Intracellular NAD+ content and ADP-ribose polymerase activity of serum-stimulated baby hamster kidney fibroblasts.

Abstract

We have examined a number of events relating to ADP-ribose metabolism during serum-stimulated growth of BHK-21/C13 fibroblasts. Both the intracellular NAD+ content and the ADP-ribose polymerase activity were found to increase after serum stimulation of cells that were previously arrested by growth in low-serum medium. NAD+ content increased about two-fold, reaching a maximum of 4.2 nmol/microgram of DNA 8 hr after serum steK-21/C13 fibroblasts. Both the intracellular NAD+ content and the ADP-ribose polymerase activity were found to increase after serum stimulation of cells that were previously arrested by growth in low-serum medium. NAD+ content inreased about two-fold, reaching a maximum of 4.2 nmol/microgram of DNA 8 hr after serum step-up. The polymerase exhibited a sharp rise in activity, reaching a peak at about 5 hr after step-up; the activity declined below initial values by 10 hr, and then increased again to reach a plateau at 20 hr. We also report evidence which suggests a possible effect of ADP-ribosylation on the activity of DNA-dependent RNA polymerase I. The activity of this enzyme is diminished in isolated nuclei, and in a subsequent (NH4)2SO4 extract, when the nuclei are incubated with NAD+, the substrate for poly(ADP-ribose) polymerase. This inhibitory effect on the RNA polymerase is abolished when nuclei are incubated also with nicotinamide, a powerful inhibitor of the poly(ADP-ribose) polymerase.