Poly synthetase immobilized enzyme

Immobilization was carried out using a homogeneous preparation of poly(ADP-ribose) synthetase obtained from calf thymus [6] and commercially available BrCN-activated Sepharose 4B. For successful immobilization of active enzyme, it was important to block a majority (% two-thirds) of the active groups of the gel by pretreatment with dithiothreitol. The treated gel was mixed with the enzyme solution in the presence of 0.35 M KCl, 0.2 mAf dithiothreitol, 20% glycerol, and 0.1 A/K phosphate buffer (pH 8.0). Figure 1 shows the time course of immobilization of the enzyme activity. The decrease in the soluble enzyme activity reflects mainly immobilization of the enzyme, whereas the decrease in the immobilized enzyme activity is indicative of inactivation of the enzyme. In order to avoid the increase of inactive... 

Fig. 1. Time course of immobilization of poly(ADP-ribose) synthetase. Immobilization was carried out as described in the text at 0-4°C. At intervals, aliquots were taken out and examined for enzyme activities remaining soluble ( — ) or bound to the gel (x—x)...

In the present study, by making use of immobilized enzyme preparations, we presented evidence for the intrinsic NAD glycohydrolase activity associated with poly(ADP-ribose) synthetase and its manifestation by immobilization, and for the intramolecular mechanism of auto-poly(ADP-ribosyl)ation, including initiation, elongation, and branching of the polymer. 

In the absence of an exogenous acceptor, the immobilized synthetase catalyzed auto-modification, that is, poly(ADP-ribose) synthesis on gel-bound enzyme molecules (Table 1). The product polymer was a mixed population with a variety of chain lengths as revealed by polyacrylamide gel electrophoresis. The polymer had branching at a frequency of about once every 40 ADP-ribose residues. The average chain size was calculated as vlOO ADP-ribose residues/polymer molecule in a 1 h incubation at 25°C. 

---