Molecular interactions between poly(ADP-ribose) polymerase (PARP I) and topoisomerase I (Topo I): identification of topology of binding.

The molecular interactions of poly(ADP-ribose) polymerase I (PARP I) and topoisomerase I (Topo I) have been determined by the analysis of physical binding of the two proteins and some of their polypeptide components and by the effect of PARP I on the enzymatic catalysis of Topo I. Direct association of Topo I and PARP I as well as the binding of two Topo I polypeptides to PARP I are demonstrated. The effect of PARP I on the 'global' Topo I reaction (scission and religation), and the activation of Topo I by the 36 kDa polypeptide of PARP I and catalytic modifications by poly(ADP-ribosyl)ation are also shown. The covalent binding of Topo I to circular DNA is activated by PARP I similar to the degree of activation of the 'global' Topo I reaction, whereas the religation of DNA is unaffected by PARP I. The geometry of PARP I-Topo I interaction compared to automodified PARP I was reconstructed from direct binding assays between glutathione S-transferase fusion polypeptides of Topo I and PARP I demonstrating highly selective binding, which was correlated with amino acid sequences and with the 'C clamp' model derived from X-ray crystallography.

Comparative in vitro studies on native and recombinant human cationic trypsins. Cathepsin B is a possible pathological activator of trypsinogen in pancreatitis.

Hereditary pancreatitis, an autosomal dominant disease is believed to be caused by mutation in the human trypsinogen gene. The role of mutations has been investigated by in vitro studies using recombinant rat and human trypsinogen (TG). In this study we compare the enzymatic properties and inhibition by human pancreatic secretory trypsin inhibitor (hPSTI) of the native, postsynthetically modified and recombinant cationic trypsin, and found these values practically identical. We also determined the autolytic stability of recombinant wild type (Hu1Asn21) and pancreatitis-associated (Hu1Ile21) trypsin. Both forms were equally stable. Similarly, we found no difference in the rate of activation of the two zymogens by human cationic and anionic trypsin. Mesotrypsin did not activate either form. The rate of autocatalytic activation of Hu1Asn21 TG and Hu1Ile21 TG was also identical at pH 8 both in the presence and absence of Ca2+. At pH 5 Hu1Ile21 TG autoactivated about twice as fast as Hu1Asn21 TG. The presence of physiological amount of hPSTI completely prevented autoactivation of both zymogens at pH 8 and at pH 5 as well. Cathepsin B readily activated both zymogens although Hu1Ile21 TG was activated about 2.5-3 times as fast as Hu1Asn21 TG. The presence of hPSTI did not prevent the activation of zymogens by cathepsin B. Our results underlie the central role of cathepsin B in the development of different forms of pancreatitis.