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)

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

In wild-type mycelial cultures of Neurospora crassa under Pi-limited conditions, alkaline phosphatase, cyclic phosphodiesterases I, II, III, and IV, 5'-nucleotidase, acid and alkaline nucleases, RNase N1, and a newly detected endonuclease were secreted into the culture media. These enzymes were either not produced or were produced in very reduced levels in mutants nuc-1, -2, -3, -4, -5, -6, and -7 and cpd-4. The proteins were examined by polyacrylamide gel electrophoresis in a manner which allowed the identification of each of them.
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PMID:Characterization of Pi-repressible enzymes secreted in culture media by Neurospora crassa wild-type cells and null-type mutants. 282 Sep 43

Under phoA promoter control, TaqI endonuclease was overproduced to 5% of Escherichia coli cellular proteins. This was achieved by fusing the endonuclease gene to the first four codons of the alkaline phosphatase signal sequence. For maximal overproduction (30% of cellular proteins), a putative 14-bp hairpin within the endonuclease coding sequence was replaced with degenerate codons. In addition, TaqI methylase was required to protect host DNA. The endonuclease was purified in sufficient amounts for crystallization.
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PMID:Overproduction, purification and crystallization of TaqI restriction endonuclease. 284 31

DNA-dependent ATPase IV has been purified to near homogeneity from the Novikoff rat hepatoma. The enzyme is devoid of DNA polymerase, RNA polymerase, exonuclease, endonuclease, phosphomonoesterase, 3'- or 5'-phosphodiesterase, polynucleotide kinase, protein kinase, topoisomerase, helicase or DNA reannealing activities at a detection level of 10(-5) to 10(-7) relative to the ATPase activity. The enzyme is a monomer of Mr 110,000, has a sedimentation coefficient of 5.9 S, a Stokes radius of 40 A and a frictional coefficient of 1.32. In the presence of Mg2+ ion and a polynucleotide effector, ATPase IV hydrolyzes either ATP or dATP to the nucleoside diphosphate plus Pi. Other ribo- or deoxyribonucleoside triphosphates are not substrates. ATPase IV utilizes double-stranded DNA and single-stranded DNA as effector; however, it does not utilize poly(dT). The Km for dsDNA or ssDNA is 2.2 microM (nucleotide). A variety of ATP analogues were found to be competitive inhibitors of ATPase IV.
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PMID:Purification and enzymological characterization of DNA-dependent ATPase IV from the Novikoff hepatoma. 296 5

Various factors influencing the detection of human cytomegalovirus (HCMV) in infected cells by DNA-DNA hybridization have been investigated. Employing the Hind III O fragment of HCMV AD169 labelled with 32P, we found that detection sensitivity was highly influenced by the method employed for extraction of DNA from infected cells. Excision of the Hind III O fragment from the vector by restriction endonuclease digestion prior to 32P-labelling further improved the detection capability of the probe. Similarly, cytomegalovirus (CMV) DNA detection employing biotin-labelled probes and streptavidin/alkaline phosphatase in the hybridot assay was also highly dependent on the method of DNA extraction prior to hybridization. Finally, we describe an in situ assay employing a biotin-labelled probe and fluorescein-conjugated avidin to detect CMV DNA in cultured cells.
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PMID:Detection of cytomegalovirus by DNA-DNA hybridization employing probes labelled with 32-phosphorus or biotin. 299 36

The catalytic properties of the HhaII restriction endonuclease were studied using plasmid pSK11 DNA containing a single 5'-G-A-N-T-C HhaII cleavage site as substrate. Reactions were followed by two methods: 1) gel electrophoretic analysis of nicked circular and linear DNA products, or 2) release of 32P-labeled inorganic phosphate from specifically labeled HhaII sites in a reaction coupled with bacterial alkaline phosphatase. The enzyme is optimally active at 37 degrees C in 10 mM Tris-HCl (pH 9.1) and 4-10 mM MgCl2 without added NaCl. Activity is stabilized by the presence of 2-mercaptoethanol and 0.2% Triton X-100 or 50 microgram/ml bovine serum albumin. At enzyme concentrations below 10 nM and using pSK11 as substrate, initial kinetic rates were dependent on the order of mixing of reactants. A lag of 3-4 min was observed if enzyme or substrate was added last. Preincubation of substrate and enzyme followed by initiation of the reaction with MgCl2 or preincubation of the enzyme with nonspecific DNA followed by initiation with substrate eliminated or reduced the lag, respectively, and speeded up the reactions. Under a wide range of reaction conditions, nicked pSK11 DNA accumulated early, while linear molecules appeared later, suggesting that HhaII cleaves one strand at a time in separate binding events. The apparent Km for covalently closed pSK11 DNA molecules was approximately 17 nM, and the turnover number for the conversion of covalent to nicked sites was 1.1 single strand scissions/min. Pre-steady state kinetic analysis indicated that cleavage of the first phosphodiester bond in a site is first order with a rate constant of about 0.8 min-1, while cleavage of the second phosphodiester bond is first order with a rate constant of about 0.2 min-1.
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PMID:Catalytic properties of the HhaII restriction endonuclease. 299 12

A sensitive enzyme-linked immunosorbent capture assay using biotin and streptavidin (capture B/SA ELISA) was developed using type-specific monoclonal antibodies for typing of herpes simplex virus. Rabbit anti-herpes simplex virus immunoglobulin G was used as the capturing antibody, and biotin-linked type-1-specific mouse monoclonal antibody or rabbit type-1- or type-2-specific polyclonal antibody served as the detecting antibody. The captured antigen was detected by an ELISA with alkaline phosphatase-conjugated streptavidin, which reacted with biotin molecules on the detector antibody. The capture B/SA ELISA was compared with other methods for efficiency and reliability in typing. Results obtained by restriction endonuclease digestion of the radiolabeled viral genome were used to determine the type (1 or 2) of clinical isolates. These results were then used as a reference for determining the accuracy of the capture B/SA ELISA, as well as that of the immunofluorescence method, both of which are easily adaptable for use in the clinical laboratory. The three methods were in perfect agreement. It was determined that both the capture B/SA ELISA and the immunofluorescence method using monoclonal antibodies provided typing results with 100% specificity and 100% sensitivity and thus were accurate and reliable. However, the ELISA was the method of choice because of its simplicity, rapidity, and use of nonradioisotopic reagents.
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PMID:Typing of herpes simplex virus by capture biotin-streptavidin enzyme-linked immunosorbent assay and comparison with restriction endonuclease analysis and immunofluorescence method using monoclonal antibodies. 302 48

The potentiation of radiation damage, which can be accomplished by the inhibition of repair, is estimated from published studies of repair deficient mutants. Sensitization factors as high as 10 have been achieved. Because it has previously been suggested that the most probable lethal lesion is a DNA double strand break (DSB), it is not surprising that cells deficient in repairing this type of damage are the most radiosensitive. The structures of DNA DSBs and other Locally Multiply Damaged Sites (LMDS) (involving both single strand breaks (SSB) and base damage sites) are reviewed, together with the processes by which cells may attempt to repair these lesions. Repair processes occur in competition with damage fixation, again, mechanisms of damage fixation are predicted from studies in model systems. A strategy for inhibiting the repair processes is devised that consists of holding the first SSB constituent of the LMDS open by repairing in the presence of deoxynucleoside analogues, such as ara-C, so that there is a higher probability of the formation of a DSB upon cleavage of the second site (on the other strand) by hydrolysis of a labile bond or by endonuclease cleavage of a base damaged site. To achieve preferential sensitization of tumor vs. normal tissue it may be possible to take advantage of the deficiency in alkaline phosphatase in tumor vs. normal vasculature, that is, in analogy with treatment with WR-2721. The deoxynucleoside analogue would be delivered together with the phosphate ester (deoxynucleotide) of the correct deoxynucleoside, for example, ara-C, in the presence of deoxycytidine monophosphate (dCMP). Higher alkaline phosphatase levels in normal tissue capillaries would hydrolyse the dCMP to deoxycytidine, which competes effectively with ara-C in repair replication.
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PMID:Mechanisms of DNA repair and their potential modification for radiotherapy. 352 85

We describe a system to generate cDNA or genomic libraries from DNA segments that have blunt termini. Background and rearrangement levels are low, but efficiencies are high and the procedural times very short. T4 ligase in the presence of polyethylene glycol produces high Mr oligomers of vector and insert. These concatemers are reduced to vector-insert monomers at a high frequency by subsequent cleavage with a restriction endonuclease, which recognises the insert rarely, if at all, and the vector once. The monomers are recircularised under standard ligation conditions prior to transformation. Thus insertion conditions are optimised independently of those for recircularisation. All reading frames for expression libraries are generated by short BAL 31 cleavage followed by the blunt-end cloning procedure. Similarly, genomic expression libraries can be made by BAL 31 or mung-bean nuclease treatment after cleavage with DNase I is the presence of Mn2+. The technique is suitable for any DNA segment that is blunt-ended or can be made so. When the vector is treated with alkaline phosphatase, recombinants are generated at a frequency greater than 90% and have single inserts. Yields are 3-5 X 10(6) colony-forming units per micrograms of insert.
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PMID:Rapid and efficient method for cloning of blunt-ended DNA fragments. 359 40

phi X174 RFI DNA treated with bleomycin (BLM) under conditions permitting nicking does not serve as a template-primer for Escherichia coli DNA polymerase I. Purified exonuclease III from E. coli and extracts from wild-type E. coli strains are able to convert the BLM-treated DNA to suitable template-primer, but extracts from exonuclease III deficient strains are not. Brief digestion by exonuclease III is enough to create the template-primer, suggesting that the exonuclease III is converting the BLM-treated DNA by a modification of 3' termini. The exonucleolytic rather than the phosphatase activity of exonuclease III appears to be involved in the conversion. Comparative studies with micrococcal nuclease indicate that BLM-created nicks do not have a simple 3'-P structure. Bacterial alkaline phosphatase does not convert BLM-treated DNA to template-primer. The endonuclease VI activity associated with exonuclease III does not incise DNA treated with BLM under conditions not allowing nicking, in contrast to DNA with apurinic sites made by acid treatment, arguing that conversion does not require the endonuclease VI action on uncleaved sites.
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PMID:Synthesis by DNA polymerase I on bleomycin-treated deoxyribonucleic acid: a requirement for exonuclease III. 616 81


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