Microbiology Department, Faculty of Science, Zagazig University, Egypt.
Enzyme Microb Technol. 2012 Sep 10;51(4):200-10. doi: 10.1016/j.enzmictec.2012.06.004. Epub 2012 Jun 28.
Methionine starvation can powerfully modulate DNA methylation, cell cycle transition, polyamines and antioxidant synthesis of tumor cells, in contrary to normal ones. Aspergillus flavipesl-methioninase was previously characterized by our studies, displaying affordable biochemical properties comparing to Pseudomonas putida enzyme (ONCASE). Thus, the objective of current study was to evaluate the catalytic properties of Af-METase in New Zealand rabbits, exploring its antitumor efficacy. In vivo, Af-METase (40.8 U/ml) have T(1/2) 19.8 h, elimination constant 0.088 U/h and apparent volume distribution 85 U/ml. Also, Af-METase has two maxima one at A(280 nm) (apo-enzyme) and at A(420 nm) (internal Schiff base of PLP), unlike control plasma (without enzyme). The two peaks of absorption spectra were detected maximally at 15 min then the absorbance at 420 nm was subsequently decreased with circulation time, due to dissociation of the co-enzyme. The A₂₈₀/₄₂₀ ratio was increased from 1.69 to 5.81 with circulation time from 15 to 30 h. Rabbits plasma methionine was depleted from 18.7 μM (control) to 8.8 μM after 1h of enzyme injection and completely omitted after 2 h till 19 h, assuming the sustainability of negligible levels of methionine (< 2 μM) in plasma of rabbits, for about 17 h. Upon infusion of PLP, the T(½) of Af-METase was significantly prolonged by 3.2 fold, assuming the fully reconstitution of the enzyme. The holo-AfMETase still retained its co-enzyme, completely, till 33 h of PLP infusion. From spectral studies, the internal aldimine linkage of apo-Af-METase was constructed upon PLP infusion, with fully catalytic structure after less than 4h of its infusion, the A₂₈₀/₄₂₀ ratio being not relatively changed till 45 h. After 25 days of last enzyme dose, the titer of IgG was increase by about 1.66 fold comparing to control (without enzyme). However, IgM was not detected along the tested challenge points. In vitro, plasma anti-Af-METase neutralizing antibodies (NAb) were assessed, with no significant reduction on activity of Af-METase by Nab. All the hematological parameters were in normal range, otherwise, the RBCs titer and platelet level was slightly increased, after 25 days of Af-METase injection, comparing to control. There is no obvious negative effect on chemistry of liver, kidney, glucose, lipids, and other electrolytes. Additionally, the anticancer activity of Af-METase was evaluated against five types of human cancer cell lines, in vitro. The enzyme showed a powerful activity against prostate (PC3), liver (HEPG2) and breast (MCF7) cancers, with IC₅₀ 0.001 U/ml, 0.26 U/ml and 0.37 U/ml, respectively.
蛋氨酸饥饿可以强烈调节肿瘤细胞的 DNA 甲基化、细胞周期转变、多胺和抗氧化剂合成，与正常细胞相反。黄曲霉 methioninase 以前被我们的研究所表征，与假单胞菌酶（ONCASE）相比，具有可负担的生化特性。因此，本研究的目的是评估 Af-METase 在新西兰兔中的催化特性，探索其抗肿瘤功效。在体内，Af-METase（40.8 U/ml）的 T（1/2）为 19.8 h，消除常数为 0.088 U/h，表观体积分布为 85 U/ml。此外，Af-METase 有两个最大值，一个在 A（280nm）（脱辅基酶），另一个在 A（420nm）（PLP 的内部希夫碱），与对照血浆（无酶）不同。吸收光谱的两个峰值在 15 分钟时最大，然后随着循环时间的延长，在 420nm 处的吸光度随后降低，这是由于辅酶的解离。A₂₈₀/₄₂₀ 比值从 1.69 增加到 5.81，循环时间从 15 小时增加到 30 小时。酶注射 1 小时后，兔血浆蛋氨酸从 18.7 μM（对照）耗竭至 8.8 μM，2 小时后完全耗尽，直至 19 小时，这表明兔血浆中蛋氨酸（<2 μM）的水平可维持约 17 小时的低水平。在输注 PLP 后，Af-METase 的 T（½）延长了 3.2 倍，假设酶完全重组。全酶 holo-AfMETase 仍然完全保留其辅酶，直到 PLP 输注 33 小时。从光谱研究来看，apo-Af-METase 的内部希夫碱连接在 PLP 输注后形成，在其输注不到 4 小时后就具有完全的催化结构，A₂₈₀/₄₂₀ 比值直到 45 小时才相对不变。在最后一次酶剂量后 25 天，与对照组（无酶）相比，IgG 的效价增加了约 1.66 倍。然而，在测试的挑战点中未检测到 IgM。在体外，评估了血浆抗 Af-METase 中和抗体（NAb），NAb 对 Af-METase 的活性没有显著降低。所有血液学参数均在正常范围内，否则，与对照组相比，注射 Af-METase 25 天后，RBC 浓度和血小板水平略有升高。对肝脏、肾脏、葡萄糖、脂质和其他电解质的化学性质没有明显的负面影响。此外，还评估了 Af-METase 对五种类型的人癌细胞系的体外抗癌活性。该酶对前列腺（PC3）、肝脏（HEPG2）和乳腺（MCF7）癌具有强大的活性，IC₅₀ 分别为 0.001 U/ml、0.26 U/ml 和 0.37 U/ml。
