Evidence for an inhibitory effect exerted by yeast NMN adenylyltransferase on poly(ADP-ribose) polymerase activity.
We have previously reported for the first time the purification to homogeneity of the enzyme NMN adenylyltransferase (EC 2.7.7.1) from yeast and its major molecular and catalytic properties. The homogeneous enzyme was found to be a glycoprotein containing 2% carbohydrate and 1 mol of adenine residue and 2 mol of phosphate covalently bound per mole of protein. Such a stoichiometry, apparently consistent with that of ADP-ribose, prompted us to further investigate the possibility that NMN adenylyltransferase could be subjected to poly(ADP-ribosylation) in vitro in a reconstituted system. Poly(ADP-ribose) polymerase was purified to homogeneity from bull testis by means of a rapid procedure involving two batchwise steps on DNA-agarose and Reactive Blue 2 cross-linked agarose and a column affinity chromatography step on 3-aminobenzamide-Sepharose; the optimal conditions for the poly(ADP-ribosylation) of exogenous substrates were determined. When pure NMN adenylyltransferase was incubated in the presence of the homogeneous poly(ADP-ribose) polymerase, a marked inhibition of the polymerase was observed, both in the presence and in the absence of histones, while the activity of NMN adenylyltransferase was not affected. The inhibition could not be prevented by increasing the concentrations of either DNA or NAD. Mg2+ did not affect the activity or the inhibition. The significance of such a phenomenon is at present unknown, but it may be of biological relevance in view of the close topological and metabolic relationship between the two enzymes.
Ruggieri
S
S
Dipartimento di Biologia Cellulare, Università di Camerino, Italy.
Gregori
L
L
Natalini
P
P
Vita
A
A
Emanuelli
M
M
Raffaelli
N
N
Magni
G
G
D000818:Animals; D002417:Cattle; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000494:pharmacology*; D009713:Nucleotidyltransferases / Q000494:pharmacology*; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D013737:Testis / Q000201:enzymology; D015003:Yeasts / Q000187:drug effects / Q000201:enzymology*
D006657:Histones; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011064:Poly Adenosine Diphosphate Ribose; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D009713:Nucleotidyltransferases; D009612:Nicotinamide-Nucleotide Adenylyltransferase; D008274:Magnesium