Terminal deoxynucleotidyltransferase of 60,000 daltons from mouse, rat, and calf thymus. Purification by immunoadsorbent chromatography and comparison of peptide structures.
For rapid simple purification of terminal deoxynucleotidyltransferase from limited amounts of tissues, we have developed an immunoadsorbent column chromatographic method using antiterminal transferase antibody-conjugated Sepharose 4B. The column specifically adsorbed all mammalian terminal deoxynucleotidyltransferase (terminal transferases) tested and, in all cases, nearly homogeneous enzymes were recovered at extremely high yields of activity and protein. By this method, we first succeeded in purifying rodent enzymes from rat or mouse thymus, which enzymes were comprised of a single polypeptide chain (Mr = 60,000). The enzyme purified from calf thymus by the same procedure showed the two well known subunits (alpha: Mr = 10,000 and beta: Mr = 32,000). However, the calf preparation purified in the presence of protease inhibitors exhibited several polypeptides with molecular weights ranging from Mr = 42,000 to Mr = 60,000, but did not contain the two-subunit form. From peptide mapping analyses, it was evident that each of the high molecular weight polypeptides contained sequences of both of the two low molecular weight subunits. These results indicate that the two subunits (alpha and beta) of the calf thymus enzyme reported earlier may be proteolytic products derived from a single polypeptide of Mr = 60,000, which may be the native form. It was noted that an extensive homology existed in primary structure of the enzymes from three species of mammals.
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002918:Chymotrypsin; D004254:DNA Nucleotidyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D007164:Immunosorbents; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D013045:Species Specificity; D013950:Thymus Gland / Q000201:enzymology*; D014357:Trypsin
D007164:Immunosorbents; D046911:Macromolecular Substances; D010446:Peptide Fragments; D004254:DNA Nucleotidyltransferases; D002918:Chymotrypsin; D014357:Trypsin