Plasmapheresis in multiple sclerosis

Abstract

Kinetic and template studies have been performed with 1-β-D-arabinofuranosylcytosine 59-triphosphate (araCTP), dCTP, and calf thymus DNA polymerase. Using denatured DNA as the template, the estimated apparent <i>K</i>_m values for araCTP and dCTP were about the same; however, the estimated apparent <i>V</i>_max value for dCTP was about 8 times greater than the apparent <i>V</i>_max for araCTP. Both araCTP and dCTP appeared to complete for the same catalytic site of DNA polymerase. When poly(dA-dT) was used as the template, araCTP did not produce any detectable inhibition of the incorporation of [³H]dTTP into acid-insoluble material. DNA Polymerase catalyzed the incorporation of [³H]araCTP into poly dC:poly dG, but not into poly(dA-dT). DNA containing [α-³²P]arabinofuranosylcytosine 59-monophosphate ([α-³²P]araCMP) was synthesized enzymatically, using denatured DNA and DNA polymerase. Following the digestion of this [³²P]DNA with micrococcal nuclease and spleen phosphodiesterase to deoxynucleoside 39-monophosphates, the radioactivity from [α-³²P]araCMP appeared in 39-dAMP, 39-dGMP, 39-dTMP, and 39-dCMP, suggesting that DNA polymerase could catalyze the formation of a phosphodiester bond between araCMP and each of the deoxynucleotides present in the DNA template. The data from studies on the rate of release of [α-³²P]araCMP and [³H]dCMP from DNA by snake venom phosphodiesterase and the incorporation of araCTP and dCTP into denatured, sonicated DNA are consistent with the proposal that the incorporation of araCTP into DNA in the reaction catalyzed by DNA polymerase produces termination of polynucleotide chain growth.