TY - JOUR
T1 - Monochloramine Impairs Caspase-3 Through Thiol Oxidation and Zn2+ Release
AU - Kohler, Jonathan E.
AU - Mathew, Jeff
AU - Tai, Kaniza
AU - Blass, Amy L.
AU - Kelly, Edward
AU - Soybel, David I.
N1 - Funding Information:
This work was funded by NIH R01 DK069929 (to D.I.S.), NIH T32 DK 007754 (to J.M. and J.E.K.) and by an American College of Surgeons Resident Research Fellowship (to J.E.K.). We thank Melissa Beshire for technical assistance with the assays.
PY - 2009/5/1
Y1 - 2009/5/1
N2 - Background: Caspase-3, a pro-apoptotic enzyme, represents a class of proteins in which the active site contains reduced thiol (S-H) groups and is modulated by heavy metal cations, such as Zn2+. We explored the effects of the thiol oxidant monochloramine (NH2Cl) on caspase-3 activity within cells of isolated rabbit gastric glands. In addition, we tested the hypothesis that NH2Cl-induced alterations of caspase-3 activity are modulated by oxidant-induced accumulation of Zn2+ within the cytoplasm. Materials and methods: Isolated gastric glands were prepared from rabbit mucosa by collagenase digestion. Caspase-3 activity was measured colorimetrically in suspensions of healthy rabbit gastric glands, following exposure to various concentrations of NH2Cl with or without the zinc chelator TPEN [tetrakis-(2-pyridylmethyl)ethylene diamine] for 1 h, and re-equilibration in Ringer's solution for 5 h. Conversion of procaspase-3 to active caspase-3 was monitored by Western blot. Results: Monochloramine inhibited caspase-3 activity in a dose-dependent fashion. At concentrations of NH2Cl up to 100 μm, these effects were prevented if TPEN was given concurrently and were partly reversed if TPEN was given 1 h later. Caspase-3 activity was preserved by concurrent treatment with a thiol-reducing agent, dithiothreitol. Conclusions: At pathologically relevant concentrations, NH2Cl impairs caspase-3 activity through oxidation of its thiol groups. Independently from its thiol oxidant effects on the enzyme, NH2Cl-induced accumulation of Zn2+ in the cytoplasm is sufficient to restrain endogenous caspase-3 activity. Our studies suggest that some bacterially generated oxidants, such as NH2Cl, impair host pathways of apoptosis through release of Zn2+ from endogenous pools.
AB - Background: Caspase-3, a pro-apoptotic enzyme, represents a class of proteins in which the active site contains reduced thiol (S-H) groups and is modulated by heavy metal cations, such as Zn2+. We explored the effects of the thiol oxidant monochloramine (NH2Cl) on caspase-3 activity within cells of isolated rabbit gastric glands. In addition, we tested the hypothesis that NH2Cl-induced alterations of caspase-3 activity are modulated by oxidant-induced accumulation of Zn2+ within the cytoplasm. Materials and methods: Isolated gastric glands were prepared from rabbit mucosa by collagenase digestion. Caspase-3 activity was measured colorimetrically in suspensions of healthy rabbit gastric glands, following exposure to various concentrations of NH2Cl with or without the zinc chelator TPEN [tetrakis-(2-pyridylmethyl)ethylene diamine] for 1 h, and re-equilibration in Ringer's solution for 5 h. Conversion of procaspase-3 to active caspase-3 was monitored by Western blot. Results: Monochloramine inhibited caspase-3 activity in a dose-dependent fashion. At concentrations of NH2Cl up to 100 μm, these effects were prevented if TPEN was given concurrently and were partly reversed if TPEN was given 1 h later. Caspase-3 activity was preserved by concurrent treatment with a thiol-reducing agent, dithiothreitol. Conclusions: At pathologically relevant concentrations, NH2Cl impairs caspase-3 activity through oxidation of its thiol groups. Independently from its thiol oxidant effects on the enzyme, NH2Cl-induced accumulation of Zn2+ in the cytoplasm is sufficient to restrain endogenous caspase-3 activity. Our studies suggest that some bacterially generated oxidants, such as NH2Cl, impair host pathways of apoptosis through release of Zn2+ from endogenous pools.
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U2 - 10.1016/j.jss.2008.05.021
DO - 10.1016/j.jss.2008.05.021
M3 - Article
C2 - 19118843
AN - SCOPUS:63649135592
SN - 0022-4804
VL - 153
SP - 121
EP - 127
JO - Journal of Surgical Research
JF - Journal of Surgical Research
IS - 1
ER -