It has been shown that nuclei isolated from wheat seeds can incorporate [3H] NAD into an acid insoluble material which was subsequently shown to be poly (ADP-Ribose). The poly (ADP-Ribose) polymerase responsible for this incorporation has been fully characterised, the enzyme having a pH optimum of pH 7.9 and an apparent temperature optimum of 17°C. The enzyme requires free -SH groups for activity, with an optimal rate at a concentration of 10 mM mercaptoethanol, magnesium also plays an important role in the enzyme activity, with the optimal Mg2+ concentration being 2 mM. The polymerase is 100% inhibited by 5 mM DTNB, and this inhibition cannot be reversed by the addition of mercaptoethanol. Contrary to expectation the 100% inhibition of the polymerase by 0.5 mM Hg2+ is completely reversible upon addition of ? mercaptoethanol. Two km were found for poly (ADP-Ribose) polymerase in wheat, 7.4 x 10-5 M and 5.7 x 10-6 M, and both nicotinamide and 3-amino benzamide were shown to be inhibitory, however the inhibition did not follow normal Michaelis-Menten kinetics. Activity was unaffected by the addition of DNA but was inhibited by AMP, cyclic ANP, ATP, and GTP. The polymerase was shown to be unstable, the rate of decay following an Arrhenius relationship. The average chain length of poly (ADP-Ribose) formed by isolated wheat nuclei at an NAD concentration of 2.4 muM was 2.51 +/- 0.09. The isolated wheat nuclei were also shown to contain poly (ADP- Ribose) glycohydrolase activity, which was partially charaiterised. The degradation has pH optima at pH 6.4 and pH 6, and has an apparent temperature optimum of 34°C. The enzyme activity was shown to be independent of addend inercaptoethanol and magnesium, however addition of DTNB caused some inhibition of the glyrohydrolase. It was found that the addition of the polyamines spermine, spermidine and putrescine to isolated wheat nuclei stimulated the formation of [3H] poly (ADP-Ribose). Plant histones were extracted from the nuclear preparations and histones H1, H2A, H2B and possibly H3 were shown to be modified. The stimulation was shown to be independent of fluctuations in the poly (ADP-Ribose) glycohydrolase activity, changes in the average chain length of polymer synthesised, or increased specific labelling of any one histone. The increased ADP-Ribosylation was in fact due to a general increase in histone modification. Finally the activities of poly (ADP-Ribose) polymerase and glycohydrolase were shown to vary during germination of isolated wheat embryos. The polymerase activity did not vary significantly during the time studied, however, over the same period of time (up to 12 hours) the glycohydrolase activity showed significant changes. After 6 hours of germination at least 96% of the glycohydrolase activity was lost, this loss being tentatively associated with the presence of an inhibitor of poly (ADP-Ribose) glycohydrolase produced in vivo.
|Date of Award||1980|