Direct Photoaffinity Labeling Research Articles

Direct photoaffinity labeling of proteins with adenosine 3‘-[32P]phosphate 5‘-phosphosulfate. Atractyloside inhibits labeling of a Mr = 34,000 protein in an adrenal medullary Golgi fraction.

Direct photoaffinity labeling with radioactively labeled adenosine 3'-phosphate 5'-phosphosulfate (PAPS) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography was used to identify PAPS binding proteins in a Golgi membrane preparation of bovine adrenal medulla. [3'-32P]PAPS was synthesized from adenosine 5'-phosphosulfate (APS) and [gamma-32P]ATP using APS kinase prepared from yeast and was purified by reverse-phase ion pair high performance liquid chromatography. Upon irradiation with UV light, [3'-32P]PAPS, as well as [35S]PAPS under conditions which minimized sulfotransferase-catalyzed incorporation of 35SO4 from [35S]PAPS into proteins, bound selectively to a 34-kDa protein of the Golgi membrane preparation. PAPS binding to the 34-kDa protein was strongly inhibited by the presence of 50 microM atractyloside. The 34-kDa PAPS binding protein therefore appears to be similar to the mitochondrial ATP/ADP translocator with regard to both molecular weight and inhibition by atractyloside of adenine nucleotide binding. Photoaffinity labeling will be useful in the purification and functional identification of the 34-kDa protein.

Nucleoside triphosphate binding to DNA polymerase III holoenzyme of Escherichia coli. A direct photoaffinity labeling study.

The physical basis of ATP binding and activation of DNA polymerase III holoenzyme was studied by an ultraviolet irradiation cross-linking technique. ATP and dATP were photocrosslinked to the alpha, tau, gamma, and delta subunits of holoenzyme; photocrosslinking of dATP was competitively inhibited by ATP. No photocrosslinking was observed with GTP or CTP, nor did GTP, CTP, or UTP inhibit cross-linking of ATP. ADP and adenosine 5'-O-(3-thio)-triphosphate, both potent inhibitors of ATP activation of holoenzyme, inhibited cross-linking of ATP to tau, gamma, and delta subunits, but not to the alpha subunit, suggesting that one or more of these subunits are ATP (or dATP)-binding sites. Photocrosslinking of dTTP to the ATP-activated holoenzyme was exclusively to the epsilon subunit, the dnaQ ( mutD ) gene product; dCTP and dGTP were not photocrosslinked to any subunit. Binding of dTTP was enhanced by ATP, but by no other nucleotide (or deoxynucleotide). This binding of dTTP to epsilon, a subunit likely responsible for regulation of proofreading by the holoenzyme, may function in the control of the fidelity of replication.

Direct photoaffinity labeling of the high affinity nitrendipine-binding site in subcellular membrane fractions isolated from canine myocardium.

[3H]Nitrendipine and high intensity ultraviolet irradiation have been used to photoaffinity label the protein component of the high affinity nitrendipine-binding site in subcellular membrane fractions from canine cardiac muscle. Irradiation of isolated cardiac membranes in the presence of [3H]nitrendipine resulted in the covalent labeling of a protein component that migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 32,000. Incorporation of [3H]nitrendipine did not occur in the absence of irradiation. The photoaffinity labeling of the 32,000-Da protein by [3H]nitrendipine was inhibited by excess unlabeled nitrendipine, nifedipine, or verapamil. EDTA, ATP, and La3+, which are known to reduce high affinity nitrendipine binding, also inhibited the photoaffinity labeling of this membrane protein by [3H]nitrendipine. The 32,000-Da [3H]nitrendipine-labeled protein was found to be enriched in the ryanodine-sensitive fraction of cardiac sarcoplasmic reticulum and absent from the ryanodine-insensitive fraction of cardiac sarcoplasmic reticulum which is known to lack high affinity nitrendipine binding. Therefore, the 32,000-Da photoaffinity-labeled [3H]nitrendipine-binding protein exhibits properties identical to those expected for the protein component of the high affinity nitrendipine-binding site in isolated cardiac membranes.

Identification by direct photoaffinity labeling of an altered phosphodiesterase in a mutant S49 lymphoma cell.

Extracts of a mutant S49 lymphoma cell line, termed K30a, hydrolyze cAMP and cGMP at rates much faster than do wild type S49 extracts. This elevated phosphodiesterase activity, called K-PDE, elutes as a single peak of activity on DEAE-cellulose columns (Brothers, V. M., Walker, N., and Bourne, H. R. (1982) J. Biol. Chem. 257, 9349-9355). Direct photoaffinity labeling of K30a extracts with [32P]cGMP results in radiolabeling of a unique polypeptide, not observed in wild type extracts, which migrates in sodium dodecyl sulfate polyacrylamide gels with an Mr = 106,000. The 106-kDa band was identified as the catalytic K-PDE polypeptide based on the following observations: competitive inhibitors and substrates of K-PDE inhibit photolabeling of the 106-kDa band, indicating that [32P] cGMP photolabels the enzyme at its catalytic site; on DEAE-cellulose chromatography the polypeptide that is susceptible to photolabeling co-elutes with K-PDE activity; the 106-kDa band is detectable in extracts of WT X K30a hybrids (where WT denotes wild type) in amounts proportional to the K-PDE activity in the hybrids, but is undetectable in wild type. The hybrid phenotype strongly suggests that the K30a phenotype is not due to mutations that affect either a diffusible regulator of transcription or an enzyme that modifies K-PDE. Although wild type cells contain a minor cGMP phosphodiesterase activity distinct from the major cAMP phosphodiesterase, the wild type cGMP phosphodiesterase is not susceptible to radiolabeling with [32P]cGMP; this rules out the possibility that the K30a phenotype is caused by overexpression of a wild type phosphodiesterase. We conclude that the K30a mutation produced expression of a new species of phosphodiesterase molecule that is not detectably expressed in the parental S49 wild type cell line.

Direct photoaffinity labeling of ribonucleotide reductase from Escherichia coli. Evidence for enhanced binding of the allosteric effector dTTP by the presence of substrates.

Direct photoaffinity labeling of ribonucleotide reductase from Escherichia coli. Evidence for enhanced binding of the allosteric effector dTTP by the presence of substrates.

Characterization of an ATPase/dATPase activity associated with the Drosophila nuclear matrix-pore complex-lamina fraction. Identification of the putative enzyme polypeptide by direct ultraviolet photoaffinity labeling.

An ATPase/dATPase activity found associated with the nuclear matrix-pore complex-lamina fraction isolated from embryos of Drosophila melanogaster has been characterized. In the presence of either Ca2+ or Mg2+, this activity hydrolyzed either ATP or dATP to ADP or dADP, respectively, and Pi. Hydrolysis was optimal from pH 6.5-7.2, did not require either Na+ or K+, and was not significantly inhibited by NaF, ouabain, quercetin, Na3VO4, CTP, or GTP. In contrast, hydrolysis was inhibited by N-ethylmaleimide, EDTA, and cordycepin 5'-triphosphate. In all respects tested, hydrolysis of ATP was indistinguishable from that of dATP and when incubated in the same reaction mixture, each was linearly competitive with the other. Based upon these properties, a series of direct UV photoaffinity labeling experiments was performed. Using alpha-[32P]dATP, alpha-[32P]ATP, or gamma-[32P]ATP, only a single polypeptide (Mr approximately 174,000) was photolabeled in a manner completely consistent with the enzymology of ATP and dATP hydrolysis; cell fractionation studies revealed a predominantly or exclusively nuclear localization. A polypeptide with virtually the identical mobility on sodium dodecyl sulfate-polyacrylamide gels was similarly identified as the major photolabeled species in nuclear envelope fractions obtained from chickens, opossums, rats, and guinea pigs. Thus, it seems probable that this 174-kilodalton polypeptide constitutes at least the active site-containing subunit of the major insoluble ATPase/dATPase found in structural protein subfractions prepared from higher invertebrate as well as vertebrate nuclei.

Direct photoaffinity labeling of the catalytic site of mouse ribonucleotide reductase by CDP.

Ribonucleotide reductase reduces all four ribonucleoside diphosphates to the deoxyribonucleotides required for DNA synthesis. The enzyme is composed of two nonidentical subunits, M1 and M2. The 89-kilodalton M1 subunit contains at least two allosteric sites which, by binding nucleotide effectors, regulate the catalytic activity and substrate specificity of the enzyme. We now show that in addition, protein M1 contains a substrate-binding (catalytic) site which is specifically photolabeled after UV irradiation in the presence of the natural substrate, [32P]CDP. The photolabeling of protein M1 by [32P]CDP required the presence of the second subunit, protein M2, and ATP, the positive allosteric effector for CDP reduction. The negative effectors, dATP, dGTP, and dTTP, inhibited the photolabeling of wild type protein M1. Deoxy-ATP did not inhibit the labeling of a mutant protein M1 that is resistant to feedback inhibition by dATP. In addition, hydroxyurea and 4-methyl-5-aminoisoquinoline thiosemicarbazone, two inhibitors of ribonucleotide reductase which affect protein M2, also inhibited the [32P]CDP labeling of protein M1. These data provide new insights into the role and interaction of the two ribonucleotide reductase subunits, proteins M1 and M2, and the mechanism of action of the allosteric effectors.

Identification of cyclic guanosine monophosphate-binding proteins in Drosophila by direct photoaffinity labeling

A procedure for direct photoaffinity labeling with [ 32P]cGMP has been used to identify cGMP-binding proteins in Drosophila. This method provides better sensitivity and resolution than previously described direct methods, because the proteins can be visualized by autoradiography following sodium dodecyl sulfate-gel electrophoresis. Labeling is observed with cGMP concentrations as low as 4 × 10 −8 m and is specific for cGMP. The sensitivity of the technique is sufficient to permit detection of cGMP-binding proteins in crude extracts. With this technique a single cytoplasmic cGMP-binding protein of subunit M r 108,000 has been identified in Drosophila embryos and cultured cells.

Direct photoaffinity labeling of an allosteric site of subunit protein M1 of mouse ribonucleotide reductase by dATP. Evidence for two independent binding interactions within the allosteric specificity site.

The M1 subunit of ribonucleotide reductase contains two kinds of allosteric sites, the activity site and the specificity site, which regulate the overall catalytic activity and the substrate specificity of the enzyme, respectively. The effector nucleotides, dGTP and dTTP, bind only to the specificity site; dATP and ATP bind to both sites. Partially purified protein M1 was photolabeled specifically after UV irradiation in the presence of [32P]dATP. The labeling occurred exclusively at the allosteric specificity site as evidenced by 1) total inhibition of the labeling by dGTP and dTTP, 2) normal photoincorporation of [32P]dATP by mutant protein M1 molecules that lack a functional activity site, and 3) coidentity of one-dimensional peptide maps of protein M1 labeled with either [32P]dATP or [32P]dTTP. A mutant protein M1 that is resistant to normal regulation by dGTP and dTTP (indicating an alteration in the allosteric specificity site) showed normal photoincorporation of [32P]dATP (but not [32P]dTTP). This labeling was not inhibited by dGTP or dTTP. Our data suggest that this mutation has altered the binding of dGTP and dTTP but not dATP (or ATP) at the specificity site. Thus, by the combination of genetic and photolabeling techniques, two independent nucleotide binding interactions occurring within this one complex regulatory domain can be distinguished.

Direct photoaffinity labeling of an allosteric site on subunit protein M1 of mouse ribonucleotide reductase by dTTP.

The protein M1 subunit of ribonucleotide reductase contains at least two allosteric nucleotide binding sites that control the capacity of the enzyme to reduce ribonucleotides to the deoxyribonucleotides required for DNA synthesis. Direct photoaffinity labeling of partially purified protein M1 from mouse T-lymphoma (S49) cells was observed after UV irradiation in the presence of dTTP at 0 degrees C. The relative molar incorporation of nucleotide per subunit was 4-8%. Competition experiments showed that the dTTP was bound to an allosteric domain genetically and kinetically defined as the substrate specificity site of the enzyme. An altered protein M1 isolated from a thymidine-resistant mutant cell line showed significantly decreased photoincorporation of dTTP, consistent with the fact that its CDP reductase activity is resistant to feedback inhibition by dTTP. Specific photolabeling of several other proteins with pyrimidine and purine nucleotides was also found, indicating the general usefulness of direct photoaffinity labeling in the study of enzymes involved in nucleotide and nucleic acid metabolism.

Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins.

The heavy chains of Acanthamoeba myosins. IA, IB and II, turkey gizzard myosin, and rabbit skeletal muscle myosin subfragment-1 were specifically labeled by radioactive ATP, ADP, and UTP, each of which is a substrate or product of myosin ATPase activity, when irradiated with UV light at 0 degrees C. With UTP, as much as 0.45 mol/mol of Acanthamoeba myosin IA heavy chain and 1 mol/mol of turkey gizzard myosin heavy chain was incorporated. Evidence that the ligands were associated with the catalytic site included the observations that reaction occurred only with nucleotides that are substrates or products of the ATPase activity; that the reaction was blocked by pyrophosphate which is an inhibitor of the ATPase activity; that ATP was bound as ADP; and that label was probably restricted to a single peptide following limited subtilisin proteolysis of labeled Acanthamoeba myosin IA heavy chain and extensive cleavage with CNBr and trypsin of labeled turkey gizzard myosin heavy chain.

Direct and enzyme-mediated photoaffinity labeling of membrane-associated actin in Dictyostelium discoideum.

Direct and enzyme-mediated photoaffinity labeling of membrane-associated actin in Dictyostelium discoideum.

Direct photoaffinity labeling of the primary region of the ouabain binding site of (Na + + K +)-ATPase with [ 3H]ouabain, [ 3H]digitoxin and [ 3H]digitoxigenin

The tritiated cardiotonic steroids, ouabain, digitoxin, and digitoxigenin are shown to photolabel the large polypeptide but not the glycoprotein or proteolipid component of the (Na + + K +)-ATPase when they are bound to the inhibitory site and exposed to light of 220 or 254 nm. The extent of photolabeling is low, less than 1%, and is limited by photocross-linking of the enzyme. The mechanism of photoincorporation does not appear to be either photolysis of the lactone ring in ouabain or photolysis of tryptophan or tyrosine residues in the polypeptide.