Binding of nucleotide spin probes to AMV reverse transcriptase and metal ions

• Published in: FEBS letters 1982-03, Vol.139 (2), p.185-189
• Main Authors: Warwick-Koochaki, P.E., Langemeier, P.W., Toppin, C.R., Bobst, A.M.
• Format: Article
• Language: English
• Subjects: AMV, avian myeloblastosis virus; Avian Leukosis Virus - enzymology; avian myeloblastosis virus; Avian Myeloblastosis Virus - enzymology; Cyclic N-Oxides; DNA polymerase (RNA-dependent); dTTP; dTTP, thymidine 5'-triphosphate; E.S.R; Electron Spin Resonance Spectroscopy; ESR, electron spin resonance; Manganese; pDUTT and pppDUTT, 5'-mono- and triphosphate analog of DUTT, N-[1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl]-O-[1-β-D-2'-deoxyribofuranosyluracil-5-yl]-thioglycolamide; pppDUMPT, 5'-tir-phosphate analog of DUMPT, N-[1-oxyl-2,2,6,6-tetramethyl- 4-piperridinyl]-S-[1-β-D-2'-deoxyribofuranosyluracil-5-yl]-3-mercaptopropionamide; pppDUTA, 5'-triphosphate analog of N-[1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl]-N-[1-β-D-2'-deoxyribofuranosyluracil-5-yl] amine; Protein Binding; RNA-Directed DNA Polymerase - metabolism; Spin Labels; spin probes; Zinc
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(82)80847-8

This study concerns the binding properties of some spin-labeled analogs of dTTP and AMV reverse transcriptase or Zn super(2+), Mn super(2+) or Mg super(2+). These dTTP analogs differ with respect to the chemical composition and size of the 'leg' attaching the nitroxide radical to the base. Since several nitroxide-containing compounds with a chemcal composition similar to the spin label 'leg' of these dTTP analogs have demonstrated metal chelating potential it was of interest to determine the affinity of the analogs for various metal ions used in polymerization reactions. Specifically, the 3 objectives of these studies were: To determine the ESR the dissociation constants and number of binding sites for the spin labeled nucleotides, pppDUTT or pDUTT, and AMV reverse transcriptase; To compare by ESR the affinity of dTTP and spin-labeled nucleotides with various "legs" attaching the nitroxide to the base for Zn super(2+) from the paramagnetic properties of the nitroxide; To compare by ESR the dissociation constants for dTTP or pppDUTT and Mn super(2+) or Mg super(2+) from the paramagnetic properties of Mn super(2+).