Reconstitution and poly(ADP-ribosyl)ation of proteolytically fragmented poly(ADP-ribose) synthetase.
Calf thymus poly(ADP-ribose) synthetase (Mr = 120,000) is cleaved with papain into two fragments of M(r) = 74,000 and 46,000 and also split with chymotrypsin into two fragments of M(r) = 66,000 and 54,000. Each fragment purified to homogeneity is enzymatically inactive, but combined incubation of the 74,000 and 46,000 fragments in the presence of DNA restored 20% of the enzyme activity. In contrast, combined incubation of the 66,000 and 54,000 fragments does not restore any enzyme activity. In the former incubation, autopoly(ADP-ribosyl)ation reaction occurs exclusively on the 74,000 fragment. When each fragment is incubated with [adenine-U-14C]NAD in the presence of DNA and a catalytic amount of the native enzyme, poly(ADP-ribosyl)action occurs in the overlapped portion (22,000) of the 66,000 fragment and the 74,000 fragment. Nevertheless, the purified 22,000 fragment is a poor acceptor for poly(ADP-ribosyl)ation. The degree of poly(ADP-ribosyl)ation of the proteolytic fragments is significantly reduced by increasing NaCl concentration, probably due to the lack of the interaction between the enzyme fragments and DNA. These results, taken together, indicate that DNA is indispensable for the reconstitution of the catalytic activity as well as the poly(ADP-ribosyl)ation of the fragmented enzyme.
Kameshita
I
I
Matsuda
M
M
Nishikimi
M
M
Ushiro
H
H
Shizuta
Y
Y
D000596:Amino Acids / Q000032:analysis; D004247:DNA / Q000502:physiology; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D010206:Papain / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis* / Q000302:isolation & purification; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis*
D000596:Amino Acids; D009702:Nucleoside Diphosphate Sugars; D010446:Peptide Fragments; D009243:NAD; D011064:Poly Adenosine Diphosphate Ribose; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D010206:Papain