With a view toward the determination of nucleic acid binding domains and sites on nucleic acid he... more With a view toward the determination of nucleic acid binding domains and sites on nucleic acid helix-destabilizing (single strand-specific) proteins (HDPs), we have studied the interactions of the copolymer polynucleotide photoaffinity label, poly(adenylic, 8-azidoadenylic acid), (poly(A,8-N3A] with the T4 bacteriophage HDP, 32 protein. Poly(A,8-N3A) quenched the intrinsic tryptophan fluorescence of 32 protein in a manner similar to that observed with other polynucleotides, and the effect could be reversed by addition of sufficient NaCl. The binding affinity and site size of this noncovalent interaction of poly(A,8-N3A) with 32 protein are similar to the values obtained for poly(A) and this protein. When [3H]poly(A,8-N3A)/32 protein mixtures were irradiated at 254 nm, fluorescence quenching was not reversed by NaCl, suggesting that the label was covalently bound to the protein. Mixtures of photolabel and protein subjected to short periods of irradiation (generally 1 min, 2000 erg mm-2) formed high molecular weight complexes, which when electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels were radioactive and stained with Coomassie Blue R. Under the same conditions, [3H]poly(A) failed to label 32 protein. The radioactivity of [3H]poly(A,8-N3A)-labeled complexes subjected to micrococcal nuclease after irradiation was seen to migrate just behind the free 32 protein monomer on SDS-polyacrylamide gels, indicating that portions of the photolabel not in direct contact with protein were accessible to this enzyme. By several criteria, we conclude that 32 protein was photolabeled specifically at its single-stranded nucleic acid binding site. Single-stranded nucleic acids with affinities for protein greater than that of poly(A,8-N3A) effectively inhibited photolabeling. The [NaCl] dependence of photolabeling monitored on SDS gels paralleled the NaCl reversal of (noncovalent) poly(A,8-N3A)-32 protein binding. Photolabeling reached a plateau after 1-2 min. The formation of high molecular weight complexes with increasing [poly(A,8-N3A)] paralleled the disappearance of free protein on SDS gels, and reached a saturation level of about 75% labeling. Several chromatographic procedures appear to be useful for the separation of the photolabeled complexes from free protein and photolabel. Limited trypsin hydrolysis of photolabeled 32 protein indicated that all the label was within the central ("III") portion of the protein. This approach should have general applicability to the identification of nucleic acid binding sites on helix-destabilizing proteins.
They have enzymatically synthesized /sup 3/H- and /sup 32/P-poly(A,8N/sub 3/A) from 8-N/sub 3/ADP... more They have enzymatically synthesized /sup 3/H- and /sup 32/P-poly(A,8N/sub 3/A) from 8-N/sub 3/ADP and radiolabeled ADP, and have used this polynucleotide to photoaffinity label T4 gene 32 protein, as well as several other helix-destabilizing proteins (HDPs). Irradiation of /sup 32/P-/sup 3/H-poly(A,N/sub 3/A) mixtures for short durations produces a covalent complex, seen as a high molecular weight, radioactive band on SDS-polyacrylamide gels. Preliminary experiments on other HDPs, from prokaryotic, eukaryotic and animal viral sources, show analogous results. Several successful control experiments indicate that this system is suitable for binding site localization on /sup 32/P. Single-stranded nucleic acids competitively inhibit photolabeling. The effect of NaCl on photolabeling parallels the salt-dependence of /sup 32/P-poly(A,N/sub 3/A) binding. Photolabeling reaches a plateau after approx.1 min, and the formation of the high molecular weight complex parallels the reduction of free /sup 32/P on SDS gels. Staph. nuclease digestion of crosslinked complexes produces a diffuse, radioactive band on SDS gels, migrating just behind free /sup 32/P. When these digested complexes are subjected to reverse-phase HPLC on a C3 Ultrapore column, the nucleic acid /sup 32/P-label is seen to coelute with protein. They are currently employing RP-HPLC methods to locate the label on tryptic peptides of nuclease-digested complexes.
Light-activated hydrolysis of cyclic GMP is achieved through the photoexcitation of rhodopsin, a ... more Light-activated hydrolysis of cyclic GMP is achieved through the photoexcitation of rhodopsin, a process which then triggers the replacement of GDP for GTP by a retinal guanosine 5′-triphosphatase referred to as ‘transducin’. The transducin-GTP complex then switches on the phosphodiesterase [Fung, Hurley & Stryer (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 152-156]. The bovine transducin consists of an alpha-subunit (39000 Mr), which is a GTP-binding component, together with a beta-(37000 Mr) and a gamma-subunit (10000 Mr). We have purified retinal transducin from cow, pig, chick and frog. The enzyme specific activities and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic profiles indicate that this enzyme is similar in all species except the frog. Whereas the bovine, pig and chick transducins consist of major 37000- and 39000-Mr components, that of the frog consists of a single 75000-Mr component. Labelling of the GTP-binding components with the photoaffinity label 8-azidogu...
Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthe... more Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking [125I]PF-M (see Figure 1 for structure) to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifical...
Previous studies have identified the guanine and adenine binding domains of the GTP and ADP bindi... more Previous studies have identified the guanine and adenine binding domains of the GTP and ADP binding sites of GDH. In this study the peptide sequences within or near to the terminal phosphate‐binding domains of the GTP and ADP binding sites of bovine liver glutamate dehydrogenase (GDH) were identified using photoaffinity labeling with the benzophenone nucleotide derivatives, [γ‐32P]GTPγBP and [γ‐32P]ATPγBP. Without activating light, GTPγBP exhibited inhibiting effects on the GDH reaction similar to GTP; ATPγBP, as expected, produced activating effects similar to those of ADP. Photoinsertion into GDH by both probes exhibited saturation effects in agreement with the respective kinetic effects. Specificity of labeling was supported by specific and effective reduction of photoinsertion of [γ‐32P]GTPγBP and [γ‐32P]ATPγBP into GDH by GTP and ADP, respectively. Using a combination of immobilized Fe3+‐chelate affinity chromatography and reversed‐phase HPLC, photolabeled peptides located with...
8-azido-ATP, when used in the 0.2-5 vM concentration range, fulfills the criteria for a specific ... more 8-azido-ATP, when used in the 0.2-5 vM concentration range, fulfills the criteria for a specific photoaffinity label for the (Ca+Mg)ATPase of sarcoplasmic reticulum. It is a substrate for the enzyme. It is a mixed inhibitor of ATPase activity. When photolyzed at 0" it is an inhibitor of ATPase activity. promoted The photoinduced binding of 8-azido-ATP to the (Ca+Mg)ATPase is by Ca2 The dependence of the labeling of the (Ca+Mg)ATPase on 8-azido-ATP, Ca2+ and Mg2 concentrations strongly suggests that 2 classes of sites are labeled. When lo-60 PM 8-azido-ATP was used to label sarcoplasmic reticulum, proteins in addition to the (Ca+Mg)ATPase were labeled.
Subcellular fractions from tobacco (N. rustica) leaves were photolabelled with the UDPG analog, (... more Subcellular fractions from tobacco (N. rustica) leaves were photolabelled with the UDPG analog, ({sup 32}P)-N{sub 3} UDPG. Two stromal polypeptides (M{sub r} = 42 and 21 kD) were the major photolabelled chloroplast polypeptides. UDPG protected the 42 but not the 21 ...
ABSTRACT The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymati... more ABSTRACT The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from [gamma-32P]2-azidoATP and [alpha-32P]8-azidoATP by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p3A4[32P]pCp from RNase L with affinity equivalent to p3A3 (IC50 = 2 X 10(-9) M in radiobinding assays). The 8-azido photoprobe also activates RNase L to hydrolyze poly(U) [32P]pCp 50% at 7 X 10(-9) M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p3A3 activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10(-9) M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The [gamma-32P]2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the [alpha-32P]8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p3A3 allosteric binding site was obtained as follows.(ABSTRACT TRUNCATED AT 250 WORDS)
The photoaffinity probes [ Y-~~P ] 2-azidoATP (2-N3ATP) and [~x-~~P]8-azido-ATP (8-N3ATP) were us... more The photoaffinity probes [ Y-~~P ] 2-azidoATP (2-N3ATP) and [~x-~~P]8-azido-ATP (8-N3ATP) were used to investigate the binding of ATP to highly purified 2-5A synthetase. 2-N3ATP and 8-N3ATP are substrates for 2-5A synthetase [Suhadolnik, R.
Biochemical and Biophysical Research Communications, 1987
Nucleotide binding sites on DNA-dependent RNA polymerase from E. coli have been studied by photoa... more Nucleotide binding sites on DNA-dependent RNA polymerase from E. coli have been studied by photoaffinity labeling with a GTP analog [gamma-32P]-8-AzidoGTP and a guanosine-3'-diphosphate-5'-diphosphate analog, 8-Azidoguanosine-3'-phosphate-5'-85'-32P]phosphate. The guanosine diphosphate photoprobe labeled the beta, beta', and sigma subunits with the sigma subunit being most heavily labeled. The GTP photoprobe also labeled the beta, beta', sigma subunits but the beta' subunit was most heavily labeled. In competition experiments guanosine-3'-diphosphate-5'-diphosphate decreased photolabeling by 8-Azidoguanosine-3'-phosphate-5'-[5'-32P]phosphate better than GTP, while the opposite was true for photolabeling with [gamma-32P]8- AzidoGTP. The guanosine diphosphate photoprobe inhibited transcription on E. coli DNA with Ki of ca. 150 microM. Present studies suggest a unique ppGpp binding site distinct from substrate binding site(s) and this photoprobe may be used to localize this binding site(s).
A photoaffinity analog of cyclic AMP, 8-azidoadenosine-3',5'-monophosphate (8-N,cAMP), and a line... more A photoaffinity analog of cyclic AMP, 8-azidoadenosine-3',5'-monophosphate (8-N,cAMP), and a linear gradient sodium dodecyl sulfate-polyacrylamide gel system have been used to photolabel and separate possible cyclic AMP binding sites in whole cells of the sarcoma 37 line. At least four sites within the whole sarcoma 37 cell bind l~2P]8-N,cAMP such that they are saturated at 1.0 pM concentration. These four binding moieties appear to have measurably different affinities for 8-N,cAMP and the number of total sites labeled per cell differs with each binding moiety. Cell fractionation allows separation of some of the photolabeled sites and gives an indication as to the geographical location of these binding sites within the whole cell. Enrichment by cell fractionation allows additional photolabeled sites to be observed.
Guanosine triphosphate (GTP) is an absolute requirement for tubulin polymerization in situ. The n... more Guanosine triphosphate (GTP) is an absolute requirement for tubulin polymerization in situ. The nucleotide photoaffinity probe 8‐azidoguanosine 5′‐triphosphate (8N3GTP) has been shown to be a biological mimic of GRP in this system and, also, an effective active site probe of the exchangeable GRP binding site. Using [32P]8N3GTP we demonstrate that the exchangeable GTP site of the beta subunit of tubulin is available to added guanine nucleotide in normal aged brain homogenates, whereas it is variably unavailable in Alzheimer's diseased brain. Inability of 8N3GTP to photolabel beta tubulin appears to be associated with neurofibrillary tangle density. These results support the hypothesis that microtubule formation is abnormal in brains affected by Alzheimer's disease.
The causes of autism and neurodevelopmental disorders are unknown. Genetic and environmental risk... more The causes of autism and neurodevelopmental disorders are unknown. Genetic and environmental risk factors seem to be involved. Because of an observed increase in autism in the last decades, which parallels cumulative mercury expo-sure, it was proposed that autism may be in part caused by mercury. We review the evidence for this proposal. Several epidemiological studies failed to find a cor-relation between mercury exposure through thimerosal, a preservative used in vaccines, and the risk of autism. Recently, it was found that autistic children had a higher mercury exposure during pregnancy due to maternal dental amalgam and thimerosal-containing immunoglobulin shots. It was hypothesized that children with autism have a decreased detoxification capacity due to genetic polymor-phism. In vitro, mercury and thimerosal in levels found several days after vaccina-tion inhibit methionine synthetase (MS) by 50%. Normal function of MS is crucial in biochemical steps necessary for brain develo...
Sucrose phosphate synthase (SPS) was isolated from spinach leaves by precipitation with polyethyl... more Sucrose phosphate synthase (SPS) was isolated from spinach leaves by precipitation with polyethylene glycol, ion-exchange and hydrophobic interaction chromatography, and rate zonal centrifugation. The enzyme was purified more than 600-fold to a specific activity of 57 mumol/min/mg protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that a 120-kDa polypeptide was enriched through purification and was the major polypeptide in the final SPS preparation. The 120-kDa polypeptide was photoaffinity labeled with the substrate analog, 5-azidouridine [beta-32P]5'-diphosphate-glucose ([beta-32P]5-N3UDP-Glc). Covalent incorporation of 5-N3UDP-Glc into the 120-kDa polypeptide exhibited an apparent Kd of 74 microM, similar to the apparent Ki for inhibition of SPS activity by unphotolyzed 5-N3UDP-Glc. Competition experiments showed that photolabeling of the 120-kDa polypeptide by 5-N3UDP-Glc was reduced in the presence of UDP-Glc, exhibiting an apparent Ki value that was similar to the apparent Km (UDP-Glc) of 2.9 mM for the purified enzyme. The relative molecular mass of the SPS holoenzyme was 253,000, and the isoelectric point of the 120-kDa subunit was 5.2. The data confirmed the identity of the 120-kDa polypeptide as the SPS subunit, established the structure of the active enzyme as a dimer, and demonstrated active-site labeling of SPS by a photoaffinity analog of the substrate.
The radioactive, photoactivatable labeling probe [β-32 P]5-azidouridine 5′-diphosphoglucose has r... more The radioactive, photoactivatable labeling probe [β-32 P]5-azidouridine 5′-diphosphoglucose has recently been shown to label a 62-kDa protein in crude homogenates and in partially purified enzyme preparations without photoactivation. Here, we report that a portion of this ...
With a view toward the determination of nucleic acid binding domains and sites on nucleic acid he... more With a view toward the determination of nucleic acid binding domains and sites on nucleic acid helix-destabilizing (single strand-specific) proteins (HDPs), we have studied the interactions of the copolymer polynucleotide photoaffinity label, poly(adenylic, 8-azidoadenylic acid), (poly(A,8-N3A] with the T4 bacteriophage HDP, 32 protein. Poly(A,8-N3A) quenched the intrinsic tryptophan fluorescence of 32 protein in a manner similar to that observed with other polynucleotides, and the effect could be reversed by addition of sufficient NaCl. The binding affinity and site size of this noncovalent interaction of poly(A,8-N3A) with 32 protein are similar to the values obtained for poly(A) and this protein. When [3H]poly(A,8-N3A)/32 protein mixtures were irradiated at 254 nm, fluorescence quenching was not reversed by NaCl, suggesting that the label was covalently bound to the protein. Mixtures of photolabel and protein subjected to short periods of irradiation (generally 1 min, 2000 erg mm-2) formed high molecular weight complexes, which when electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels were radioactive and stained with Coomassie Blue R. Under the same conditions, [3H]poly(A) failed to label 32 protein. The radioactivity of [3H]poly(A,8-N3A)-labeled complexes subjected to micrococcal nuclease after irradiation was seen to migrate just behind the free 32 protein monomer on SDS-polyacrylamide gels, indicating that portions of the photolabel not in direct contact with protein were accessible to this enzyme. By several criteria, we conclude that 32 protein was photolabeled specifically at its single-stranded nucleic acid binding site. Single-stranded nucleic acids with affinities for protein greater than that of poly(A,8-N3A) effectively inhibited photolabeling. The [NaCl] dependence of photolabeling monitored on SDS gels paralleled the NaCl reversal of (noncovalent) poly(A,8-N3A)-32 protein binding. Photolabeling reached a plateau after 1-2 min. The formation of high molecular weight complexes with increasing [poly(A,8-N3A)] paralleled the disappearance of free protein on SDS gels, and reached a saturation level of about 75% labeling. Several chromatographic procedures appear to be useful for the separation of the photolabeled complexes from free protein and photolabel. Limited trypsin hydrolysis of photolabeled 32 protein indicated that all the label was within the central ("III") portion of the protein. This approach should have general applicability to the identification of nucleic acid binding sites on helix-destabilizing proteins.
They have enzymatically synthesized /sup 3/H- and /sup 32/P-poly(A,8N/sub 3/A) from 8-N/sub 3/ADP... more They have enzymatically synthesized /sup 3/H- and /sup 32/P-poly(A,8N/sub 3/A) from 8-N/sub 3/ADP and radiolabeled ADP, and have used this polynucleotide to photoaffinity label T4 gene 32 protein, as well as several other helix-destabilizing proteins (HDPs). Irradiation of /sup 32/P-/sup 3/H-poly(A,N/sub 3/A) mixtures for short durations produces a covalent complex, seen as a high molecular weight, radioactive band on SDS-polyacrylamide gels. Preliminary experiments on other HDPs, from prokaryotic, eukaryotic and animal viral sources, show analogous results. Several successful control experiments indicate that this system is suitable for binding site localization on /sup 32/P. Single-stranded nucleic acids competitively inhibit photolabeling. The effect of NaCl on photolabeling parallels the salt-dependence of /sup 32/P-poly(A,N/sub 3/A) binding. Photolabeling reaches a plateau after approx.1 min, and the formation of the high molecular weight complex parallels the reduction of free /sup 32/P on SDS gels. Staph. nuclease digestion of crosslinked complexes produces a diffuse, radioactive band on SDS gels, migrating just behind free /sup 32/P. When these digested complexes are subjected to reverse-phase HPLC on a C3 Ultrapore column, the nucleic acid /sup 32/P-label is seen to coelute with protein. They are currently employing RP-HPLC methods to locate the label on tryptic peptides of nuclease-digested complexes.
Light-activated hydrolysis of cyclic GMP is achieved through the photoexcitation of rhodopsin, a ... more Light-activated hydrolysis of cyclic GMP is achieved through the photoexcitation of rhodopsin, a process which then triggers the replacement of GDP for GTP by a retinal guanosine 5′-triphosphatase referred to as ‘transducin’. The transducin-GTP complex then switches on the phosphodiesterase [Fung, Hurley & Stryer (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 152-156]. The bovine transducin consists of an alpha-subunit (39000 Mr), which is a GTP-binding component, together with a beta-(37000 Mr) and a gamma-subunit (10000 Mr). We have purified retinal transducin from cow, pig, chick and frog. The enzyme specific activities and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic profiles indicate that this enzyme is similar in all species except the frog. Whereas the bovine, pig and chick transducins consist of major 37000- and 39000-Mr components, that of the frog consists of a single 75000-Mr component. Labelling of the GTP-binding components with the photoaffinity label 8-azidogu...
Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthe... more Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking [125I]PF-M (see Figure 1 for structure) to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifical...
Previous studies have identified the guanine and adenine binding domains of the GTP and ADP bindi... more Previous studies have identified the guanine and adenine binding domains of the GTP and ADP binding sites of GDH. In this study the peptide sequences within or near to the terminal phosphate‐binding domains of the GTP and ADP binding sites of bovine liver glutamate dehydrogenase (GDH) were identified using photoaffinity labeling with the benzophenone nucleotide derivatives, [γ‐32P]GTPγBP and [γ‐32P]ATPγBP. Without activating light, GTPγBP exhibited inhibiting effects on the GDH reaction similar to GTP; ATPγBP, as expected, produced activating effects similar to those of ADP. Photoinsertion into GDH by both probes exhibited saturation effects in agreement with the respective kinetic effects. Specificity of labeling was supported by specific and effective reduction of photoinsertion of [γ‐32P]GTPγBP and [γ‐32P]ATPγBP into GDH by GTP and ADP, respectively. Using a combination of immobilized Fe3+‐chelate affinity chromatography and reversed‐phase HPLC, photolabeled peptides located with...
8-azido-ATP, when used in the 0.2-5 vM concentration range, fulfills the criteria for a specific ... more 8-azido-ATP, when used in the 0.2-5 vM concentration range, fulfills the criteria for a specific photoaffinity label for the (Ca+Mg)ATPase of sarcoplasmic reticulum. It is a substrate for the enzyme. It is a mixed inhibitor of ATPase activity. When photolyzed at 0" it is an inhibitor of ATPase activity. promoted The photoinduced binding of 8-azido-ATP to the (Ca+Mg)ATPase is by Ca2 The dependence of the labeling of the (Ca+Mg)ATPase on 8-azido-ATP, Ca2+ and Mg2 concentrations strongly suggests that 2 classes of sites are labeled. When lo-60 PM 8-azido-ATP was used to label sarcoplasmic reticulum, proteins in addition to the (Ca+Mg)ATPase were labeled.
Subcellular fractions from tobacco (N. rustica) leaves were photolabelled with the UDPG analog, (... more Subcellular fractions from tobacco (N. rustica) leaves were photolabelled with the UDPG analog, ({sup 32}P)-N{sub 3} UDPG. Two stromal polypeptides (M{sub r} = 42 and 21 kD) were the major photolabelled chloroplast polypeptides. UDPG protected the 42 but not the 21 ...
ABSTRACT The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymati... more ABSTRACT The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from [gamma-32P]2-azidoATP and [alpha-32P]8-azidoATP by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p3A4[32P]pCp from RNase L with affinity equivalent to p3A3 (IC50 = 2 X 10(-9) M in radiobinding assays). The 8-azido photoprobe also activates RNase L to hydrolyze poly(U) [32P]pCp 50% at 7 X 10(-9) M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p3A3 activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10(-9) M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The [gamma-32P]2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the [alpha-32P]8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p3A3 allosteric binding site was obtained as follows.(ABSTRACT TRUNCATED AT 250 WORDS)
The photoaffinity probes [ Y-~~P ] 2-azidoATP (2-N3ATP) and [~x-~~P]8-azido-ATP (8-N3ATP) were us... more The photoaffinity probes [ Y-~~P ] 2-azidoATP (2-N3ATP) and [~x-~~P]8-azido-ATP (8-N3ATP) were used to investigate the binding of ATP to highly purified 2-5A synthetase. 2-N3ATP and 8-N3ATP are substrates for 2-5A synthetase [Suhadolnik, R.
Biochemical and Biophysical Research Communications, 1987
Nucleotide binding sites on DNA-dependent RNA polymerase from E. coli have been studied by photoa... more Nucleotide binding sites on DNA-dependent RNA polymerase from E. coli have been studied by photoaffinity labeling with a GTP analog [gamma-32P]-8-AzidoGTP and a guanosine-3'-diphosphate-5'-diphosphate analog, 8-Azidoguanosine-3'-phosphate-5'-85'-32P]phosphate. The guanosine diphosphate photoprobe labeled the beta, beta', and sigma subunits with the sigma subunit being most heavily labeled. The GTP photoprobe also labeled the beta, beta', sigma subunits but the beta' subunit was most heavily labeled. In competition experiments guanosine-3'-diphosphate-5'-diphosphate decreased photolabeling by 8-Azidoguanosine-3'-phosphate-5'-[5'-32P]phosphate better than GTP, while the opposite was true for photolabeling with [gamma-32P]8- AzidoGTP. The guanosine diphosphate photoprobe inhibited transcription on E. coli DNA with Ki of ca. 150 microM. Present studies suggest a unique ppGpp binding site distinct from substrate binding site(s) and this photoprobe may be used to localize this binding site(s).
A photoaffinity analog of cyclic AMP, 8-azidoadenosine-3',5'-monophosphate (8-N,cAMP), and a line... more A photoaffinity analog of cyclic AMP, 8-azidoadenosine-3',5'-monophosphate (8-N,cAMP), and a linear gradient sodium dodecyl sulfate-polyacrylamide gel system have been used to photolabel and separate possible cyclic AMP binding sites in whole cells of the sarcoma 37 line. At least four sites within the whole sarcoma 37 cell bind l~2P]8-N,cAMP such that they are saturated at 1.0 pM concentration. These four binding moieties appear to have measurably different affinities for 8-N,cAMP and the number of total sites labeled per cell differs with each binding moiety. Cell fractionation allows separation of some of the photolabeled sites and gives an indication as to the geographical location of these binding sites within the whole cell. Enrichment by cell fractionation allows additional photolabeled sites to be observed.
Guanosine triphosphate (GTP) is an absolute requirement for tubulin polymerization in situ. The n... more Guanosine triphosphate (GTP) is an absolute requirement for tubulin polymerization in situ. The nucleotide photoaffinity probe 8‐azidoguanosine 5′‐triphosphate (8N3GTP) has been shown to be a biological mimic of GRP in this system and, also, an effective active site probe of the exchangeable GRP binding site. Using [32P]8N3GTP we demonstrate that the exchangeable GTP site of the beta subunit of tubulin is available to added guanine nucleotide in normal aged brain homogenates, whereas it is variably unavailable in Alzheimer's diseased brain. Inability of 8N3GTP to photolabel beta tubulin appears to be associated with neurofibrillary tangle density. These results support the hypothesis that microtubule formation is abnormal in brains affected by Alzheimer's disease.
The causes of autism and neurodevelopmental disorders are unknown. Genetic and environmental risk... more The causes of autism and neurodevelopmental disorders are unknown. Genetic and environmental risk factors seem to be involved. Because of an observed increase in autism in the last decades, which parallels cumulative mercury expo-sure, it was proposed that autism may be in part caused by mercury. We review the evidence for this proposal. Several epidemiological studies failed to find a cor-relation between mercury exposure through thimerosal, a preservative used in vaccines, and the risk of autism. Recently, it was found that autistic children had a higher mercury exposure during pregnancy due to maternal dental amalgam and thimerosal-containing immunoglobulin shots. It was hypothesized that children with autism have a decreased detoxification capacity due to genetic polymor-phism. In vitro, mercury and thimerosal in levels found several days after vaccina-tion inhibit methionine synthetase (MS) by 50%. Normal function of MS is crucial in biochemical steps necessary for brain develo...
Sucrose phosphate synthase (SPS) was isolated from spinach leaves by precipitation with polyethyl... more Sucrose phosphate synthase (SPS) was isolated from spinach leaves by precipitation with polyethylene glycol, ion-exchange and hydrophobic interaction chromatography, and rate zonal centrifugation. The enzyme was purified more than 600-fold to a specific activity of 57 mumol/min/mg protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that a 120-kDa polypeptide was enriched through purification and was the major polypeptide in the final SPS preparation. The 120-kDa polypeptide was photoaffinity labeled with the substrate analog, 5-azidouridine [beta-32P]5'-diphosphate-glucose ([beta-32P]5-N3UDP-Glc). Covalent incorporation of 5-N3UDP-Glc into the 120-kDa polypeptide exhibited an apparent Kd of 74 microM, similar to the apparent Ki for inhibition of SPS activity by unphotolyzed 5-N3UDP-Glc. Competition experiments showed that photolabeling of the 120-kDa polypeptide by 5-N3UDP-Glc was reduced in the presence of UDP-Glc, exhibiting an apparent Ki value that was similar to the apparent Km (UDP-Glc) of 2.9 mM for the purified enzyme. The relative molecular mass of the SPS holoenzyme was 253,000, and the isoelectric point of the 120-kDa subunit was 5.2. The data confirmed the identity of the 120-kDa polypeptide as the SPS subunit, established the structure of the active enzyme as a dimer, and demonstrated active-site labeling of SPS by a photoaffinity analog of the substrate.
The radioactive, photoactivatable labeling probe [β-32 P]5-azidouridine 5′-diphosphoglucose has r... more The radioactive, photoactivatable labeling probe [β-32 P]5-azidouridine 5′-diphosphoglucose has recently been shown to label a 62-kDa protein in crude homogenates and in partially purified enzyme preparations without photoactivation. Here, we report that a portion of this ...
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Papers by Boyd Haley