Modification of plasma membrane protein cysteine residues by ADP-ribose in vivo.

Proteins can be post-translationally modified by ADP-ribose. Previously, two classes of ADP-ribosyl protein linkages have been detected in vivo which have chemical properties indistinguishable from ADP-ribosyl arginine and ADP-ribosyl glutamate or aspartate. Reported here is the detection of a third class of endogenous ADP-ribosyl protein linkage. This class is chemically indistinguishable from ADP-ribose linked to cysteine residues by a thioglycosidic bond. The distribution of ADP-ribosyl cysteine residues was studied in subcellular fractions of rat liver. Proteins modified on cysteine were detected only in the plasma membrane fraction. Pertussis toxin is known to disrupt signal transduction of ADP-ribosylation of cysteine residues of plasma membrane GTP binding proteins. The results described here raise the interesting possibility that the endogenous modification of plasma membrane protein cysteine residues may be involved in signal transduction.

Effect of nicotinamide analogues on recovery from DNA damage in C3H10T1/2 cells.

The effects of nicotinamide analogues on cellular recovery following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment have been characterized in the transformable cell line, C3H10T1/2. The recovery of cell division potential was measured under conditions which allow simultaneous quantification of intracellular levels of poly(adenosine diphosphate ribose), nicotinamide adenine dinucleotide, and rates of RNA, DNA, and protein synthesis. 3- Methoxybenzamide (MBA), 3-aminobenzamide, and benzamide, which are effective inhibitors of adenosine diphosphate ribosyltransferases , blocked recovery of cell division following treatment with 34 microM MNNG, while the noninhibitors , 3- methoxybenzoate and benzoate, had no effect. In the presence of MBA, cells progressively lost the ability to resume cell division during the first 24 to 36 hr following DNA damage. The intracellular levels of poly(adenosine diphosphate ribose) increased approximately 7-fold within 20 min following MNNG treatment, and 1 mM MBA inhibited this increase by approximately 82%. In the presence of MBA, a dramatic decrease in the rate of DNA synthesis occurred approximately 16 hr after MNNG treatment, while RNA and protein synthesis continued at rates similar to those in cells treated with MNNG alone.

Mono- and poly(ADP-ribose) metabolism following DNA damage.

Newly developed chemical methods have been applied to study ADP-ribosyl transferase reactions in intact cells following DNA damage. The intracellular levels of NAD and protein-bound monomeric and polymeric ADP-ribose residues were measured in cultured human cells following UV irradiation and in cultured mouse cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). UV irradiation of cells caused a rapid increase in the levels of poly(ADP-ribose). Individual molecules of the polymer are present only transiently and the overall rate of conversion of NAD to poly(ADP-ribose) is proportional to the cellular content of DNA strand breaks. Treatment of cells with MNNG also causes a rapid increase in the levels of both monomers and polymers of ADP-ribose. Non-toxic levels of members of two different classes of ADP-ribosyl transferase inhibitors prevent recovery of cell division following treatment of C3H10T1/2 cells with MNNG, while closely related compounds that are not inhibitory have no effect. These studies demonstrate that DNA damage results in a rapid perturbation of ADP-ribose metabolism and suggest that ADP-ribosyl transferase activity is necessary for cellular recovery from DNA damage.