, Megasphera elsdenii flavodoxin, yeast Old Yellow Enzyme, Aspergillus niger, glucose oxidase, and pig kidney D-amino acid oxidase, and the effect of exposure to visible light has been determined. Only small extents of covalent attachment of the flavin to the protein were found with flavodoxin, D-amino acid oxidase, and Old Yellow Enzyme; much more extensive labeling was obtained with glucose oxidase and riboflavin-binding protein. In addition to their photoreactivity, 8-azidoflavins have been found to be converted to 8-aminoflavins by reaction with sulfite or upon reduction. Similar reactions occur with 8-hydroxylamino-, 8-(O-methyl)hydroxylamino-, and 8-hydrazinoflavins, which serve as models for possible flavin-protein covalent linkages which could be formed in the photolabeling procedure. Some of the properties of these flavins, which were obtained by reaction of 8-F-flavin with the corresponding nucleophiles, are also described.

Affinity Labels↗
PubMed1988

Photoaffinity labeling of functional states of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor was subjected to photoaffinity labeling in different conformational and functional states. The photolabel used was the ion-channel blocker [3H]-TPMP+. A procedure is described for isolating labeled delta-polypeptide chains from the receptor complex by preparative SDS-polyacrylamide gel electrophoresis. The photolabel was localized in the primary structure of the delta-chain. The site of labeling was found to be identical when photoaffinity labeling was performed in the resting, desensitized, or antagonist state, respectively.

Affinity Labels↗
PubMed1986

Photoaffinity labeling of alpha 1-adrenergic receptors of rat heart.

The photoaffinity probe [125I]aryl azidoprazosin was used to examine structural aspects of rat left ventricular alpha 1-adrenergic receptor. Autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins from photoaffinity-labeled membranes revealed a specifically labeled protein of mass 77 kDa. Adrenergic drugs competed with the photoaffinity probe for binding to the receptor in a manner expected of an alpha 1-adrenergic antagonist. Because the autoradiographic pattern was unaltered by incubating labeled membranes in gel sample buffer containing high concentrations of reducing agents, the binding component of the cardiac alpha 1-adrenergic receptor appears to be a single polypeptide chain. The photoaffinity probe specifically labeled a single protein of approximately 68 kDa in membranes of cardiac myocytes prepared from rat left ventricles. The role played by sulfhydryls in receptor structure and function was also studied. Dithiothreitol (DTT) inhibited [3H]prazosin binding to left ventricular membranes and altered both the equilibrium dissociation constant and maximal number of [3H]prazosin-binding sites but not the ability of the guanine nucleotide guanyl-5'-yl imidodiphosphate to decrease agonist affinity for the receptors. When photoaffinity-labeled membranes were incubated with 40 mM DTT for 30 min at room temperature, two specifically labeled proteins of 77 and 68 kDa were identified. The DTT-induced conversion of the 77-kDa protein to 68 kDa was irreversible with washing, but the effect of DTT on [3H]prazosin binding was reversible. Both 77- and 68-kDa proteins were observed with liver membranes even in the absence of reducing agent. We suggest that the DTT-induced conversion of the 77-kDa protein to 68 kDa is due to enhancement in protease activity by the reductant. These results document that the cardiac alpha 1-adrenergic receptor is a 77-kDa protein, similar in mass to the receptor in liver and other sites. Proteolysis likely accounts for lower Mr forms of this receptor found in cardiac myocytes and in previous publications on hepatic alpha 1-receptors.

Affinity Labels↗
PubMed1986

Characterization of androgen receptors after photoaffinity labelling with [3H]methyltrienolone (R1881).

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in rat prostate, in a human transplantable prostatic adenocarcinoma (PC-82) and in calf uterus. Androgen receptors preparations were partially purified either via differential chromatography on 2',5'-ADP-Sepharose (rat prostate), via anion exchange fast protein liquid chromatography (rat prostate and PC-82) or via DNA-cellulose chromatography (calf uterus). Purification factors obtained with the three different methods were: 245, 75 and 40 respectively. Photolabelling of receptor preparations was performed via irradiation with a high pressure mercury lamp either before or after partial purification. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of approx 95 kD. The covalent attachment of [3H]R1881 to the 95 kD protein could be completely suppressed by a 200-fold molar excess of dihydrotestosterone. In rat prostate cytosol an androgen receptor with a molecular mass of approx 50 kD could be photoaffinity labelled with R1881. A similar size was found for the androgen receptor in the human prostatic adenocarcinoma. Our results show that photoaffinity labelling of androgen receptors with [3H]R1881 as ligand can be applied for characterization of partial purified androgen receptor preparations.

Affinity Labels↗
PubMed1989

S-(4-azidophenacyl)[35S]glutathione photoaffinity labeling of rat liver plasma membrane-associated proteins.

A method for the synthesis of the glutathione conjugate S-(4-azidophenacyl)[35S]glutathione is described. The compound was used for photoaffinity labeling of proteins present in canalicular membrane vesicles (CMV), sinusoidal membrane vesicles (SMV), mitochondria and microsomes from rat liver. Most of the radioactivity introduced by photoaffinity labeling of CMV appeared in the 25-29 kDa range. Further labeled proteins were observed in bands at 37, 105 and about 120 kDa. 79% of the 25-29 kDa associated radioactivity was recovered in the supernatant after extensive revesiculation (washing) of the vesicles, together with the 37 kDa protein. CMV and SMV contained glutathione S-transferase (GST) activity which in CMV was decreased by 75% by washing. Photolabeling of a mixture of purified basic GST subunits from rat liver resulted in a band pattern at 25-29 kDa similar to that in the membrane preparations. Isoelectric focusing of the CMV indicated the presence of basic soluble GST subunits. S-Hexylglutathione-Sepharose affinity chromatography showed reversible binding of photolabeled proteins at 25-29 kDa. Difference photoaffinity labeling with GSSG, S-hexylglutathione, taurocholate and phenylmethylsulfonyl fluoride decreased the radioactivity bound by GST, but not that introduced into the 105 kDa protein band present in CMV. It is concluded that membrane-associated basic GST isoenzymes are present in standard membrane vesicle preparations. In the cell, the function may be transport of GST-bound compounds across the membrane and protection of the membranes against electrophiles.

Affinity Labels↗
PubMed1984

Photoaffinity labeling of the Sarcoma 180 cell surface by daunomycin.

We have used photoaffinity labeling to investigate the distribution and function of daunomycin binding sites in Sarcoma 180 cells. When native daunomycin is irradiated at 366 or 488 nm in the presence of cells, the drug is irreversibly incorporated into cellular molecules. The cellular acceptor for the photoincorporation cannot be extracted by chloroform-methanol nor can it be degraded by DNase. However, the drug acceptor is susceptible to trypsin digestion. These results show that the photoincorporation site is composed of protein but not of lipid or DNA. Furthermore, the fact that photoincorporation proceeds equally well at 0 degrees (where drug does not accumulate inside the cells) as compared to 37 degrees (where free drug concentrates in the cells) suggests that the labeling reaction occurs principally at the cell surface. The photolabeling process is not highly specific since it is not saturable at high drug concentrations and cannot be competed for by unlabeled daunomycin. When 2 X 10(5) daunomycin molecules are incorporated per Sarcoma 180 cell, the cells can still accumulate free drug. This result suggests that the photolabeling reaction does not occur at the drug transport locus. Photoincorporation of daunomycin also does not affect the viability of Sarcoma 180 cells, as judged by a cloning assay. Thus, there is probably no surface receptor for the drug which mediates cytotoxicity when occupied. This result is as expected from previous work predicting that the mechanism of daunomycin involves disruption of some generalized membrane property like fluidity. However, in a series of Sarcoma 180 sublines selected for increasing resistance to daunomycin, the photoincorporation increases in direct proportion to drug sensitivity. Consequently, daunomycin appears to be capable of photoaffinity labeling a cell surface protein which, although not directly involved in the mechanism of cytotoxicity is implicated in the expression of drug resistance.

Animals↗
PubMed1987

Binding studies and photoaffinity labeling identify two classes of phencyclidine receptors in rat brain.

Binding and photoaffinity labeling experiments were employed in order to differentiate 1-(1-phenylcyclohexyl)piperidine (PCP) receptor sites in rat brain. Two classes of PCP receptors were characterized and localized: one class binds [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) with high affinity (Kd = 10-15 nM) and the other binds the ligand with a relatively low affinity (Kd = 80-100 nM). The two classes of sites have different patterns of distribution. Forebrain regions are characterized by high-affinity sites (hippocampus greater than frontal cortex greater than thalamus greater than olfactory bulb greater than hypothalamus), but some parts (e.g., hippocampus, hypothalamus) contain low-affinity sites as well. In the cerebellum only low-affinity sites were detected. Binding sites for [3H]PCP and for its photolabile analogue [3H]azido-PCP showed a regional distribution similar to that of the [3H]TCP sites. The neuroleptic drug haloperidol did not block binding to either the high- or the low-affinity [3H]TCP sites, whereas Ca2+ inhibited binding to both. Photoaffinity labeling of the PCP receptors with [3H]AZ-PCP indicated that five specifically labeled polypeptides of these receptors (Mr 90,000, 62,000, 49,000, 40,000, and 33,000) are unevenly distributed in the rat brain. Two of the stereoselectively labeled polypeptides (Mr 90,000 and 33,000) appear to be associated with the high- and low-affinity [3H]TCP-binding sites; the density of the Mr 90,000 polypeptide in various brain regions correlates well with the localization of the high-affinity sites, whereas the density of the Mr 33,000 polypeptide correlates best with the distribution of the low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1989

Mapping the benzodiazepine photoaffinity-labelling site with sequence-specific gamma-aminobutyric acidA-receptor antibodies.

The gamma-aminobutyric acidA (GABAA) receptor purified from adult bovine cerebral cortex was photoaffinity-labelled with the agonist benzodiazepine [3H]flunitrazepam and the radioactivity shown to be coincident with a band with Mr 53,000 that was recognized by three anti-(GABAA receptor alpha 1 subunit sequence)-specific antibodies. Complete and limited CNBr cleavage of the purified photoaffinity-labelled receptor was carried out. The products of this reaction were analysed for radioactivity, for immunoreactivity with anti-[alpha 1-(1-15)-peptide], anti-[alpha 1-(324-341)-peptide] and anti-[alpha 1-(413-429)-peptide] polyclonal antibodies and for carbohydrate by biotinylated concanavalin A lectin overlay. Complete CNBr cleavage gave a radioactive peptide with Mr 10,000-12,000 that was not recognized by the above-mentioned specific antisera. By using the deduced amino acid sequence of the alpha 1 subunit [Schofield, Darlison, Fujita, Burt, Stephenson, Rodriguez, Rhee, Ramachandran, Reale, Glencorse, Seeburg & Barnard (1987) Nature (London) 328, 221-227], it is proposed that the site of the benzodiazepine-agonist photoaffinity-labelling reaction does not lie within the amino acid sequences alpha 1 1-58 and alpha 1 149-429.

Affinity Labels↗
PubMed1993

Exchangeable GTP binding site of beta-tubulin. Identification of cysteine 12 as the major site of cross-linking by direct photoaffinity labeling.

After direct photoaffinity cross-linking of [3H]GTP to the beta-subunit of tubulin, followed by tryptic digestion and alkaline phosphatase treatment, we employed cis-diol-specific boronate gel chromatography and reversed-phase high-pressure liquid chromatography to purify a peptide containing most of the covalently bound radioactivity. The sequence of this peptide corresponded to that of residues 3-19 of beta-tubulin. Residue 10 of the peptide, which is Cys-12 in beta-tubulin, could not be identified. The fast atom bombardment mass spectrum of this peptide showed the presence of a predominant species with a molecular mass of 2022 kDa (2021 kDa for the 12C variant), which is 255 Da greater than the molecular mass of the peptide. Fast atom bombardment collision-activated decomposition mass spectrometry analysis produced fragments which are consistent with the beta(3-19) peptide but having a unit of mass of 358 at position 12. Thermolysin digestion of the tryptic peptide restricted the cross-linking site to the 9-amino acid sequence, I(L)QAGQXGNQ. The molecular mass of this peptide was 1174 kDa, which is equal to the mass of the beta(7-15) peptide containing an extra group of mass 255. To explain the molecular masses of the two labeled peptides, which are 26 atomic mass units less than expected, a mechanism of photolabeling is proposed that involves opening of the guanine ring and loss of the C-6 carbonyl function as CO2.

Affinity Labels↗