Photoaffinity labeling of terminal deoxynucleotidyl transferase. 2. Identification of peptides in the nucleotide binding domain

Journal Article · Tue Jan 24 04:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00428a045· OSTI ID:5564822

Evans, R K; Beach, C M; Coleman, M S ^[1]

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1. Univ. of Kentucky, Lexington (USA)

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the nucleotide binding site by the substrate photoaffinity analogue ({gamma}-{sup 32}P)-8-azido-dATP. The {alpha} and {beta} polypeptides of photolabeled terminal transferase were resolved by high-performance liquid chromatography. The {beta} polypeptide was digested with trypsin and fractionated by reverse-phase chromatography. Two {sup 32}P-containing fractions were isolated and subjected to amino acid sequence analysis. Peptides were identified as Ile{sup 209}-Lys{sup 232} (B26) and Val{sup 233}-Lys{sup 239} (B27). Peptide B26 was further resolved into two overlapping species; one contained an additional lysine residue at the N-terminus which resulted from tryptic cleavage between Lys{sup 207} and Lys{sup 208}. In order to ensure that the sequenced peptides corresponded to the photolabeled species, the authors devised an anion-exchange procedure to isolate photolabeled peptides from the mixture. Analysis of photolabeled peptides from terminal transferase {alpha}{beta} using DEAE-cellulose chromatography followed by reverse-phase HPLC confirmed that the photolabeled species were peptides B26 and B27. Structure predictions, based on sequence data, place the two peptides identified by photolabeling in spatial proximity consistent with the participation of both in the nucleotide binding domain.

• OSTI ID: 5564822
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:2; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
ATP
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHEMICAL REACTIONS
CHROMATOGRAPHY
CROSS-LINKING
DAYS LIVING RADIOISOTOPES
ENZYMES
ISOTOPE APPLICATIONS
ISOTOPES
LABELLING
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
MOLECULAR STRUCTURE
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDES
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERIZATION
RADIOISOTOPES
SEPARATION PROCESSES
SUBSTRATES
TRACER TECHNIQUES
TRANSFERASES