ABSTRACT
Glutamine synthetase from ovine brain has a critical arginine residue at the catalytic site (Powers, S. G., and Riordan, J.F. (1975) Proc. Natl. Acad. Sci. U.S. A. 72, 2616-2620). This enzyme is now shown to be a substrate for a purified NAD:arginine ADP-ribosyltransferase from turkey erythrocyte cytosol that catalyzes the transfer of ADP-ribose from NAD to arginine and purified proteins. The transferase catalyzed the inactivation of the synthetase in an NAD-dependent reaction; ADP-ribose and nicotinamide did not substitute for NAD. Agmatine, an alternate ADP-ribose acceptor in the transferase-catalyzed reaction, prevented inactivation of glutamine synthetase. MgATP, a substrate for the synthetase which was previously shown to protect that enzyme from chemical inactivation, also decreased the rate of inactivation in the presence of NAD and ADP-ribosyltransferase. Using [32P]NAD, it was observed that approximately 90% inactivation occurred following the transfer of 0.89 mol of [32P]ADP-ribose/mol of synthetase. The erythrocyte transferase also catalyzed the NAD-dependent inactivation of glutamine synthetase purified from chicken heart; 0.60 mol of ADP-ribose was transferred per mol of enzyme, resulting in a 95% inactivation. As noted with the ovine brain enzyme, agmatine and MgATP protected the chicken synthetase from inactivation and decreased the extent of [32P]ADP-ribosylation of the synthetase. These observations are consistent with the conclusion that the NAD:arginine ADP-ribosyltransferase modifies specifically an arginine residue involved in the catalytic site of glutamine synthetase. Although the transferase can use numerous proteins as ADP-ribose acceptors, some characteristics of this particular arginine, perhaps the same characteristics that are involved in its function in the catalytic site, make it a favored ADP-ribose acceptor site for the transferase.
Subject(s)
Brain/enzymology , Glutamate-Ammonia Ligase/antagonists & inhibitors , Pentosyltransferases/metabolism , ADP Ribose Transferases , Adenosine Triphosphate/pharmacology , Agmatine/pharmacology , Animals , Cattle , Erythrocytes/enzymology , Kinetics , NAD/metabolism , TurkeysABSTRACT
The response of cells to various poly(ADP-ribose) synthetase inhibitors may vary with the cell type and inhibitor type. We have observed that most cells show an inhibition of growth, at least transiently, when exposed to synthetase inhibitors. For one cell type, there is, in contrast, a stimulation of proliferation. These results suggest that the synthetase plays different roles in different cell types or, the inhibitors also affect other cell processes that are variably represented in the different cell types. Because of these difficulties we have sought alternative methods of modulating poly(ADP-ribose) synthetase activity in intact cells. A method of inactivating the synthetase by heat shock is described and the consequences of this inactivation for the repair of single strand breaks in DNA is presented. Additionally, a method for elevating the synthetase in intact cells is given.
Subject(s)
Hot Temperature , NAD+ Nucleosidase/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Animals , Benzamides/pharmacology , Cell Line , Cells, Cultured/drug effects , Cricetinae , Female , HeLa Cells/drug effects , HeLa Cells/enzymology , Humans , In Vitro Techniques , Mammary Glands, Animal , Mice , Niacinamide/analogs & derivatives , Niacinamide/pharmacology , Ovary , Poly(ADP-ribose) Polymerase InhibitorsSubject(s)
Adenosine Diphosphate Ribose/metabolism , Nucleoproteins/metabolism , Nucleoside Diphosphate Sugars/metabolism , Animals , Antibodies , Carcinoma, Ehrlich Tumor/enzymology , Cell Nucleus/metabolism , Humans , Leukemia/enzymology , Mice , Poly(ADP-ribose) Polymerases/isolation & purification , Poly(ADP-ribose) Polymerases/metabolism , Protein Binding , Radioimmunoassay , Swine , Thymus Gland/enzymology , Tissue DistributionABSTRACT
In a search for new inhibitors of the nuclear enzyme poly(ADP-ribose) synthetase, it was found that various benzamides substituted in the 3-position were the most inhibitory compounds found to date. Two of the benzamides, 3-aminobenzamide and 3-methoxybenzamide, were found to be competitive inhibitors, with Ki values or less than 2 microM.
