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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activity of RNA-dependent DNA polymerase (RDDP) from avian myeloblastosis virus (AMV), either in purified form or in virus lysates, was increased by phosphorylation. Stability of RDDP in lysates buffered with phosphate was much greater (no loss of activity in 48 hours at 4 degrees) than that in lysates buffered with Tris-Cl (76% loss). Activity lost in the Tris-buffered extracts was completely restored by phosphorylation. The findings suggested that AMV RDDP activity is influenced by the degree of phosphorylation of the enzyme or enzyme-associated proteins and that this chemical modification is mediated by protein phosphokinase and phosphoprotein phosphatase present in crude extracts of purified AMV. Application of these results provided the basis of procedures whereby RDDP can be recovered in significantly higher yield and purity than formerly.
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PMID:Influence of phosphate on activity and stability of reverse transcriptase from avian myeloblastosis virus. 6 81

Procedures were established for the isolation and partial purification of DNA polymerase, RNA polymerase and poly(A) polymerase activities from the cytoplasm and nuclei of NIH-Swiss mouse embryos. Based on the elution pattern of these enzyme activities from DEAE-cellulose and phosphocellulose columns in Tris-HCl buffer, pH 8.0, the apparent basicities of the enzymes can be arranged as follows: cytoplasmic(C) poly(A) polymerase greater than (C)DNA polymerase beta greater than (C)DNA polymerase alpha and nuclear(N) poly(A) polymerase greater than (N)DNA polymerase greater than (N)RNA polymerase I greater than (N)RNA polymerase II. Twenty rifamycins, including rifamycin B, rifamycin S, rifamycin SV, and rifamycin SV derivatives, were examined for their ability to inhibit the above mentioned nucleic acid polymerizing enzymes and Simian sarcoma virus type I (SSV-1) reverse transcriptase. Rifamycin SV 3'-formyldiphenylhydrazone, rifamycin SV 3'-formyl-n-octyloxime (AF/013) and rifamycin SV 3'-formyldiphenylmethyloxime (AF/05) inhibited all the tested enzyme activities. Rifamycin SV 3'-formylpropylphenyloxime (AF/015) inhibited cellular nucleic acid polymerase activities but not SSV-1 DNA polymerase activity. Rifamycin SV 3'-formyldinitrophenylhydrazone (AF/DNFL) strongly inhibited reverse transcriptase activity but did not inhibit cellular DNA polymerase activities. AF/DNFI slightly inhibited RNA and poly(A) polymerase activities. Rifamycin SV 3'-formyldipropylhydrazone (AF/DPI) and 2,6-dimethyl-4-N-benzyldemethyl-rifampicin (AF/ABDMP) slightly inhibited reverse transcriptase activity but did not inhibit cellular nucleic acid polymerase activities. Active rifamycin derivatives inhibited enzyme reactions by interacting with the enzyme proteins. Nascent polynucleotide chain elongation continued although at a reduced rate in the presence of inhibitor. The addition of increasing concentrations of nonionic detergent (Triton X-100) to rifamycin-inhibited enzyme reactions fully restored enzyme activities. The presence of highly lipophilic 3'-side chains on active rifamycins and the reversibility of enzyme inhibition by Triton X-100 suggest that the tested nucleic acid polymerizing enzymes may have hydrophobic regions with which inhibitory rifamycins interact.
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PMID:Interaction of rifamycins with mammalian nucleic acid polymerizing enzymes. 6 93

DNA complementary to ovalbumin mRNA and covalently bound to cellulose (cDNA-cellulose) was synthesized using avian myeloblastosis virus RNA-directed DNA polymerase. High concentrations of actinomycin D (200 migrogram/ml) were required to produce 97% inhibition of double-stranded DNA synthesis, but mRNA transcription was only slightly inhibited (14%). The conditions used for binding of mRNA to cDNA-cellulose permitted complete hybridization of ovalbumin mRNA in 10 min while stable poly(A):(dT) hybrids failed to form. The temperature at which 50% of the ovalbumin mRNA activity was eluted from cDNA-cellulose was 62 degrees in 0.01 M Tris.HCl. When a batchwise procedure of hybridization and elution was used, the total recovery of ovalbumin mRNA activity applied to the cDNA-cellulose was greater than 98%, indicating little if any degradation of mRNA. Ovalbumin mRNA activity eluted in each chromatographic run was 50 to 70% of that originally used for the synthesis of the cDNA-cellulose. When total polysomal RNA was subjected to chromatography, the bound fraction consisted of ovalbumin mRNA, rRNA, and material behaving like fragments of ovalbumin mRNA. Applying this fraction to cDNA-cellulose a second time eliminated the rRNA but not the presumptive fragments. Ovalbumin mRNA purified either once or twice was enriched between 43- and 56-fold over polysomal RNA in translational activity.
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PMID:Chromatography of ovalbumin messenger ribonucleic acid on complementary deoxyribonucleic acid-cellulose. 7 8

We have partially purified and characterized two separate DNA polymerase activities associated with Epstein-Barr virus (EB virus). One activity is present in EB virus producer cell lines but not in nonproducer or negative cell lines. It adheres more strongly to DEAE-cellulose than any host cell enzymes, eluting at 210 to 270 mM potassium phosphate buffer. Further elution from phosphocellulose and sedimentation in glycerol gradients yields an enzyme purified 900-fold with an S value of 8.3. The second DNA polymerase activity co-purifies with EB viral particles, elutes at low salt from DEAE-cellulose (40 to 60 mM potassium phosphate buffer) and phosphocellulose (100 mM), and has an S value of 9.5 on glycerol gradient sedimentation. These two enzymes are referred to for convenience as the EB virus-induced DNA polymerase and the EB virion-associated DNA polymerase. The EB virus-induced polymerase can be distinguished from host alpha, beta, and the virion-associated polymerase in 1) being resistant to salt inhibition, 2) having a more basic pH optima in Tris buffer (pH 9.5), and 3) having a 10-fold lower saturating concentration for the activated DNA template. The EB virion-associated polymerase is distinguished from host alpha, beta, and the EB virus-induced polymerase, because it cannot utilize synthetic deoxy- and ribohomopolymer primer-templates in place of the activated calf thymus DNA template in DNA polymerase assays. Neither of the EB virus-associated polymerases can copy the ribohomopolymers dT10poly(rA) or dG12-18(poly(rC) efficiently and therefore can be distinguished from host gamma polymerase and reverse transcriptase. The activity of the EB virus-induced and virion-associated polymerases are unaffected both by antibody to alpha polymerase, and by antiserum with high antibody titers to EB early antigen and viral capsid antigen.
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PMID:Two Epstein-Barr virus-associated DNA polymerase activities. 21 39

Although reverse transcriptase has been the subject of intensive investigation, minimal information is available regarding the physical properties of the enzyme. The basic hydrodynamic properties of avian myeloblastosis virus reverse transcriptase in solution were measured by both sedimentation velocity and equilibrium measurements in two buffer systems. In a 0.3 M potassium phosphate buffer system, pH 7.8, the enzyme sedimented as a homogenous particle with a sedimentation coefficient of (7.1 +/- 0.3) S with a weight-average molecular weight, Mw, of (1.52 +/- 0.05) x 10(5). Since the enzyme consists of an alpha and beta subunit of equal molar ratio with Mw of 6.3 x 10(4) and 9.4 x 10(4), respectively, it was concluded that the enzyme exists as an alpha beta heterodimer in this buffer system. In a Tris buffer system, pH 7.9, containing 0.46 M NaCl and 4% glycerol, the native enzyme also sedimented as a homogeneous particle with an apparent sedimentation coefficient of (10.1 +/- 0.5) S, without considering the effect of glycerol on solvent-protein interaction. Based on the results of Gekko and Timasheff (Gekko, K., and Timasheff, S. N. (1981) Biochemistry 20, 4667-4676) and the polarity of the enzyme, it was estimated that there is significant solvent-protein interaction even at 4% glycerol leading to a value of -0.06 g/g in the preferential solvent interaction parameter. When the solvent effect was taken into consideration, the value for s020,w increased from 10.1 to 11.9 S, implying that the native enzyme dimerizes in the presence of 4% glycerol. The combined results of gel filtration and sedimentation velocity showed that the dimerization of the enzyme to form (alpha beta)2 is favored at 20 degrees C with the alpha beta form predominating at 4 degrees C. The secondary structure of the reverse transcriptase was measured by circular dichroism. Results showed that the enzyme consists of (16 +/- 2)% alpha-helix, (24 +/- 2)% beta-sheet, (24 +/- 2)% beta-turn, and (36 +/- 4)% undefined structures.
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PMID:Avian myeloblastosis virus reverse transcriptase. Effect of glycerol on its hydrodynamic properties. 170 51

Polyspermine-ribonuclease (Mr approximately 17 000) and the enzyme transcriptase from Rauscher-leukaemia virus (Mr approximately 70 000) form a complex Mr approx. 160 000) such that the molar ratio of polyspermine-ribonuclease to reverse transcriptase is 5:1. The most favourable condition for complex-formation is in a solution consisting of 0.01 M-Tris/HCl buffer, pH 7.5, 0.25 M-KCl and 1 mM-Mn2+ at 37 degrees C. The association of the two enzymes retains full RNAase activity, but reverse-transcriptase activity is completely inhibited when ribonuclease-sensitive polymers such as (dG)12 x (rC)n or viral 70S RNA are used as primer templates.
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PMID:Complexing reverse transcriptase with polyspermine-ribonuclease. 616 6

Structural and biochemical evidence strongly supports a heterodimeric (p66p51) active form for human immunodeficiency virus-1 reverse transcriptase (RT). Heterodimer stability was examined by sedimentation analysis as a function of temperature and ionic strength. Using NONLIN regression software, monomer-dimer-trimer and monomer-dimer-tetramer association models gave the best fit to the analytical ultracentrifuge sedimentation equilibrium data. The heterodimer is the predominant form of RT at 5 degrees C, with a dimerization Ka value of 5.2 x 10(5) M-1 for both models. Ka values of 2.1 x 10(5) and 3.8 x 10(5) M-1 were obtained for the respective association models at 20 degrees C. RT in 50 and 100 mM Tris, pH 7.0, completely dissociates at 37 degrees C and behaves as an ideal monomeric species. The dissociation of RT as a function of increasing temperature was also observed by measuring the decrease in sedimentation velocity (sw,20). If the stabilization of the heterodimer was due primarily to hydrophobic interactions we would anticipate an increase in the association from 21 degrees C to 37 degrees C. The opposite temperature dependence for the association of RT suggests that electrostatic and hydrogen bond interactions play an important role in stabilizing heterodimers. To examine the effect of ionic strength on p66p51 association we determined the changes in sw,20 as a function of NaCl concentration. There is a sharp decrease in sw,20 between 0.10 and 0.5 M NaCl, leading to apparent complete dissociation. The above results support a major role for electrostatic interactions in the stabilization of the RT heterodimer.
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PMID:Human immunodeficiency virus-1 reverse transcriptase heterodimer stability. 753 May 41

We and others have described methods to label specific nucleic acid sequences in fixed cells by reverse in situ transcription (IST). They are simple alternatives to the tedious steps of in situ hybridization with labeled probes. We have favored use of thermostable DNA polymerases after heat denaturation of template secondary structure, accompanied by synthesis of cDNA from an annealed primer, but the approach has been limited by the low reverse transcriptase (RT) activity of Taq polymerase and delayed detection methods. We have improved the technique by the use of recombinant Thermus thermophilus (rTth) DNA polymerase and fluorescein-12-dUTP (FIST). Jurkat T lymphocytes were stimulated with ionomycin + phorbol myristate acetate to produce interleukin-2 (IL-2) mRNA in vitro overnight. They were cytospun onto slides and fixed in 70% ethanol + 30% DEPC-treated water, acetone, and air-dried. The slides were placed on a temperature-controlled heating block, and the cell spot was covered with a plastic coverslip. The temperature was raised to 95 degrees C, and 5-10 microliters of modified Perkin-Elmer/Cetus rTth RT reaction mix was injected under the edge of the coverslip. Each 10 microliters of mix in DEPC-water contained 10 mM Tris-HCl, pH 8.3, 90 mM KCl, 1 mM MnCl2, 1 mM dithiothreitol, 10 U placental ribonuclease inhibitor, 0.125 mM dA,C,GTPs, 0.1 mM fluorescein-12-dUTP, 2 U rTth DNA polymerase, and 4 pM 22-mer oligonucleotide primer, which spanned the second intron of IL-2. After 3 min at 95 degrees C, 1 min at 50 degrees C and 10 min at 72 degrees C, the slides were washed in 0.5 x phosphate-buffered saline, pH 7.0, at 42 degrees C, in 70% ethanol, 100% ethanol, and air-dried. The cells were mounted in antifade solution (2% n-propyl gallate in 70% glycerol), and could be viewed immediately by fluorescence microscopy. Image analysis showed that stimulated Jurkat cells were brighter than uninduced controls or those treated with RNase or without polymerase or primer. FIST appears to be useful for the detection of specific mRNAs in single cells.
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PMID:In situ transcription with Tth DNA polymerase and fluorescent nucleotides. 798 81

We have devised a single-step method that enables purification of HIV-1 recombinant reverse transcriptase directly from bacterial lysates in less than 2 h. Clarified lysates are applied to commercial Q- and S-matrix cartridge columns connected in series. The columns are washed with low-salt buffer to remove unbound protein, then the Q column is removed and reverse transcriptase is eluted from the S column using a salt gradient. The purification has been carried out with both medium-pressure and high-pressure chromatographic systems. Purifications are carried out at room temperature near neutral pH, providing enzyme with high DNA polymerase specific activity. A crucial aspect of the procedure is the use of Tris buffer, a buffer that is normally incompatible in cation-exchange methods. The method is applicable for the purification of the p51/p66 heterodimer and the p5l and p66 homodimer forms of reverse transcriptase. We have used this method to purify wild-type reverse transcriptase and several recombinant proteins containing mutations correlated with dideoxynucleoside drug resistance.
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PMID:Single-step purification of recombinant wild-type and mutant HIV-1 reverse transcriptase. 917 79

The effect of 44 different metal ions (Ag+, Al3+, As(O-)2, Au3+, Ba2+, Be2+, Bi3+, Cd2+, Ce3+, CO2+, Cr(O2-)4, Cr3+, Cs+, Cu2+, Fe3+, Fe2+, Ga3+, Ge4+, Hg2+, Ir4+, La3+, Li+, Mn2+, MO6+, Ni2+, OS4+, Pb2+, Pt4+, Rb+, Rh3+, Sb5+, Se(O2-)4, Se(O2-)3, Sn2+, Sr2+, Th4+, T1+, U(O2+)2, V(O-)3, VO2+, W(O2-)4, Y3+, Zn2+, and Zr4+) on the activity of the reverse transcriptase (RT) of the human immunodeficiency virus (HIV-1) was investigated in vitro. For this study, the RT activity assay was carried out by means of an enzyme-linked immunosorbent assay (ELISA) kit, using the template/primer hybrid poly(A) oligo(dT)15, which required some modifications: (1) possible interfering metal chelators (such as EDTA) in the original lysis buffer were avoided, and a new buffer (50 mM Tris-NO3, pH 7.8) was used throughout; (2) an amount of 2 ng of RT per well was considered to be optimal after checking the linearity of the reaction with increasing amounts of enzyme; (3) an incubation temperature of 37 degrees C and an incubation time of 1 h were chosen after preliminary studies in a wide range of temperature and time. At an incubation temperature > or = 40 degrees C, there was a dramatic loss of enzymatic activity. In addition, when RT alone was preincubated for 1 h at 5 degrees C, 25 degrees C, and 37 degrees C, there was a large (83%) loss of activity at 37 C as compared to that at 5 degrees C. These results are indicative of enzyme thermolability, which is higher in the absence of substrates. The effect of metal ions on RT activity was tested using two different metal salt concentrations (10(-4) M and 10(-5) M). Under such experimental conditions, the presence of five metal ions (Pt4+, Ag+, Rh3+, Zn2+, and Hg2+) decreased the RT activity in a dose-response fashion. The observed order of effectiveness with respect to inhibition was Pt4+ > Ag+ > Rh3+ > Zn2+ = Hg2+. Estimated mean inhibitory concentrations (IC50) were 7.8 microM for (NH4)2PtCl6, 14.1 microM for AgNO3, 46.8 microM for RhCl3, 53.7 microM for Zn(SO)4, and 56.2 microM for Hg(NO3)2. Because these data are of the same order of magnitude as the corresponding values related to other RT inhibitors used in anti-AIDS therapy, metal compounds or their derivatives could give an interesting contribution in the development of new RT inhibitors for clinical use.
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PMID:Effects of trace metal compounds on HIV-1 reverse transcriptase: an in vitro study. 1032 22


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