Amani M, Moosavi-Movahedi A A, Floris G, Longu S, Mura A, Moosavi-Nejad S Z, Saboury A A, Ahmad F
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
Protein J. 2005 Apr;24(3):183-91. doi: 10.1007/s10930-005-7842-5.
The thermal stability of copper/quinone containing amine oxidases from Euphorbia characias latex (ELAO) and lentil seedlings (LSAO) was measured in 100 mM potassium phosphate buffer (pH 7.0) following changes in absorbance at 292 nm. ELAO was shown to be about 10 degrees C more stable than LSAO. The dissociative thermal inactivation of ELAO was studied using putrescine as substrate at different temperatures in the range 47-70 degrees C, and a "conformational lock" was developed using the theory pertaining to oligomeric enzyme. Moreover ELAO was shown to be more stable towards denaturants than LSAO, as confirmed by dodecyl trimethylammonium bromide denaturation curves. A comparison of the numbers of contact sites in inter-subunits of ELAO relative to LSAO led us to conclude that the higher stability of ELAO to temperature and towards denaturants was due to the presence of larger number of contact sites in the conformational lock of the enzyme. This study also gives a putative common mechanism for thermal inactivation of amine oxidases and explains the importance of C-terminal conserved amino acids residues in this class of enzymes.
在100 mM磷酸钾缓冲液（pH 7.0）中，通过监测292 nm处吸光度的变化，测定了来自大戟乳胶（ELAO）和扁豆幼苗（LSAO）的含铜/醌胺氧化酶的热稳定性。结果表明，ELAO比LSAO的稳定性高约10℃。在47-70℃范围内的不同温度下，以腐胺为底物研究了ELAO的解离热失活，并利用寡聚酶相关理论建立了“构象锁”。此外，十二烷基三甲基溴化铵变性曲线证实，ELAO比LSAO对变性剂更稳定。比较ELAO和LSAO亚基间接触位点的数量，我们得出结论，ELAO对温度和变性剂具有更高的稳定性，是由于该酶构象锁中存在更多的接触位点。这项研究还给出了胺氧化酶热失活的一个推测性共同机制，并解释了这类酶中C端保守氨基酸残基的重要性。
