Skip to main content
OSTI.GOVU.S. Department of Energy Office of Scientific and Technical Information
U.S. Department of Energy
Office of Scientific and Technical Information
 

Active site characterization of terminal deoxynucleotidyl transferase (TdT): modification with (deoxy) nucleotide photoaffinity analogs

Conference · · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
OSTI ID:5334571
; ; ;
TdT is a non-template directed DNA polymerase. Photoaffinity analogues of (deoxy)NTP (8-N/sub 3/dATP, (..gamma..-/sup 32/P)8-N/sub 3/dATP, (..gamma..-/sup 32/P)8-N/sub 3/ATP) were used in the analysis of the nucleotide-binding site(s) of calf TdT ..cap alpha beta.. dimer form. Under assay conditions 8-N/sub 3/dATP was shown to be a competitive inhibitor of dATP polymerization (K/sub dATP/approx.125..mu..M; K/sub i/(8-N/sub 3/dATPapprox.260..mu..M). Optimal conditions for photoincorporation of (..gamma..-/sup 32/P) 8-N/sub 3/dATP were determined (pH 6.0, 100..mu..M Ca/sup +2/, 1-3 min preincubation, 20 sec photolysis). TdT was not active under these conditions due to the Ca/sup +2/. However, good protection against photoinsertion of (..gamma..-/sup 32/P)8N/sub 3/dATP by dATP was observed. Also, K/sub ds/ of 8N/sub 3/dATP were lower than observed under assay conditions (K/sub d/8N/sub 3/ATPapprox.12 ..mu..M, K/sub d/dATPapprox.8 ..mu..M). The photolabeled protein product showed no loss in radioactive label 20 min after photolysis. Photolabeled subunits were purified using reverse-phase HPLC. Both ..cap alpha.. and ..beta.. subunits were modified to the same extent and showed approximately the same Kd for (..gamma..-/sup 32/P)8-N/sub 3/dATP. These data indicate that the active site is comprised of both ..cap alpha.. and ..beta.. peptides. The separated subunits are being subjected to proteolysis and labeled peptides isolated using reverse-phase HPLC.
Research Organization:
Univ. of Kentucky, Lexington
OSTI ID:
5334571
Report Number(s):
CONF-8606151-
Conference Information:
Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
Country of Publication:
United States
Language:
English

Similar Records

Identification of the guanosine interacting peptide of the GTP binding site of. beta. -tubulin using 8N/sub 3/GTP
Conference · Fri May 01 00:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) · OSTI ID:6029263
GTP binding to the. beta. -subunit of tubulin is greatly reduced in Alzheimers disease
Conference · Fri May 01 00:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) · OSTI ID:6122781
Photoaffinity labeling of terminal deoxynucleotidyl transferase. 2. Identification of peptides in the nucleotide binding domain
Journal Article · Mon Jan 23 23:00:00 EST 1989 · Biochemistry; (USA) · OSTI ID:5564822

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ANIMALS
ATP
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CALVES
CATTLE
CHEMICAL COMPOSITION
CHEMICAL REACTIONS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DOMESTIC ANIMALS
ENZYME ACTIVITY
ENZYMES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
MAMMALS
MEMBRANE PROTEINS
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PEPTIDES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOTOCHEMICAL REACTIONS
PHOTOLYSIS
POLYMERIZATION
PROTEINS
PURIFICATION
RADIOISOTOPES
RECEPTORS
RUMINANTS
SEPARATION PROCESSES
TRACER TECHNIQUES
TRANSFERASES
VERTEBRATES