Properties of a DNA polymerase from purified nuclei and DNA-synthesizing complexes of human spermatozoa.
A DNA polymerase was isolated from human spermatozoa. In one procedure, spermatozoa were decapitated with detergent, the heads purified and then lysed with dithiothreitol, trypsin and deoxyribonuclease. DNA polymerase was isolated from the lysate by sedimentation through an 18% Metrizamide solution, solubilization with 0.8 M-KCl-0.5% Triton X-100 and sequential chromatography on DEAE cellulose, phosphocellulose and hydroxylapatite. Alternatively, the heads of intact spermatozoa, untreated with detergent, were lysed as above; the subsequent Metrizamide pellet fraction was isolated and further fractionated by gel filtration and buoyant density centrifugation. The enzyme in this fraction was solubilized with KCl-Triton X-100. Characterization by velocity centrifugation and phosphocellulose chromatography revealed that it possessed properties indistinguishable from those of the enzyme purified from isolated sperm nuclei. The DNA polymerase had an apparent molecular weight of 79,000-89,000, Mn2+ (1 mM) was the preferred divalent cation and ativity was inhibited by concentrations of potassium phosphate greater than 10 mM. The synthetic template preferences of the enzyme were dT12-18 . poly rA > poly(dA-dT) > dT12-18 . poly dA; no activity was observed with dG12-18 . poly rC or dT10.
D002467:Cell Nucleus / Q000201:enzymology; D002498:Centrifugation; D002853:Chromatography, Liquid; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008297:Male; D013077:Sperm Head / Q000201:enzymology; D013094:Spermatozoa / Q000201:enzymology*; D013698:Templates, Genetic
D004259:DNA-Directed DNA Polymerase