Comparative characterisation of poly(ADP-ribose) polymerase-1 from two mammalian species with different life span.
DNA damage induced in higher eukaryotes by alkylating agents, oxidants or ionising radiation triggers the synthesis of protein-conjugated poly(ADP-ribose) catalysed by poly(ADP-ribose) polymerase-1 (PARP-1). Previously, cellular poly(ADP-ribosyl)ation capacity has been shown to correlate positively with the life span of mammalian species [Proc. Natl. Acad. Sci. USA 89 (1992) 11,759-11,763]. Here, we have tested whether this correlation results from differences in kinetic parameters of the enzymatic activity of PARP-1. We therefore compared recombinant enzymes, expressed in a baculovirus system, from rat and man as two mammalian species with extremely divergent life span. In standard activity assays performed in the presence of histones as poly(ADP-ribose) acceptors both enzymes showed saturation kinetics with [NAD(+)]. The kinetic parameters (k(cat), k(m) and k(cat)/k(m)) of the two enzymes were not significantly different. However, in assays assessing the auto-poly(ADP-ribosyl)ation reaction, both enzymes displayed second-order kinetics with respect to [PARP-1], and up to two-fold higher specific activity was observed for human versus rat PARP-1. We conclude that the correlation of poly(ADP-ribosyl)ation capacity with life span is not reflected in the kinetic parameters, but that subtle differences in primary structure of PARP-1 from mammalian species of different longevity may control the extent of the automodification reaction.
Beneke
S
S
Abteilung Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Alvarez-Gonzalez
R
R
Bürkle
A
A
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D004249:DNA Damage; D006801:Humans; D007700:Kinetics; D008136:Longevity / Q000502:physiology*; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013045:Species Specificity
D011994:Recombinant Proteins; D011065:Poly(ADP-ribose) Polymerases