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PubMed1993

Identification of the nucleotide binding site of HIV-1 reverse transcriptase using dTTP as a photoaffinity label.

We have utilized UV-induced cross-linking of [methyl-3H]dTTP to identify the nucleotide binding site on heterodimeric HIV-1 reverse transcriptase (RT). RT was derivatized by irradiating a solution containing [methyl-3H]dTTP and purified recombinant RT for 10 min. The UV-induced cross-linking reaction between dTTP and RT is linear with time of UV exposure up to 10 min, and it has been determined previously that dTTP cross-linking is half-maximal at 90 microM [Cheng, N., Painter, G. R., & Furmann, P.A. (1991) Biochem. Biophys. Res. Commun. 174, 785-789]. Under these reaction conditions, only the 66-kDa subunit of the 66-kDa/51-kDa RT heterodimer was labeled with dTTP. The [methyl-3H]dTTP-labeled RT was fragmented with trypsin and endoproteinase Asp-N, and peptides were purified on reversed phase HPLC. The peptide covalently linked to [methyl-3H]dTTP was subjected to amino acid sequence analysis. The sequencing data localized the nucleotide binding site of RT to Lys-73 in the vicinity of several mutation sites linked to antiviral drug resistance. Since most effective anti-AIDS compounds are inhibitors of RT, information about its dNTP binding site may make it possible to understand the basis for the antiviral activity of nucleoside analogs such as AZT, ddI, and ddC. This information may also be useful for a more rationally based design of anti-HIV agents.

Affinity Labels↗
PubMed1993

Photoaffinity labeling of the vesamicol receptor of cholinergic synaptic vesicles.

On the basis of the high-affinity vesamicol analog 4-aminobenzovesamicol (ABV), a tritiated, arylazido ligand (azidoABV) of the vesamicol receptor (VR) in cholinergic synaptic vesicles was synthesized. azidoABV is an inhibitor of acetylcholine (AcCh) active transport, and it binds to the VR with higher affinity than vesamicol. The rate of dissociation of azidoABV from synaptic vesicles is 0.058 +/- 0.003 min-1 at 20 degrees C (about 3-fold slower than that of vesamicol), and the equilibrium dissociation constant is 2 nM (about 4-fold lower than that of vesamicol). Photolysis of [3H]azidoABV in the presence of a stoichiometric excess of the VR led to incorporation of 28% of the radiolabel, of which 57% was blocked by 50 microM vesamicol. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the labeled vesicles revealed, after autofluorography, specific labeling over a broad molecular weight range that extended from about 50 to 200 kDa. This labeling pattern was essentially the same as that obtained with an azido analog of AcCh that was used to label the AcCh transporter (Rogers, G.A., & Parsons, S. M. (1992) Biochemistry 31, 5770-5777). In addition, about 6% of the radioligand that was specifically incorporated into proteins with M(r) greater than 12 kDa labeled four polypeptides that corresponded to bands in the Coomassie image at M(r) = 23, 33, 35, and 38 kDa. The results suggest that the VR exists as part of a complex system of subunits.

Acetylcholine↗
PubMed1992

Photoaffinity labeling study of the interaction of calmodulin with the plasma membrane Ca2+ pump.

Bovine brain calmodulin was labeled with synthetic peptides corresponding to the calmodulin-binding domain of the erythrocyte plasma membrane Ca(2+)-ATPase. One 20-amino acid peptide and two 28-amino acid peptides were used, carrying L-4'-(1-azi-2,2,2-trifluoroethyl)phenylalanine residues in position 9 (peptides C20W* and C28W*) and position 25 (peptide C28WC*), respectively. The localization of the contact regions between calmodulin and the N- and C-terminal portions of the peptides was the aim of this study. The three peptides were N-terminally blocked with a 3H-labeled acetyl group to facilitate the identification of labeled fragments after isolation and digestion. The binding site for phenylalanine 25 was identified in the N-terminal domain of calmodulin while the phenylalanine derivative in position 9 labeled the C-terminal domain. Fluorescence studies using the dansylated N- and C-terminal halves of calmodulin and peptide C20W corresponding to the first 20 amino acids of the calmodulin-binding domain showed that only the C-terminal lobe of calmodulin had high affinity for the peptide (KD in the nanomolar range).

Affinity Labels↗
PubMed1994

Photoaffinity labeling of cytochrome P450 2B4: capture of active site heme ligands by a photocarbene.

Spiro[adamantane-2,2'-diazirine], which produces adamantyl carbene upon photolysis, binds tightly to P450 2B4 (KS = 3.2 microM), giving a normal substrate binding difference spectrum. Irradiation of 2-[3H]adamantane diazirine at 365 nm in the presence of native, ferric P450 2B4 resulted in first-order photolysis (t1/2 = 1.8 min). The main product was 2-[3H]adamantanol, with about 6% of the radioactivity covalently bound to P450 2B4. With the ferrous carbonyl form of P450 2B4, 2-adamantanol production decreased and protein labeling increased to 12%. When ferric cyanide 2B4 was used, 2-adamantanecarbonitrile was formed in addition to 2-adamantanol. The nitrile appears to have resulted from capture of the iron-bound cyanide ligand by the carbene. The use of multiple cycles of photolysis increased the percentage of protein labeling to 76%. Photolabeling was inhibited by known 2B4 substrates and inhibitors. Also, N-demethylation of benzphetamine and generation of a substrate binding difference spectrum by benzphetamine were both inhibited stoichiometrically with the fraction of radiolabeled protein. The labeled protein was permanently converted to the high-spin state, as indicated by the characteristic change in the absorbance spectrum, demonstrating irreversible occupation of the substrate binding site by the adamantyl residue. Mild acid hydrolysis of radiolabeled 2B4 at the five Asp-Pro bonds generated a 2-kDa peptide which carried 78% of the radioactivity. These results are interpreted as the result of the active site carbene reacting by three competing pathways: capture of the heme sixth ligand to yield either 2-adamantanol or 2-adamantanecarbonitrile, capture of an unbound active site water molecule to yield adamantanol, and covalent attachment to a protein residue. Thus, the P450 2B4 active site appears to contain at least one unbound water molecule in addition to the heme aquo sixth ligand, even when substrate is present.

Adamantane↗
PubMed1994

Identification of Trp-371 as the main site of specific photoaffinity labeling of corticosteroid binding globulin using delta 6 derivatives of cortisol, corticosterone, and progesterone as unsubstituted photoreagents.

Immunopurified human corticosteroid binding globulin (CBG) was photolabeled with delta 6-[3H]cortisol, delta 6-[4-14C]cortisol, delta 6-[3H]corticosterone, and delta 6-[3H]progesterone. The maximal levels of specific incorporation, as estimated with tritiated photoreagents, were 0.21, 0.14, and 0.08 mol of label/mol of CBG, respectively. Tryptic cleavage of photolabeled CBG gave in all cases a major radioactive peptide that was no longer detectable when a 100-fold molar excess of cortisol was added to the photoreagents. Edman sequencing of tryptic peptides photolabeled with delta 6-[3H]cortisol or delta 6-[3H]corticosterone showed that these peptides correspond to residues 357-378 of the human CBG sequence. The major peak of radioactivity of these peptides was eluted at the 15th cycle (Trp-371). The radioactive tryptic peptides photolabeled with the four steroid photoreagents were subcleaved with alpha-chymotrypsin. The major part of radioactivity was recovered in the T-[*X]-S-S-L-F hexapeptide 370-375 (major peptide) and in the D-H-F-T-[*X]-S-S-L-F nonapeptide 367-375, at the second and fifth Edman cycles, respectively, whereas no PTH derivative could be identified at these cycles, thus suggesting Trp-371 as the main site of photolabeling for all tested photoreagents. Mass spectrometry of tryptic peptides photolabeled with delta 6-[3H]cortisol and delta 6-[3H]corticosterone and of chymotryptic peptides photolabeled with delta 6-[3H]cortisol, delta 6-[3H]corticosterone, and delta 6-[3H]progesterone showed molecular masses corresponding to the addition of delta 6-steroid photoreagents to the peptide.

Affinity Labels↗
PubMed1991

Photoaffinity labeling of the erythropoietin receptor and its identification in a ligand-free form.

Pure human recombinant erythropoietin (EP) was acylated through a primary amino residue with a cross-linking reagent, N-[[3-[[4-[(p-azido-m-[125I]iodophenyl)azo]benzoyl]amino] propanoyl]oxy]-succinimide (Denny-Jaffe reagent), which is photoreactive and cleavable at the azo residue. The resulting conjugated hormone (DJ-EP) was purified from unmodified EP by reverse-phase high-pressure liquid chromatography and maintained its capacity to bind to receptors for EP on erythroid progenitor cells. The receptor for EP was previously identified as two related proteins of 100 and 85 kDa molecular mass by chemical cross-linking to 125I-EP. Recently, D'Andrea and co-workers [(1989) Cell 57, 277-285] cloned a cDNA that codes for a protein of 55-66 kDa, which is thought to be the EP receptor. In this report, cross-linking to the receptor through the monofunctional DJ-EP labeled the same 140- and 125-kDa molecular mass bands (100- and 85-kDa proteins) cross-linked with 125I-EP and disuccinimidyl suberate. Furthermore, cleavage of the azo bond of the DJ-EP receptor complex by sodium dithionite (80 degrees C, 5 min) demonstrated that proteins of 105 and 90 kDa were labeled in ligand-free form by DJ-EP. This result demonstrates that artifactual cross-linking of multiple proteins or other artifacts of cross-linking do not explain the difference in molecular mass of the EP receptor identified by cross-linking and the receptor identified by expression cloning.

Acylation↗
PubMed1991

Photoaffinity labeling the substance P receptor using a derivative of substance P containing p-benzoylphenylalanine.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe8 residue of SP. [Phe8(pBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the 125I-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (Bmax = 0.2 pmol/mg of membrane protein) with an affinity KD = 0.4 nM. The binding of 125I-[Phe8(pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound 125I-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of Mr = 53,000 and 46,000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on 125I-[Phe8(pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. In addition, the labeling of the two polypeptides was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate, a nonhydrolyzable analogue of GTP. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies: the Mr = 46,000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1991

Photoaffinity labeling of the primer binding domain in murine leukemia virus reverse transcriptase.

We have labeled the primer binding domain of murine leukemia virus reverse transcriptase (MuLV RT) by covalently cross-linking 5' end labeled d(T)8 to MuLV RT, using ultraviolet light energy. The specificity and the functional significance of the primer cross-linking reaction were demonstrated by the fact that (i) other oligomeric primers, tRNAs, and also template-primers readily compete with radiolabeled d(T)8 for the cross-linking reaction, (ii) under similar conditions, the competing primers and template-primer also inhibit the DNA polymerase activity of MuLV RT to a similar extent, (iii) substrate deoxynucleotides have no effect, and (iv) the reaction is sensitive to high ionic strength. In order to identify the primer binding domains/sites in MuLV RT; tryptic digests prepared from the covalently cross-linked MuLV RT and [32P]d(T)8 complexes were resolved on C-18 columns by reverse-phase HPLC. Three distinct radiolabeled peptides were found to contain the majority of the bound primer. Of these, peptide I contained approximately 65% radioactivity, while the remainder was associated with peptides II and III. Amino acid composition and sequence analyses of the individual peptides revealed that peptide I spans amino acid residues 72-80 in the primary amino acid sequence of MuLV RT and is located in the polymerase domain. The primer cross-linking site appears to be at or near Pro-76. Peptides II and III span amino acid residues 602-609 and 615-622, respectively, and are located in the RNase H domain. The probable cross-linking sites in peptides II and III are suggested to be at or near Leu-604 and Leu-618, respectively.

Affinity Labels↗
PubMed1982

Photoaffinity labeling of Escherichia coli ribosomes by an aryl azide analogue of puromycin. Evidence for the functional site specificity of labeling.

The photoincorporation of p-azido[3H]puromycin [6-(dimethylamino)-9-[3'-deoxy-3'-[(p-azido-L-phenylalanyl)amino]-beta-D-ribofuranosyl]purine] into specific ribosomal proteins and ribosomal RNA [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry (preceding paper in this issue)] is decreased in the presence of puromycin, thus demonstrating that labeling is site specific. The magnitudes of the decreases in incorporation into the major labeled 50S proteins found on addition of different potential ribosome ligands parallel the abilities of these same ligands to inhibit peptidyltransferase. This result provides evidence that p-azidopuromycin photoincorporation into these proteins occurs at the peptidyltransferase center of the 50S subunit, a conclusion supported by other studies of ribosome structure and function. A striking new finding of this work is that puromycin aminonucleoside is a competitive inhibitor of puromycin in peptidyltransferase. The photoincorporation of p-azidopuromycin is accompanied by loss of ribosomal function, but photoincorporated p-azidopuromycin is not a competent peptidyl acceptor. The significance of these results is discussed. Photolabeling of 30S proteins by p-azidopuromycin apparently takes place from sites of lower puromycin affinity than that of the 50S site. The possible relationship of the major proteins labeled, S18, S7, and S14, to tRNA binding is considered.

Affinity Labels↗
PubMed1983

Specific photoaffinity labeling of the digitalis binding site of the sodium and potassium ion activated adenosinetriphosphatase induced by energy transfer.

A ouabain p-aminobenzenediazonium derivative with a high specific radioactivity has been synthesized from ouabain and used as a photolabel for the (sodium plus potassium)-activated adenosinetriphosphatase from Electrophorus electricus electric organ and from dog kidney. In the dark it binds reversibly to the digitalis receptor site, with binding characteristics comparable to those of ouabain. The photoactivation of the ouabain derivative to produced covalent labeling of the receptor was obtained by energy transfer from a tryptophan residue in the (Na+,K+)ATPase to the ouabain p-aminobenzenediazonium molecule bound at the active site. The great advantage of this procedure compared to previous methods is that free molecules of the photoactivatable derivative are not photodecomposed. Analysis of the photolabeled polypeptides on sodium dodecyl sulfate gel electrophoresis showed that over 90% of the total radioactivity incorporated was found in the large molecular weight alpha-chain of the kidney enzyme (Mr 93 000). The same specific labeling of the alpha-subunit was obtained with a crude microsomal fraction from Electrophorus electricus. A mild tryptic fragmentation of the subunit into two peptide fragments of Mr 58 000 and 41 000, respectively, shows that the digitalis receptor is located in the N-terminal 41 000 fragment.

Affinity Labels↗
PubMed1983

Analysis of the adrenal angiotensin II receptor with the photoaffinity labeling method.

The angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, AT) receptor of bovine adrenocortex has been investigated with photosensitive analogues of AT. In a first series of experiments, we have shown that isolated cortical cells secrete aldosterone in a permanent and specific manner if they have been photolyzed in the presence of the photolabel [Sar1,(4'-N3)Phe8]AT. This permanent stimulation is in contrast to the smooth muscle assays where under similar conditions a permanent and specific block was always observed. It is assumed that the irreversible occupation of the AT receptor produces this effect. In a second type of experiment, we have shown that the AT binding site on adrenocortical membranes can be specifically and irreversibly occupied under similar conditions and that this occupation can be prevented in a competitive manner by the presence of nonphotosensitive hormone. Using a radioactive label, [Sar1,(3'-125I)Tyr4,(4'-N3)Phe8]AT, we have identified the AT receptor as a 300-kDa protein by means of gel filtration under nonreducing and nondenaturating conditions. Under reducing and denaturing conditions, a subunit of 60 kDs was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The AT receptor is proposed to be a 300-kDa protein with one binding subunit of 60 kDa.

Adrenal Cortex↗
PubMed1984

Photoaffinity labeling of the alpha 1-adrenergic receptor using an 125I-labeled aryl azide analogue of prazosin.

alpha 1-Adrenergic receptor probes, which can be radioiodinated to yield high specific activity radioligands, have been synthesized and characterized. 2-[4-(4-Amino-benzoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquin azoline (CP63,155), an arylamine analogue of the selective alpha 1-adrenergic antagonist prazosin, and its iodinated derivative, 2-[4-(4-amino-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP63,789), bind reversibly and with high affinity (KD = 1 nM and 0.6 nM, respectively) to rat hepatic membrane alpha 1-adrenergic receptors. Conversion of [125I]CP63,789 to the aryl azide yields a photolabile derivative, 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP65,526), which prior to photolysis binds competitively and with high affinity (KD = 0.3 nM). Binding of [125I]CP63,789 and [125I]CP65,526 (prior to photolysis) is rapid and saturable. Both ligands identify similar alpha 1-adrenergic receptor binding site concentrations as the parent probe, [3H]prazosin. Specific binding by these iodinated ligands is stereoselective and inhibited by a variety of adrenergic agents with a specificity typical of the alpha 1-adrenergic receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of [125I]CP65,526-labeled rat hepatic membranes reveal major protein species with molecular weights of 77K, 68K and 59K. Each protein binds adrenergic ligands with stereoselectivity and with a specificity typical of the alpha 1-adrenergic receptor. Inclusion of multiple protease inhibitors during membrane preparation prior to SDS-PAGE does not alter the labeling of these peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1985

Ribosome structure: binding site of macrolides studied by photoaffinity labeling.

The macrolide antibiotics carbomycin A, niddamycin, and tylosin have been radioactively labeled by reducing their aldehyde group at the C-18 position. Dihydro derivatives with specific activities around 2.5 Ci/mmol can be obtained that, although partially affected in their activity, still bind to the ribosomes with high affinity. The presence in the chemical structure of these antibiotics of alpha-beta-unsaturated ketone groups makes them photochemically reactive, and by irradiation above 300 nm, covalent incorporation of the radioactive dihydro derivatives into ribosomes has been achieved. The covalent binding seems to take place at the specific binding sites for macrolides as deduced from binding saturation studies and competition experiments with unmodified drugs. Analysis of the ribosomal components labeled by the drugs indicated that most radioactivity is associated with the proteins L27, L2, and L28 when 50S subunits are labeled, and with L27, L2, L32/33, S9, and S12 in the case of 70S ribosomes. These results agree well with a model of macrolides' mode of action that assumes an interaction of the drug at the peptidyl transferase P site that would block the exit channel for the growing peptide chain.

Affinity Labels↗
PubMed1985

Direct photoaffinity labeling of tubulin with guanosine 5'-triphosphate.

Irradiation of tubulin in the presence of [3H]GTP or [3H]GDP at 254 nm led to the covalent incorporation of nucleotide into the protein. The specific nature of the labeling was shown in the following manner: with tubulin depleted of exchangeable nucleotide, the amount of labeling increased to a plateau value as the [3H]GTP concentration was increased, with saturation being reached at a ratio of approximately 1.5; the same amount of labeling was obtained with GTP/tubulin ratios of 1 and 100; [3H]GMP was not incorporated into the dimer, nor did GMP inhibit the incorporation of [3H]GTP; [3H]ATP was not incorporated; [3H]GTP incorporation did not occur into denatured tubulin or into serum albumin. When [alpha-32P]GTP was used in the irradiation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the carboxymethylated protein demonstrated that the incorporated label was associated with the beta subunit. The radiation treatment did cause changes in the tubulin molecule resulting in a decrease in assembly competence and in sulfhydryl groups, but these effects were minimized when a large excess of GTP was present during irradiation. Labeling of tubulin in the assembled state was much less than that observed in the free state.

Adenosine Triphosphate↗
PubMed1985

Photoaffinity labeling of the tetrabenazine binding sites of bovine chromaffin granule membranes.

An azido derivative of tetrabenazine, a specific inhibitor of the monoamine carrier of chromaffin granule membranes, has been synthesized. In the dark, this compound, 3H-labeled N-(3-isobutyl-9,10-dimethoxy-1,2,3,4,6,7-hexahydro-11bH-benzo [a]quinolizin-2-yl)-4-[(4-azido-2-nitrophenyl)amino]butanamide+ ++ ([3H]TBA), bound reversibly to purified chromaffin granule membranes. Centrifugation through SP-Sephadex columns was used to separate bound and free [3H]TBA. This technique gave low levels of nonspecific binding and allowed recovery of [3H]TBA-membrane complexes. Scatchard analysis of the data indicated one class of sites with an equilibrium dissociation constant KD of 50 nM and a density of sites of 40-50 pmol/mg of protein, consistent with reported densities of reserpine and dihydrotetrabenazine binding sites. Competition experiments showed that TBA and tetrabenazine bound to the same site. Irradiation at 435 nm of [3H]TBA-membrane mixtures induced some irreversible binding of the probe to membranes. After irreversible binding of TBA, the number of dihydrotetrabenazine binding sites was decreased, indicating that the probe was covalently bound to the monoamine carrier. [3H]TBA-membrane complexes isolated by centrifugation through SP-Sephadex columns were irradiated, and their radioactivity was analyzed by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. A polypeptide with a molecular weight of 70 000 was labeled. This polypeptide was different from dopamine beta-hydroxylase, and it was not adsorbed on concanavalin A-Sepharose. It is proposed that the monoamine carrier of chromaffin granule membrane has an oligomeric structure, involving a 45K subunit [Gabizon, R., Yetinson, T., & Schuldiner, S. (1982) J. Biol. Chem. 257, 15145] and a 70K subunit.

Adrenal Medulla↗
PubMed1985

Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes.

Pactamycin, an inhibitor of the initial steps of protein synthesis, has an acetophenone group in its chemical structure that makes the drug a potentially photoreactive molecule. In addition, the presence of a phenolic residue makes it easily susceptible to radioactive labeling. Through iodination, one radioactive derivative of pactamycin has been obtained with biological activities similar to the unmodified drug when tested on in vivo and cell-free systems. With the use of [125I]iodopactamycin, ribosomes of Escherichia coli have been photolabeled under conditions that preserve the activity of the particles and guarantee the specificity of the binding sites. Under these conditions, RNA is preferentially labeled when free, small ribosomal subunits are photolabeled, but proteins are the main target in the whole ribosome. This indicates that an important conformational change takes place in the binding site on association of the two subunits. The major labeled proteins are S2, S4, S18, S21, and L13. These proteins in the pactamycin binding site are probably related to the initiation step of protein synthesis.

Antibiotics, Antineoplastic↗
PubMed1985

Puromycin photoaffinity labels small- and large-subunit proteins at the A site of the Drosophila ribosome.

[3H]Puromycin covalently incorporates into the protein and to a much lesser extent into the RNA components of Drosophila ribosomes in the presence of 254-nm light. The photoincorporation reaction takes place with a small number of large- (L2 and L17) and small- (S8 and S22) subunit proteins as determined by two-dimensional gel analysis. More quantitative one-dimensional gel results show that puromycin reacts with each of these proteins in a functional site specific manner. The small percentage of the total labeling that occurs with rRNA also appears to be site specific. The rRNA labeling arises from a puromycin-mediated cross-linking of ribosomal protein and rRNA. Ionic conditions shift the pattern of puromycin-labeled ribosomal proteins. These results suggest that puromycin can occupy two distinct sites on Drosophila 80S ribosomes. The pattern of ribosomal proteins labeled by puromycin is affected by the presence of other antibiotics such as emetine, anisomycin, and trichodermin.

Affinity Labels↗