Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An experiment was designed to investigate the reaction mechanism of AP (apurinic or apyrimidinic) DNA endonucleases (APcI, APcII, APcIII) purified from rat liver chromatin. Sulfhydryl compounds (2-mercaptoethanol, dithiothreitol) brought about optimal activities of AP DNA endonucleases and N-ethylmaleimide or HgCl2 inhibited the enzyme activities, indicating the presence of sulfhydryl group at or near the active sites of the enzymes. Mg2+ was essential and 4mM of Mg2+ was sufficient for the optimal activities of AP DNA endonucleases. Km values of APcI, APcII and APcIII for the substrate (E. coli chromosomal AP DNA) were 0.53, 0.27 and 0.36 microM AP sites, respectively. AMP was the most potent inhibitor among adenine nucleotides tested and the inhibition was uncompetitive with respective to the substrate. The Ki values of APcI, APcII and APcIII were 0.35, 0.54 and 0.41mM, respectively. The degree of nick translation of AP DNAs nicked by APcI, APcII and APcIII with Klenow fragment in the presence and absence of T4 polynucleotide kinase or alkaline phosphatase were the same, suggesting that all 3 AP DNA endonucleases excise the phosphodiester bond of AP DNA strand to release 3-hydroxyl nucleotides and 5-phosphomonoester nucleotides.
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PMID:Studies on rat liver nuclear DNA damaged by chemical carcinogen (3'-Me DAB) and AP DNA endonuclease. II. Kinetic properties of AP DNA endonucleases in rat liver chromatin. 171 Sep

A 32-P-postlabeling assay has been developed that permits detection of several radiogenic base and sugar lesions of DNA at the femtomole level. The technique is based on the inability of DNase I and snake venom phosphodiesterase to cleave the internucleotide phosphodiester bond immediately 5' to the site of damage so that complete digestion of irradiated DNA with these nucleases and alkaline phosphatase yields lesion-bearing "dinucleoside" monophosphates. Because these fragments contain an unmodified nucleoside at the 5'-end of each molecule, they can be readily phosphorylated by T4 polynucleotide kinase and [gamma-32P]ATP and analyzed by polyacrylamide gel electrophoresis and reverse-phase HPLC. We observed a linear induction of total damage in DNA irradiated with 5-50 Gy. Virtually no damage was detected when the DNA was irradiated in solution containing 1 M DMSO, implicating hydroxyl radicals in the formation of these lesions. Evidence for the presence of thymine glycols and phosphoglycolate groups came from (i) a comparison of the radiation-induced products with those produced by OsO4 and KMnO4 and (ii) incubation of irradiated DNA with Escherichia coli endonuclease III and exonuclease III before analysis by the postlabeling procedure. This was confirmed by comigration of the radiogenic products with chemically synthesized markers. G values of 0.0022 and 0.0105 mumol J-1 were obtained for thymine glycol and phosphoglycolate production, respectively. The identity of the 5'-nucleotide of each isolated compound was obtained by nuclease P1 digestion. This analysis of nearest-neighbor bases to thymine glycols and phosphoglycolates indicated a nonrandom interaction between radiation-induced hydroxyl radicals and DNA.
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PMID:32P-postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and phosphoglycolate termini. 184 59

We have detected the in situ activities of DNA glycosylase, endonuclease, exonuclease, DNA polymerase, and DNA ligase using a novel polyacrylamide activity gel electrophoresis procedure. DNA metabolizing enzymes were resolved through either native or SDS-polyacrylamide gels containing defined 32P-labeled oligonucleotides annealed to M13 DNA. After electrophoresis, these enzymes catalyzed in situ reactions and their [32P]DNA products were resolved from the gel by a second dimension of electrophoresis through a denaturing DNA sequencing gel. Detection of modified (degraded or elongated) oligonucleotide chains was used to locate various enzyme activities. The catalytic and physical properties of Novikoff hepatoma DNA polymerase beta were found to be similar under both in vitro and in situ conditions. With 3'-terminally matched and mismatched [32P]DNA substrates in the same activity gel, DNA polymerase and/or 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I (large fragment), DNA polymerase III (holoenzyme), and exonuclease III were detected and characterized. In addition, use of matched and mismatched DNA primers permitted the uncoupling of mismatch excision and chain extension steps. Activities first detected in nondenaturing activity gels as either multifunctional or multimeric enzymes were also identified in denaturing activity gels, and assignment of activities to specific polypeptides suggested subunit composition. Furthermore, DNA substrates cast within polyacrylamide gels were successfully modified by the exogenous enzymes polynucleotide kinase and alkaline phosphatase before and after in situ detection of E. coli DNA ligase activity, respectively. Several restriction endonucleases and the tripeptide (Lys-Trp-Lys), which acts as an apurinic/apyrimidinic endonuclease, were able to diffuse into gels and modify DNA. This ability to create intermediate substrates within activity gels could prove extremely useful in delineating the steps of DNA replication and repair pathways.
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PMID:Characterization of DNA metabolizing enzymes in situ following polyacrylamide gel electrophoresis. 200 53

Two generally applicable [35S]phosphorothioate postlabeling procedures for the HPLC analysis of polycyclic aromatic hydrocarbon (PAH)-DNA adducts have been developed based upon [32P]phosphate postlabeling assays described by Gupta and Randerath et al. In one procedure, benzo[a]pyrene (B[a]P)-modified DNA was digested to nucleoside 3'-phosphates by micrococcal nuclease and spleen phosphodiesterase and the adducted nucleotides were extracted with 1-butanol. The adducted nucleoside-3'-phosphates were 5'-thiophosphorylated by T4 polynucleotide kinase (T4PNK) and adenosine 5'-O-(3-[35S]thiotriphosphate) to yield [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts. Although thiophosphorylation of B[a]P-DNA adducts was slower than the corresponding phosphorylation reaction, similar recoveries of the postlabeled adducts were achieved with longer incubation times and higher concentrations of T4PNK. A major advantage of this procedure over the 32P-postlabeling procedure is that the resistance of phosphorothioates to degradation by phosphatases allows selective removal of the unlabeled 3'-phosphate from the [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts by brief treatment with alkaline phosphatase. [35S]B[a]P-nucleoside-5'-phosphorothioate adducts were also prepared using a nuclease P1/prostatic acid phosphatase DNA degradation method. For anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-modified DNA, overall adduct recoveries were substantially higher with the nuclease P1/prostatic acid phosphatase method (48-51%) than with the micrococcal nuclease/spleen phosphodiesterase/alkaline phosphatase method (22-29%). There were no significant differences in the HPLC profiles of the [35S]phosphorothioate-postlabeled adducts obtained from these two procedures. HPLC analysis of B[a]P-DNA adducts formed in B[a]P-treated hamster embryo cell cultures demonstrated the formation of two major adducts, (+)syn-BPDE-deoxyguanosine-5'-phosphorothioate and (+)anti-BPDE-deoxyguanosine-5'-phosphorothioate, along with other minor adducts. Based upon an overall adduct recovery of 20% and 0.5 mol as the detection limit of this 35S-postlabeling/HPLC assay, the sensitivity of this assay is 1 adduct/10(8) nucleotides for a 60 micrograms DNA sample. This method offers the advantages of using 35S which has a longer half-life and lower radioactive decay energy than 32P and the ability to prepare PAH-DNA adducts at the monophosphorothioate level which greatly facilitates separation of individual 35S-postlabeled PAH-DNA adducts by HPLC.
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PMID:Detection and identification of benzo[a]pyrene-DNA adducts by [35S]phosphorothioate labeling and HPLC. 202 54

Previous work has shown that 7,12-dimethylbenz[a]anthracene (DMBA)-DNA adducts can be converted by 32P-postlabeling to different types of radiolabeled derivatives (nucleoside 3',5'-bisphosphates, nucleoside 5'-monophosphates and dinucleotides), and that the 32P-labeled 3',5'-bisphosphate derivatives can be further characterized by cross-referencing with 3H-labeled nucleoside DMBA adducts, for which structural information is available. This work has now been extended by TLC comparisons of 5'-monophosphate and 3',5'-bisphosphate DMBA adducts. To this end, DMBA-modified DNA was enzymatically hydrolyzed to 3'-monophosphates (Xp + Np) or to dinucleotides (XpN), and these digestion products were 32P-postlabeled by published procedures to yield 3',5'-bisphosphate (*pXp) or 5'-monophosphate (*pX) adducts. Individual *pXp and *pX fractions were isolated from polyethyleneimine (PEI)-cellulose TLC maps and chromatographically compared after enzymatic 3'-dephosphorylations of the 3',5'-bisphosphate (*pXp) derivatives (*pXp----*pX + Pi). Four reactions were standardized and employed for this purpose: (i) 3'-dephosphorylation by extensive digestion with nuclease P1; (ii) 3'-dephosphorylation catalyzed by polynucleotide kinase; (iii) partial dephosphorylation by bacterial alkaline phosphatase; and (iv) partial dephosphorylation by prostatic acid phosphatase. Individual DMBA adducts displayed marked differences with regard to their susceptibility to enzymatic dephosphorylation. The three major and most minor postlabeled 5'-monophosphate DMBA adducts were cross-referenced this way with 3',5'-bisphosphate and nucleoside adducts, so that specific dihydrodiol epoxide-nucleoside 5'-monophosphate adducts can now be identified and measured by 32P-postlabeling.
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PMID:High-resolution TLC mapping and characterization of 32P-postlabeled monophosphate 7,12-dimethylbenz[a]anthracene-DNA adducts. 210 82

We have examined the capacity of bacteriophage T4 polynucleotide kinase (EC 2.7.1.78) to phosphorylate the partially depurinated products of d-ApA, namely, d-SpA and d-ApS (where S represents an apurinic deoxyribose group). It was observed that the enzyme acted only on the latter isomer. Since molecules of this type (d-NpS) are the sole apurinic site containing products resulting from the combined digestion of lightly depurinated DNA by snake venom phosphodiesterase and calf alkaline phosphatase [Weinfeld, M., Liuzzi, M., & Paterson, M. C. (1989) Nucleic Acids Res. 17, 3735-3745], we were able to devise a postlabeling assay for these biologically important DNA lesions. The method offers several advantages, including (a) elimination of the need for prelabeled DNA, (b) high (femtomole range) sensitivity, and (c) nearest-neighbor analysis of bases 5' to apurinic/apyrimidinic sites. Using this assay, we obtained a value for the rate of depurination of form I pRSVneo plasmid DNA, incubated at pH 5.2 at 70 degrees C, of approximately 3.3 apurinic sites per plasmid molecule per hour. This value compares favorably with previously published data of others, acquired by alternative approaches. The rate of depurination of poly(dA), treated in a similar fashion, was found to be approximately 1 base per 10(3) nucleotides per hour.
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PMID:Response of phage T4 polynucleotide kinase toward dinucleotides containing apurinic sites: design of a 32P-postlabeling assay for apurinic sites in DNA. 215 12

Phage T4 polynucleotide kinase (EC 2.7.1.78) proved incapable of catalyzing the phosphorylation of thymidylyl-(3'----5')-thymidine containing either a cis-syn-cyclobutane pyrimidine dimer (d-T less than p greater than T) or a 6-4'-[pyrimidin-2'-one]pyrimidine photoproduct (d-T[p]-T), and similarly the UV-modified compounds of (dT)3 bearing either photoproduct at their 5'-end (d-T less than p greater than TpT and d-T[p]TpT). In contrast, the 3'-structural isomers of these trinucleotides (d-TpT less than p greater than T and d-TpT[p]T) were phosphorylated at the same rate as the parent compound. These phosphorylatable lesion-containing oligonucleotides are quantitatively released from UV-irradiated poly(dA):poly(dT) by enzymatic hydrolysis with snake venom phosphodiesterase and alkaline phosphatase (Liuzzi, M., Weinfeld, M., and Paterson, M. C. (1989) J. Biol. Chem. 264, 6355-6363). By combining this digestion regimen with phosphorylation by polynucleotide kinase and [gamma-32P]ATP, pyrimidine dimers were quantitated at the fmol level following exposure of poly(dA):poly(dT) and herring sperm DNA to biologically relevant UV fluences. The rate of dimer induction in the synthetic polymer, approximately 10 dimers/10(6) nucleotides/Jm-2, was in close agreement with that obtained by conventional methods. Dimers were induced at one-fourth of this rate in the natural DNA. Further treatment of the phosphorylated oligonucleotides derived from irradiated herring sperm DNA with nuclease P1 released the labeled 5'-nucleotide, thus permitting analysis of the nearest-neighbor bases 5' to the lesions. We observed a ratio for pyrimidine-to-purine bases of almost 6:1, implicating tripyrimidine stretches as hotspots for UV-induced DNA damage.
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PMID:Enzymatic analysis of isomeric trithymidylates containing ultraviolet light-induced cyclobutane pyrimidine dimers. II. Phosphorylation by phage T4 polynucleotide kinase. 253 80

The properties of a DNA-repair endonuclease isolated from mouse plasmacytoma cells have been further studied. It acted on ultraviolet-light-irradiated supercoiled DNA, and the requirement for a supercoiled substrate was absolute at ultraviolet light doses below 1.5 kJ m-2. At higher doses relaxed DNA could also serve as a substrate, but the activity on this DNA was due mostly to hydrolysis of ultraviolet-light-induced apurinic/apyrimidinic (AP) sites by the AP-endonuclease activity associated with the enzyme. The latter enzyme activity did not require a supercoiled form of the DNA. The enzyme also introduced nicks in unirradiated d(A-T)n. The nicked ultraviolet-light-irradiated DNA served as a substrate for DNA polymerase I, showing that the nicks contained free 3'-OH ends. Treatment of the nicked ultraviolet-light-irradiated DNA with bacterial alkaline phosphatase followed by T4 polynucleotide kinase, resulted in the phosphorylation of the 5' ends of the nicks, indicating that the nicks possessed a 5'-phosphate group; 5'- and 3'-mononucleotide analyses of the labelled DNA suggested that the enzyme introduced breaks primarily between G and T residues. The enzyme did not act on any specific region on the supercoiled DNA molecule; it produced random nicks in ultraviolet-light-modified phi X 174 replicative form I DNA. Antibodies raised against ultraviolet-light-irradiated DNA inhibited the activity. DNA adducts such as N-acetoxy-2-acetylaminofluorene and psoralen were not recognized by the enzyme. It is suggested that the enzyme has a specificity directed toward helical distortions.
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PMID:Properties of a DNA repair endonuclease from mouse plasmacytoma cells. 258 76

Polyadenylated [poly(A)+] RNA molecules have been isolated from Methanococcus vannielii by oligodeoxythymidylate-cellulose affinity chromatography at 4 degrees C. Approximately 16% of the label in RNA isolated from cultures allowed to incorporate [3H]uridine for 3 min at 37 degrees C was poly(A)+ RNA. In contrast, less than 1% of the radioactivity in RNA labeled over a period of several generations was contained in poly(A)+ RNA molecules. Electrophoretic separation of poly(A)+ RNA molecules showed a heterogeneous population with mobilities indicative of sizes ranging from 900 to 3,000 bases in length. The population of poly(A)+ RNA molecules was found to have a half-life in vivo of approximately 12 min. Polyadenylate [poly(A)] tracts were isolated by digestion with RNase A and RNase T1 after 3' end labeling of the poly(A)+ RNA with RNA ligase. These radioactively labeled poly(A) oligonucleotides were shown by electrophoresis through DNA sequencing gels to average 10 bases in length, with major components of 5, 9, 10, 11, and 12 bases. The lengths of these poly(A) sequences are in agreement with estimates obtained from RNase A and RNase T1 digestions of [3H]adenine-labeled poly(A)+ RNA molecules. Poly(A)+ RNA molecules from M. vannielii were labeled at their 5' termini with T4 polynucleotide kinase after dephosphorylation with calf intestine alkaline phosphatase. Pretreatment of the RNA molecules with tobacco acid pyrophosphatase did not increase the amount of phosphate incorporated into poly(A)+ RNA molecules by polynucleotide kinase, indicating that the poly(A)+ RNA molecules did not have modified bases (caps) at their 5' termini. The relatively short poly(A) tracts, the lack of 5' cap structures, and the instability of the poly(A)+ RNA molecules isolated from M. vannielii indicate that these archaebacterial poly(A)+ RNAs more closely resemble eubacterial mRNAs than eucaryotic mRNAs.
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PMID:Polyadenylated, noncapped RNA from the archaebacterium Methanococcus vannielii. 258 34

Satellite tobacco mosaic virus (STMV) is a plant virus with a 17-nm icosahedral particle encapsidating a 0.3 X 10(6) Mr ssRNA genome that depends on tobamoviruses for its replication. The complete nucleotide sequence of STMV RNA deduced in the experiments described here was 1059 nucleotides in length. The efficiency of labeling viral RNA with [gamma-32P]ATP using T4 polynucleotide kinase was not affected by treatment with tobacco acid pyrophosphatase and/or bacterial alkaline phosphatase, indicating that the majority of the 5' termini of encapsidated STMV RNAs were not phosphorylated. The 240 3'-terminal nucleotides of STMV RNA and either tobacco mosaic virus (TMV) U1 RNA or TMV U2/U5 RNA had greater than 65% overall sequence similarity, with two nearly identical regions of 40 and 50 bases, respectively. There were no other regions of sequence relatedness to TMV RNA. The 19 5'-terminal nucleotides of STMV RNA had greater than 65% sequence similarity with the 16 5'-terminal nucleotides of brome mosaic virus (RNA 3 and 50% sequence similarity with the 12 5'-terminal nucleotides of the Q strain of cucumber mosaic virus RNA 3. The first open reading frame (ORF) beginning at base 53 encoded a 6800 Mr protein that corresponded in size to a major in vitro translation product directed by STMV RNA. A second ORF, beginning at nucleotide 163, had the capacity to code for a protein that corresponded in size (17,500 Mr) to the other major in vitro translation product. The first 12 codons of this ORF corresponded to the sequence of the N-terminal amino acids of the capsid protein. Western-blot analysis of the in vitro translation products revealed that the 17,500 Mr protein had the same electrophoretic mobility as the authentic capsid protein; it was also antigenically related to the capsid protein, but the 6800 Mr protein was not. Time course analysis of in vitro translation demonstrated that the 6800 Mr protein was synthesized at the same time as the capsid protein and did not arise by the proteolytic cleavage of a larger precursor polypeptide. These results suggest that the genome of STMV functioned as a polycistronic messenger RNA. It has not been determined if the 6800 Mr protein is synthesized in vivo. STMV RNA had untranslated regions of 52 and 418 nucleotides at its 5' and 3' termini, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Nucleotide sequence and translation of satellite tobacco mosaic virus RNA. 271 78


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