Angiotensin converting enzyme of Thalassophryne nattereri venom.

Animal venoms are complex mixtures, including peptides, proteins (i.e., enzymes), and other compounds produced by animals in predation, digestion, and defense. These molecules have been investigated regarding their molecular mechanisms associated with physiological action and possible pharmacological applications. Recently, we have described the presence of a type of angiotensin converting enzyme (ACE) activity in the venom of Thalassophryne nattereri. It is a zinc-dependent peptidase with a wide range of effects. By removing dipeptide His-Leu from terminal C, the ACE converts angiotensinI (AngI) into angiotensin II (AngII) and inactivates bradykinin, there by regulating blood pressure and electrolyte homeostasis. The fractionation of T. nattereri venom in CM-Sepharose indicated a peak (CM2) with angiotensin-converting activity, converting AngI into Ang II. Electrophoresis on polyacrylamide gel (12%) revealed one band with 30kDa for CM2 similar in size to natterins, which are toxins with proteolytic activity found in T. nattereri venom. Mass spectrometry indicated that the protein sequence of the ACE purified from T. nattereri venom corresponds to natterin 1. The isolated protein has also demonstrated inhibition through captopril and EDTA and is characterized as a classic ACE. Thus, the isolated enzyme purified from T. nattereri venom is the first ACE isolated from fish venom.

Purification and characterization of trypsin from Luphiosilurus alexandri pyloric cecum.

Trypsin from L. alexandri was purified using only two purification processes: ammonium sulfate precipitation and anion exchange liquid chromatography in DEAE-Sepharose. Trypsin mass was estimated as 24 kDa through SDS-PAGE, which showed only one band in silver staining. The purified enzyme showed an optimum temperature and pH of 50 °C and 9.0, respectively. Stability was well maintained, with high levels of activity at a pH of up to 11.0, including high stability at a temperature of up to 50 °C after 60 min of incubation. The inhibition test demonstrated strong inhibition by PMSF, a serine protease inhibitor, and Kinetic constants km and kcat for BAPNA were 0.517 mM and 5.0 S-1, respectively. The purified enzyme was also as active as casein, as analyzed by zymography. Therefore, we consider trypsin a promising enzyme for industrial processes, owing to its stability in a wide range of pH and temperature and activity even under immobilization.