Nuclear matrix bound terminal deoxynucleotidyl transferase in rat thymus nuclei. II. Effect of ATP on free and matrix bound TdT.
Endogenous nuclease digestion of thymus nuclei from 3-4 week old rats followed by a step wise extraction with low salt, 0.5 M salt and 1 M salt removed approximately 70-85% of total nuclear terminal deoxynucleotidyl transferase (TdT) whereas approximately 15-30% of the enzyme remained tightly bound to the residual nuclear matrix. The cytoplasmic TdT as well as the bulk of nuclear TdT extracted in low salt and 0.5 M salt was found to be strongly inhibited at low concentration of ATP whereas matrix bound TdT and a significant portion of the enzyme in 1 M salt extract was completely insensitive to this nucleotide. The ATP resistant enzyme in the 1 M salt extract was unstable and slowly converted to ATP sensitive form upon prolonged preincubation on ice whereas under similar conditions it remained unaffected in the matrix bound form. These observations lead us to suggest that ATP resistant matrix bound TdT being capable of discriminating unnatural rNTPs against the natural dNTP substrates, may be the functionally organized form of the enzyme and that free TdT having lost the capability to distinguish between dNTP and rNTP may be the nonfunctional form of the enzyme in the thymus gland.
Dave
V P
VP
Dept. of Atomic Energy, Bhabha Atomic Research Centre, Bombay, India.
Patil
M S
MS
Pandey
V N
VN
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D003593:Cytoplasm / Q000187:drug effects / Q000201:enzymology; D004253:DNA Nucleotidylexotransferase / Q000032:analysis*; D007700:Kinetics; D015530:Nuclear Matrix / Q000187:drug effects / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D013950:Thymus Gland / Q000187:drug effects / Q000201:enzymology*
D000255:Adenosine Triphosphate; D004253:DNA Nucleotidylexotransferase