Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reverse transcriptase was purified from human immunodeficiency virus (HIV). It utilized the artificial primer-template poly(rA)-oligo(dT)12-18 more efficiently than activated calf thymus DNA, poly(rI)-oligo(dC)12-18, poly(rC)-oligo(dG)12-18, or poly(rCm)-oligo(dG)12-18. Maximum activity was observed at pH 7.0 to 7.6 in the presence of 5 mM MgCl2 and 100 mM KCl. 3'-Azido-3'-deoxythymidine triphosphate competed with dTTP for binding to HIV reverse transcriptase. Different kinetic constants were obtained with different primer-templates. Km and Ki values of 2.8 and 0.04 microM, respectively, were obtained with poly(rA)-oligo(dT)12-18. The corresponding values were 1.2 and 0.3 microM, respectively, with activated calf thymus DNA and 0.3 and 0.01 microM, respectively, with extracted virus and native template. Inhibition of the host cell DNA polymerases alpha and beta was considerably weaker. The Km and Ki values obtained with activated calf thymus DNA as the primer-template were 2.4 and 230 microM, respectively, for DNA polymerase alpha and 6.0 and 73 microM, respectively, for DNA polymerase beta. 3'-Azido-3'-deoxythymidine triphosphate could also serve as an alternate substrate for HIV reverse transcriptase. The resulting incorporation of 3'-azido-3'-deoxythymidine triphosphate into poly(rA)-oligo(dT)12-18 caused chain termination and premature deceleration of the reaction. The terminated primer could not be elongated when incubated with dTTP and HIV reverse transcriptase.
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PMID:3'-Azido-3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase. 244 66

The presence of terminal nucleotidyl transferase activities catalyzing the addition of AMP, CMP, GMP, and UMP residues to the 3' ends of oligonucleotide primers was detected in healthy tomato plants. These enzyme activities copurify with RNA-dependent RNA polymerase during the initial stages of purification. Their separation from RNA-dependent RNA polymerase is finally achieved by DEAE chromatography: terminal transferase activities are retained on DEAE while RNA-dependent RNA polymerase does not bind in the presence of 20 mM MgCl2. Elution by a linear gradient of 0 to 400 mM NH4Cl releases all four terminal transferase activities from the DEAE column at a concentration of 270 mM NH4Cl, thus suggesting that they may belong to one enzyme molecule; this question, however, needs further clarification. The enzyme activities are completely dependent on the presence of an RNA primer and are strongly influenced by its base composition as well as its chain length. Characterization of the respective reaction products by electrophoresis on 15% polyacrylamide sequencing gels reveals striking differences as to the number of nucleotides added to a given primer. In the case of UMP transfer to U8 or A8 and in the case of GMP transfer to A8 only 1 to 6 nucleoside monophosphates are added to the 3' terminus of the oligonucleotide primer, whereas in the case of AMP transfer to A8 or U8, the CMP transfer to A8, and the GMP transfer to U8, longer chains of minimally 30 nucleotides are added to the respective primer. After gradient elution from DEAE the transferase preparation displays no nucleolytic activity when incubated in the presence of 3H-labelled ribosomal RNA or [3H]poly(A) X poly(U). Only in the case of [3H]poly(A) and [3H]poly(U) or [3H]poly(C) 10 to 15% of the radioactivity is transferred to acid-soluble counts.
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PMID:Simultaneous presence of terminal adenylyl, cytidylyl, guanylyl, and uridylyl transferase in healthy tomato leaf tissue: separation from RNA-dependent RNA polymerase and characterization of the terminal transferases. 628 7

CHO-K1 cells were examined for their cellular responses to the P2 receptor agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (DbATP), and for the presence of mRNA for P2X receptors. Reverse transcriptase-polymerase chain reactions, using primers directed against the rat P2X subunits, detected the presence of P2X7 but not P2X1-P2X6 subunits. DbATP (EC50 approximately equal to 100 microM) evoked non-desensitizing inward currents which reversed at approximately equal to 0 mV, suggesting activation of a non-selective cation channel. ATP also evoked inward currents but was less potent than DbATP. DbATP also stimulated the accumulation of 45calcium (45Ca2+) and the DNA binding dye, YO-PRO-1, in CHO-KI cells. Both responses were inhibited by NaCl and MgCl2. In 280 mM sucrose buffer, 45Ca2+ accumulation was measurable within 10-20 s of agonist addition, whereas YO-PRO-1 accumulation was only detectable after 8 min. ATP and ATPgammaS were also agonists but were less potent than DbATP, while UTP, 2-methylthio ATP, ADP and (alphabeta)methylene ATP were inactive at concentrations up to 100 microM. DbATP increased lactate dehydrogenase release from CHO-K1 cells, suggesting cell lysis, although this effect was only pronounced after 60-90 min. These data suggest that CHO-K1 cells express an endogenous P2X7 receptor which can be activated by DbATP to cause a rapid inward current and accumulation of 45Ca2+. Prolonged receptor activation results in a delayed, increased permeability to larger molecules such as YO-PRO-1 and ultimately leads to cell lysis. Importantly, the presence of an endogenous P2X7 receptor should be considered when these cells are used to study recombinant P2X receptors.
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PMID:Identification and characterization of an endogenous P2X7 (P2Z) receptor in CHO-K1 cells. 986 47

The HeLa cell terminal uridylyltransferase (TUTase) that specifically modifies the 3'-end of mammalian U6 small nuclear RNA (snRNA) was characterized with respect to ionic dependence and substrate requirements. Optimal enzyme activity was obtained at moderate ionic strength (60 mm KCl) and depended on the presence of 5 mm MgCl2. In vitro synthesized U6 snRNA without a 3'-terminal UMP residue was not accepted as substrate. In contrast, U6 snRNA molecules containing one, two or three 3'-terminal UMP residues were filled up efficiently, generating the 3'-terminal structure with four UMP residues observed in newly transcribed cellular U6 snRNA. In this reaction, the addition of more than one UMP nucleotide depended on higher UTP concentrations. The analysis of internally mutated U6 snRNA revealed that the fill-in reaction by the U6-TUTase was not controlled by opposite-strand nucleotides, excluding an RNA-dependent RNA polymerase mechanism. Furthermore, electrophoretic mobility-shift analyses showed that the U6-TUTase was able to form stable complexes with the U6 snRNA in vitro. On the basis of these findings, a protocol was developed for affinity purification of the enzyme. In agreement with indirect labeling results, PAGE of a largely purified enzyme revealed an apparent molecular mass of 115 kDa for the U6-TUTase.
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PMID:Biochemical characterization of a U6 small nuclear RNA-specific terminal uridylyltransferase. 1260 30