Biochemistry of terminal deoxynucleotidyltransferase. Affinity labeling and identification of the deoxynucleoside triphosphate binding domain of terminal deoxynucleotidyltransferase

Journal Article · Tue Mar 15 04:00:00 EST 1988 · J. Biol. Chem.; (United States)

OSTI ID:5176135

Pandey, V; Modak, M J

Using the technique of UV-mediated cross-linking of nucleotides to their acceptor sites, we have labeled calf terminal deoxynucleotidyltransferase (TdT) with (/sup 32/P)dTTP. The specificity of dTTP cross-linking at the substrate binding site in TdT is demonstrated by the competitive inhibition of the cross-linking reaction by other deoxynucleoside triphosphates, and ATP and its analogues, requiring concentrations consistent with their kinetic constants. Tryptic peptide mapping of the (/sup 32/P)dTTP-labeled enzyme showed the presence of a single radioactive peptide fraction that contained the site of dTTP cross-linking. The amino acid composition and sequence analysis of the radioactive peptide fraction revealed it to contain two tryptic peptides, spanning residues 221-231 and 234-249. Since these two peptides were covalently linked to dTTP, the region encompassed by them constitutes a substrate binding domain in TdT. Further proteolytic digestion of the tryptic peptide-dTTP complex, using V8 protease, yielded a smaller peptide, and its analysis narrowed the substrate binding domain to 14 amino acids corresponding to residues 224-237 in the primary amino acid sequence of TdT. Furthermore, 2 cysteine residues, Cys-227 and Cys-234, within this domain were found to be involved in the cross-linking of dTTP, suggesting their participation in the process of substrate binding in TdT.

• Research Organization: Univ. of Medicine and Dentistry of New Jersey, Newark (USA)
• OSTI ID: 5176135
• Journal Information: J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 263:8; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
AMINO ACID SEQUENCE
AMINO ACIDS
ANIMALS
ATP
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOCHEMISTRY
CARBOXYLIC ACIDS
CATTLE
CHEMICAL COMPOSITION
CHEMICAL REACTIONS
CHEMISTRY
CROSS-LINKING
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ENZYMES
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLING
LIGHT NUCLEI
MAMMALS
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
NUCLEI
NUCLEOTIDES
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPTIDE HYDROLASES
PEPTIDES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERIZATION
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
RUMINANTS
SERINE PROTEINASES
SUBSTRATES
TRACER TECHNIQUES
TRANSFERASES
TRYPSIN
VERTEBRATES