Purification of High Molecular Mass Species of Calf Thymus Terminal Deoxymjcleotidyltransferase

Abstract

We have developed a simplified column chromatographic procedure for the simultaneous purification of two high molecular mass forms (58 kd and 45 kd) and a standard two subunit 44 kd from of terminal deoxynucl eot idyl transferase (TdT) from calf thymus chromatin. The procedure involves hlgh salt extraction of the chromatin fraction followed by successive chromatographles on phosphocel lulose, DEAE sephadex, and hydroxylapatite matrices. While all 3 species of TdT comigrate throughout these steps, separatlon of Individual species is achieved on a single stranded DNA agarose colunin. The combined yield of the 45 kd and 58 kd TdTs is quite high (∼8 mg/500g tlssue), 45 kd being the major species (∼60%) and the 58 kd constituting about 30%. The 44 kd species containlng two subunits usually represents under 10% of the total. All the three forms of TdT showed similar specific activlty and preference for purine deoxynucleoslde triphosphates (dNTPs). The Km for individual dNTP with all three species of TdT is quite similar and decreases in the order dCTP>dllP>dATP>dGTP. The Km for both synthetic primer and activated DNA with the 3 TdTs was, in increasing orderr two subunit 44 kd (45 kd <58 kd TdT. Both 58 kd and 45 kd TdT displayed two optima for Mn⁺⁺ (0.1 mM and 1mM) and a single sharp optimum for Mg⁺⁺ (2.5 mM). The two subunit 44 kd TdT exhibited a single but broad optimum for Mntt (1mM) and for Mg⁺⁺ (10 mM).