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

---

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

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.
...
PMID:Rapid and efficient method for cloning of blunt-ended DNA fragments. 359 40

The use of gas chromatography-mass spectrometry (GC-MS) for characterization of free radical-induced base damage to DNA is presented. Damage introduced to DNA by reactive oxygen species such as hydroxyl radicals appears to play an important role in mutagenesis, carcinogenesis and aging. Elucidation of the chemical nature of such DNA lesions is necessary for the assessment of their biological consequences and enzymatic repair. DNA exposed to radiation-generated hydroxyl radicals in aqueous solution was hydrolyzed to 2'-deoxyribonucleosides with a mixture of DNase I, venom and spleen exonucleases and alkaline phosphatase. The hydrolysate was subsequently trimethylsilylated and analyzed by GC-MS. A large number of DNA lesions were separated and identified. Mass spectra obtained were interpreted on the basis of the typical fragmentation pathways of trimethylsilylated nucleosides. The use of GC-MS with selected-ion monitoring facilitated the detection of these lesions at the very low quantities and radiation doses (below 10 Gray) that might be relevant to those in biological systems.
...
PMID:Characterization of free radical-induced damage to DNA by the combined use of enzymatic hydrolysis and gas chromatography-mass spectrometry. 378 50

A 15-kilodalton protein has been identified as a major component of the residual protein fraction of mouse epididymal/vas spermatozoal heads, demembraned by treatment with Triton X-100 and sequentially extracted with 1 M NaCl/2-mercaptoethanol/DNase I. Two-dimensional electrophoresis of that protein before and after treatment with alkaline phosphatase indicated that it is present in epididymal/vas spermatozoa as a series of five differentially phosphorylated molecules with pI 6.0-7.0. Cyto-immunofluorescence with an affinity-purified antibody to the 15-kDa protein localized that protein to a circumscribed region of the demembraned mouse sperm head mediad from the dorsal margin. By radioimmunoassay, the 15-kDa protein was shown to be sperm-unique and species-specific. The antibody was nonreactive with homogenates of meiotic spermatogenic cells and round spermatids (stages 1-11) but was reactive with a non-phosphorylated 15.5-kDa protein of elongating spermatids (stages 12-16) and testicular spermatozoa. Following alkaline phosphatase treatment, the spermatozoal 15-kDa protein migrated to the position of the spermatidal 15.5-kDa protein on a sodium dodecyl sulfate gel. Thus, we conclude that the 15-kDa protein of mouse spermatozoa is synthesized during the elongation phase of spermiogenesis (stages 12-16) and is phosphorylated in the terminal period of that phase and/or after excursion of spermatozoa from the seminiferous tubules.
...
PMID:Proteins of demembraned mouse sperm heads. Characterization of a major sperm-unique component. 388 61

13C-enriched deoxyribonucleosides have been isolated from the DNA of Algal cells grown in an atmosphere of 90% 13C-labelled carbon dioxide. The 13C enriched DNA was quantitatively hydrolysed with DNase I, snake venom phosphodiesterase I and alkaline phosphatase of intestinal mucosa. The resulting deoxyribonucleosides were separated by preparative reversed-phase high pressure liquid chromatography in 60 minutes with detection by ultraviolet absorption at 254 nm. The final products were obtained in milligram quantities in high purity and in high yield. The 1H resonances of the base and sugar protons of these deoxyribonucleosides appear as well resolved multiplets in the 600 MHz NMR spectrum, due to the extensive 1H-13C couplings. Similarly, the 13C resonances of these deoxyribonucleosides appear as multiplets in the 75.5 MHz 13C NMR spectrum, due to 13C-13C couplings. The 1H-13C and 13C-13C coupling constants were also measured and tabulated. The isotopic enrichment of 13C these deoxyribonucleosides was obtained by integration of the 1H and/or 13C NMR spectra. It was found that the enrichment varied from carbon to carbon and species to species in the range of 70-89%, suggesting differential uptake and assimilation of 90% 13CO2 during metabolism pathways. This protocol provides experimentally useful quantities of 13C-enriched deoxyribonucleosides, which may be incorporated into site-specifically labeled oligonucleotides by chemical synthesis.
...
PMID:Isolation and purification of deoxyribonucleosides from 90% 13C-enriched DNA of algal cells and their characterization by 1H and 13C NMR. 400 Sep 54

Isolation and characterization of a novel radiation-induced product, i.e., the 8-hydroxyguanine residue, produced in deoxyribonucleic acid (DNA), 2'-deoxyguanosine, and 2'-deoxyguanosine 5'-monophosphate by gamma-irradiation in aqueous solution, are described. For this purpose, gamma-irradiated DNA was first hydrolyzed with a mixture of four enzymes, i.e., DNase I, spleen and snake venom exonucleases, and alkaline phosphatase. Analysis of the resulting mixture by capillary gas chromatography-mass spectrometry after trimethylsilylation revealed the presence of a product, which was identified as 8-hydroxy-2'-deoxyguanosine on the basis of the typical fragment ions of its trimethylsilyl (Me3Si) derivative. This product was then isolated by using reversed-phase high-performance liquid chromatography. The UV and proton nuclear magnetic resonance spectra taken from the isolated product confirmed the structure suggested by the mass spectrum of its Me3Si derivative. In addition, the accurate molecular mass of the Me3Si derivative of the isolated product was determined by MS. The obtained value agreed with the theoretical molecular mass within 1 millimass unit. The yield of 8-hydroxyguanine was also measured. Its mechanism of formation is believed to involve OH radical addition to the C-8 position of guanine followed by oxidation of the radical adduct.
...
PMID:Formation of an 8-hydroxyguanine moiety in deoxyribonucleic acid on gamma-irradiation in aqueous solution. 405 10

The kinetics of the interaction of the DNA double-helix-destabilizing protein from roe-deer liver with different DNAs revealed a fast phase which is observed both by the increase in A260 of the DNA and the quenching of the protein intrinsic fluorescence. A slow phase with a smaller amplitude is only recorded by the increase of A260.--The protein contains slightly less than two phosphate groups per molecule, removal of one of which by alkaline phosphatase does not affect its activity; however, removal of both phosphates decreases the DNA-unwinding property significantly. A similar decrease in activity is also revealed upon incorporation of an additional phosphate by cAMP-dependent protein kinase I.--Results of the protection of poly[d(A--T)] from DNase I digestion by the protein are in favor of a migration of the protein along the DNA.
...
PMID:The kinetics of the interaction of a helix-destabilizing protein from roe-deer liver with DNA and the influence of phosphorylation. 625 Sep 67

The adenovirus-specific DNA-binding protein (DBP) has been shown to inhibit the hydrolysis of single-stranded DNA by a DNase isolated from KB cells, (Nass, K., and Frenkel, G.D. (1980). J. Virol. 35, 314-319). The specificity of the inhibition has now been investigated. The DBP inhibits the hydrolysis of single-stranded DNA by several different DNases (DNase II, KB DNase, S1 nuclease) under a variety of reaction conditions, but it has no effect on DNase I-catalyzed hydrolysis of single-stranded DNA. The DBP also inhibits the rate of hydrolysis of double-stranded DNA by KB DNase and DNase II, but has no effect on DNase I-catalyzed hydrolysis of this substrate. The DBP also inhibits the dephosphorylation of 5'-phosphoryl-terminated DNA by bacterial alkaline phosphatase but stimulates the phosphorylation of 5'-hydroxyl-terminated DNA by polynucleotide kinase.
...
PMID:DNase inhibition by the adenovirus DNA-binding protein exhibits specificity for the enzyme but not for the secondary structure of the DNA. 630 53

gamma-Irradiation of DNA in vitro produces two types of single strand breaks. Both types of strand breaks contain 5'-phosphate DNA termini. Some strand breaks contain 3'-phosphate termini, some contain 3'-phosphoglycolate termini (Henner, W.D., Rodriguez, L.O., Hecht, S. M., and Haseltine, W. A. (1983) J. Biol. Chem. 258, 711-713). We have studied the ability of prokaryotic enzymes of DNA metabolism to act at each of these types of gamma-ray-induced 3' termini in DNA. Neither strand breaks that terminate with 3'-phosphate nor 3'-phosphoglycolate are substrates for direct ligation by T4 DNA ligase. Neither type of gamma-ray-induced 3' terminus can be used as a primer for DNA synthesis by either Escherichia coli DNA polymerase or T4 DNA polymerase. The 3'-phosphatase activity of T4 polynucleotide kinase can convert gamma-ray-induced 3'-phosphate but not 3'-phosphoglycolate termini to 3'-hydroxyl termini that can then serve as primers for DNA polymerase. E. coli alkaline phosphatase is also unable to hydrolyze 3'-phosphoglycolate groups. The 3'-5' exonuclease actions of E. coli DNA polymerase I and T4 DNA polymerase do not degrade DNA strands that have either type of gamma-ray-induced 3' terminus. E. coli exonuclease III can hydrolyze DNA with gamma-ray-induced 3'-phosphate or 3'-phosphoglycolate termini or with DNase I-induced 3'-hydroxyl termini. The initial action of exonuclease III at 3' termini of ionizing radiation-induced DNA fragments is to remove the 3' terminal phosphate or phosphoglycolate to yield a fragment of the same nucleotide length that has a 3'-hydroxyl terminus. These results suggest that repair of ionizing radiation-induced strand breaks may proceed via the sequential action of exonuclease, DNA polymerase, and DNA ligase. The possible role of exonuclease III in repair of gamma-radiation-induced strand breaks is discussed.
...
PMID:Enzyme action at 3' termini of ionizing radiation-induced DNA strand breaks. 636 Oct 28

Oval cells and biliary epithelial cells were isolated from livers of rats fed a choline-deficient diet containing 0.1% ethionine and from normal rat livers, respectively. Nonparenchymal cell suspensions prepared from these livers by collagenase perfusion followed by digestion of undissociated tissue with 0.1% collagenase, 0.1% Pronase, and 0.004% DNase I were separated into six fractions by centrifugal elutriation. Cells in each fraction were characterized histochemically for gamma-glutamyl transpeptidase, peroxidase, alkaline phosphatase, and glucose-6-phosphatase activities, and for albumin and alpha-fetoprotein by immunocytochemical methods. Cells from Fraction 5 of the elutriation procedure had various features predicted for oval cells and were selected for further studies. The cell yield in this fraction, from each preneoplastic liver, was 5.7 X 10(7) cells, 93 +/- 2% of which were gamma-glutamyl transpeptidase positive, 6 +/- 1% peroxidase positive, 61% albumin positive, and 29% alpha-fetoprotein positive. Cells in this fraction have a median diameter of 13.1 micron and are diploid and cycling. The majority of these cells has morphological features characteristic of biliary epithelial cells, although some cells display features intermediate between duct cells and hepatocytes. Nucleic acid hybridization using specific probes revealed that these cells contain albumin and alpha-fetoprotein messenger RNAs, while hepatocytes from normal and preneoplastic liver contain only albumin messenger RNA. Biliary cells obtained from normal livers do not contain albumin messenger RNA. The large-scale purification and characterization of cell populations from preneoplastic livers is an important step in elucidating the cellular derivation of liver tumors.
...
PMID:Isolation of oval cells by centrifugal elutriation and comparison with other cell types purified from normal and preneoplastic livers. 669 43

Based on the phosphorylation of the purified actin-fragmin complex, an 80 kDa monomeric kinase (AFK) has been isolated from Physarum polycephalum. Protein chemical analysis and studies involving kinase inhibitors and effectors establish that the AFK is a unique kinase that cannot be classified so far in one of the conventional kinase families. The actin-fragmin kinase behaves as an "independent" kinase since its activity towards the actin-fragmin complex is apparently not regulated by the binding of a ligand (e.g., the cyclic-nucleotides, Ca2+, calmodulin, phosphatidylserine and diolein). Rigorous screening of the substrate specificity suggests that the actin-fragmin complex represents the only substrate for this kinase. This kinase phosphorylates the actin moiety of the actin-fragmin complex at two consecutive threonine residues which constitute one of the contact sites for DNase I (37) and which are also located at one of the proposed actin-actin contact sites along the long-pitch helix of F-actin (38, 39). The physiological importance of this phosphorylation was demonstrated by studying the effect of phosphorylation on the nucleation and the capping activity of the actin-fragmin complex using fluorescence enhancement analysis. As could be demonstrated, the nucleation of actin filaments by the actin-fragmin complex is completely abolished upon phosphorylation by the AFK. Phosphorylation of the complex also interferes with its capping activity, which becomes Ca(2+)-dependent. In addition, capping and nucleating activity is regulated in vitro by phosphoinositides, of which PIP2 displays the highest activity and specificity. PIP2 partially inhibits the nucleation and capping activity of the unphosphorylated actin-fragmin. The capping activity of the phosphorylated actin-fragmin complex was inhibited by PIP2 to a much greater extent as compared to the unphosphorylated actin-fragmin complex. Among all phospholipids tested, PIP2 displayed the highest specificity. Initial experiments with purified preparations of the PP-1, PP-2A, PP-2B, alkaline phosphatase and acid phosphatases showed that PP-1 and PP-2A phosphatases were capable of dephosphorylating the phospho actin-fragmin complex. These findings raised the question of whether these or other protein phosphatases were involved in the dephosphorylation of this substrate in vivo. To address this question, Physarum extracts were subjected to fractionation by ion exchange chromatography, and the column fractions were assayed in a variety of conditions, to identify the protein phosphatases involved in the dephosphorylation of this substrate and to identify the elution position of the major Ser/Thr protein phosphatases present in the Physarum extract.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Microfilament dynamics: regulation of actin polymerization by actin-fragmin kinase and phosphatases. 757 44


<< Previous 1 2 3 4 5 Next >>

---