Lametsch René, Lonergan Steven, Huff-Lonergan Elisabeth
Department of Food Science, University of Copenhagen, 1958 Frederiksberg C, Denmark.
Biochim Biophys Acta. 2008 Sep;1784(9):1215-21. doi: 10.1016/j.bbapap.2008.04.018. Epub 2008 May 4.
Oxidative processes have the ability to influence mu-calpain activity. In the present study the influence of oxidation on activity and autolysis of mu-calpain was examined. Furthermore, LC-MS/MS analysis was employed to identify and characterize protein modifications caused by oxidation. The results revealed that the activity of mu-calpain is diminished by oxidation with H2O2 in a reversible manner involving cysteine and that the rate of autolysis of mu-calpain concomitantly slowed. The LC-MS/MS analysis of the oxidized mu-calpain revealed that the amino acid residues 105-133 contained a disulfide bond between Cys(108) and Cys(115). The finding that the active site cysteine in mu-calpain is able to form a disulfide bond has, to our knowledge, not been reported before. This could be part of a unique oxidation mechanism for mu-calpain. The results also showed that the formation of the disulfide bond is limited in the control (no oxidant added), and further limited in a concentration-dependent manner when beta-mercaptoethanol is added. However, the disulfide bond is still present to some extent in all conditions indicating that the active site cysteine is potentially highly susceptible to the formation of this intramolecular disulfide bond.
氧化过程能够影响μ-钙蛋白酶的活性。在本研究中，考察了氧化对μ-钙蛋白酶活性和自溶作用的影响。此外，采用液相色谱-串联质谱（LC-MS/MS）分析来鉴定和表征由氧化引起的蛋白质修饰。结果显示，μ-钙蛋白酶的活性可被过氧化氢可逆性氧化而降低，该过程涉及半胱氨酸，且μ-钙蛋白酶的自溶速率随之减慢。对氧化后的μ-钙蛋白酶进行LC-MS/MS分析显示，105-133位氨基酸残基在半胱氨酸（Cys）108和半胱氨酸115之间存在二硫键。据我们所知，μ-钙蛋白酶中活性位点半胱氨酸能够形成二硫键这一发现此前尚未见报道。这可能是μ-钙蛋白酶独特氧化机制的一部分。结果还表明，在对照条件下（未添加氧化剂）二硫键的形成有限，而添加β-巯基乙醇后，其形成以浓度依赖的方式进一步受限。然而，在所有条件下二硫键仍在一定程度上存在，这表明活性位点半胱氨酸可能极易形成这种分子内二硫键。
