Purification of high molecular mass species of calf thymus terminal deoxynucleotidyltransferase.
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 deoxynucleotidyltransferase (TdT) from calf thymus chromatin. The procedure involves high salt extraction of the chromatin fraction followed by successive chromatographies on phosphocellulose, DEAE sephadex, and hydroxylapatite matrices. While all 3 species of TdT comigrate throughout these steps, separation of individual species is achieved on a single stranded DNA agarose column. The combined yield of the 45 kd and 58 kd TdTs is quite high (approximately 8 mg/5000g tissue), 45 kd being the major species (approximately 60%) and the 58 kd constituting about 30%. The 44 kd species containing two subunits usually represents under 10% of the total. All the three forms of TdT showed similar specific activity and preference for purine deoxynucleoside triphosphates (dNTPs). The Km for individual dNTP with all three species of TdT is quite similar and decreases in the order dCTP greater than dTTP greater than dATP greater than dGTP. The Km for both synthetic primer and activated DNA with the 3 TdTs was, in increasing order, two subunit 44 kd less than 45 kd less than 58 kd TdT. Both 58 kd and 45 kd TdT displayed two optima for Mn++ (0.1 mM and 1 mM) and a single sharp optimum for Mg++ (2.5 mM). The two subunit 44 kd TdT exhibited a single but broad optimum for Mn++ (1 mM) and for Mg++ (10 mM).
Department of Biochemistry, UMDNJ-New Jersey Medical School, Newark 07103.
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Pandey
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Modak
D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D002845:Chromatography; D002846:Chromatography, Affinity; D004253:DNA Nucleotidylexotransferase / Q000302:isolation & purification* / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010777:Photochemistry; D013379:Substrate Specificity; D013942:Thymine Nucleotides; D013950:Thymus Gland / Q000201:enzymology*
D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D013942:Thymine Nucleotides; D004254:DNA Nucleotidyltransferases; D004253:DNA Nucleotidylexotransferase; C024157:thymidine 5'-triphosphate