Telomerase from human leukemia cells: properties and its interaction with deoxynucleoside analogues.
Telomerase is a unique reverse transcriptase involved in the maintenance of genomic integrity. In an attempt to understand the properties of this enzyme and to study the effect of deoxynucleoside analogues, we have isolated and partially purified telomerase from the blast cells of a patient with acute myelogenous leukemia. During the course of purification of telomerase, three characteristic forms of this enzyme activity were separated. Two processive forms and one less processive form were noted. All forms of the enzyme activities could be abolished by RNase A and proteinase K treatments, implying that they are ribonucleoproteins. The major form of telomerase was characterized with respect to divalent ion requirements, effect of salt and nonionic detergents. The Km of deoxynucleoside triphosphates was determined with a modified telomerase repeat array protocol assay. Studies with deoxynucleoside analogues indicated that 3'-azido-3'deoxythymidine triphosphate is much more inhibitory than 2',3'-dideoxy 2',3'didehydrothymidine triphosphate, and the cytidine analogue ddCTP was not inhibitory. ddGTP was the most potent inhibitor among all dideoxynucleosides studied.
Pai
R B
RB
Department of Pharmacology, Yale School of Medicine, Yale University, New Haven, Connecticut 06510, USA.
Pai
S B
SB
Kukhanova
M
M
Dutschman
G E
GE
Guo
X
X
Cheng
Y C
YC
D002852:Chromatography, Ion Exchange; D003854:Deoxyribonucleotides / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011189:Potassium Chloride / Q000494:pharmacology; D012259:Ribonuclease, Pancreatic / Q000494:pharmacology; D019098:Telomerase / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000201:enzymology
D003854:Deoxyribonucleotides; D011189:Potassium Chloride; D019098:Telomerase; D012259:Ribonuclease, Pancreatic; D019286:Endopeptidase K