Chitotriosidase (ChT) is an endoglucosaminidase enzyme that cleaves chitinous substrates and has been strongly associated with innate immune activity and the ability to identify non-selftissues. This enzyme activity w...Chitotriosidase (ChT) is an endoglucosaminidase enzyme that cleaves chitinous substrates and has been strongly associated with innate immune activity and the ability to identify non-selftissues. This enzyme activity was detected and characterized the serum from the American alligator (Alligator mississippiensis) using a fluorometric probe. Alligator serum exhibited volume-dependent activity, with activity (2.1 ± 0.3 μmol/min) observed at dilutions as low as a 1:150, and maximum activity (5.2 ± 0.6 μmol/min) measured at a dilution of 1:30. Alligator serum ChT showed linear activity for approximately 20 min, at which time activity decreased exponentially, presumably due to the depletion of substrate. In addition, the ChT activity in alligator serum was temperature-dependent with low activity at 5°C, a sharp increase from 10°C - 30°C, and maximal activity from 30°C - 40°C. The activity was inhibited in the presence of water-soluble chitin, but not mannan, indicating the specificity of the enzyme. The presence of ChT in alligator serum is likely to be partially responsible for the potent innate immune system of these crocodylians, and particularly antifungal activities.展开更多
Incubation of different volumes of serum from the Komodo dragon (Varanus komodoensis) with sheep red blood cells (SRBCs) resulted in volume-dependent hemolysis, as measured spectrophotometrically at 540 nm. The hemoly...Incubation of different volumes of serum from the Komodo dragon (Varanus komodoensis) with sheep red blood cells (SRBCs) resulted in volume-dependent hemolysis, as measured spectrophotometrically at 540 nm. The hemolysis occurred rapidly, with almost 90% of the hemolytic activity occurring within 20 min of incubation. A thermal profile showed that Komodo dragon serum exhibited low activity from 5- 20℃, but exerted maximum activity at 35℃, which was substantially reduced at 40℃. The maximum activity was observed near optimal temperatures to which Komodo dragons thermoregulate. Mild heat treatment of Komodo dragon serum (56℃, 30 min) depleted the ability to hemolyze SRBCs. In addition, preincubation of Komodo dragon serum with only 5 mM EDTA or phosphate, both chelators of divalent metal ions, reduced the hemolytic activity sharply. These results indicate that the hemolytic activity was due to the presence of a potent serum complement system. Incubation of Komodo dragon serum with 5 mM EDTA and 15 mM Ca2+ or Mg2+, but not Ba2+, Zn2+, or Fe2+, completely restored activity. These results indicate that Komodo dragon serum complement activity requires the presence of Mg2+ or Ca2+. This is the first assessment of innate immune activity of a Varanid.展开更多
文摘Chitotriosidase (ChT) is an endoglucosaminidase enzyme that cleaves chitinous substrates and has been strongly associated with innate immune activity and the ability to identify non-selftissues. This enzyme activity was detected and characterized the serum from the American alligator (Alligator mississippiensis) using a fluorometric probe. Alligator serum exhibited volume-dependent activity, with activity (2.1 ± 0.3 μmol/min) observed at dilutions as low as a 1:150, and maximum activity (5.2 ± 0.6 μmol/min) measured at a dilution of 1:30. Alligator serum ChT showed linear activity for approximately 20 min, at which time activity decreased exponentially, presumably due to the depletion of substrate. In addition, the ChT activity in alligator serum was temperature-dependent with low activity at 5°C, a sharp increase from 10°C - 30°C, and maximal activity from 30°C - 40°C. The activity was inhibited in the presence of water-soluble chitin, but not mannan, indicating the specificity of the enzyme. The presence of ChT in alligator serum is likely to be partially responsible for the potent innate immune system of these crocodylians, and particularly antifungal activities.
文摘Incubation of different volumes of serum from the Komodo dragon (Varanus komodoensis) with sheep red blood cells (SRBCs) resulted in volume-dependent hemolysis, as measured spectrophotometrically at 540 nm. The hemolysis occurred rapidly, with almost 90% of the hemolytic activity occurring within 20 min of incubation. A thermal profile showed that Komodo dragon serum exhibited low activity from 5- 20℃, but exerted maximum activity at 35℃, which was substantially reduced at 40℃. The maximum activity was observed near optimal temperatures to which Komodo dragons thermoregulate. Mild heat treatment of Komodo dragon serum (56℃, 30 min) depleted the ability to hemolyze SRBCs. In addition, preincubation of Komodo dragon serum with only 5 mM EDTA or phosphate, both chelators of divalent metal ions, reduced the hemolytic activity sharply. These results indicate that the hemolytic activity was due to the presence of a potent serum complement system. Incubation of Komodo dragon serum with 5 mM EDTA and 15 mM Ca2+ or Mg2+, but not Ba2+, Zn2+, or Fe2+, completely restored activity. These results indicate that Komodo dragon serum complement activity requires the presence of Mg2+ or Ca2+. This is the first assessment of innate immune activity of a Varanid.
