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目的:3-磷酸甘油醛脱氢酶(GAPDH)是糖酵解过程中的一个关键酶,与老化和白内障发生密切相关,硫醇转移酶(TTase)可断裂晶状体蛋白质氧化形成的二硫键使硫醇化的蛋白质脱硫醇,对于维持晶状体透明性极为重要。氨甲酰化为重要的翻译后修饰。本研究观察氨甲酰化是否能诱导晶状体GAPDH和TTase的失活。方法:透明新鲜的晶状体取自2周岁健康牛,与100mmol/L氰酸钾于37℃水孵箱分别振动温育7d和12d。设透明晶状体为正常对照组。所有晶状体孵育在50mmol/LDMEM液中,然后每只晶状体去囊膜,匀浆,提取水溶性蛋白质。用分光光度计分别测量GAPDH和TTase的活性。结果:与氰酸钾孵育后GAPDH的活性与正常对照组相比显著降低(P<0.01),其中12d组与7d组相比,活性明显下降。TTase的活性在孵育7d后与对照组相比有统计学差异(P<0.05),12d组的活性与对照组相比有显著差异(P<0.01),7d组活性与12d组相比无统计学差异(P=0.19296)。结论:研究结果显示,分别与100mmol/L氰酸钾孵育7d和12d后,氨甲酰化组的GAPDH和TTase活性与对照组相比有统计学差异,证实氨甲酰化可以诱导GAPDH和TTase酶的失活,为TTase和GAPDH在白内障发病机制中的进一步研究提供了新思路。
OBJECTIVE: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in glycolysis and is closely related to aging and cataract. Thiol transferase (TTase) disrupts the formation of disulfide bonds in lens proteins Thiolated protein sweetening alcohol, for maintaining the transparency of the lens is extremely important. Carbamylation is an important post-translational modification. This study investigated whether carbamylation can induce the inactivation of lens GAPDH and TTase. Methods: The fresh and transparent lens was taken from 2-year-old healthy cow and incubated with 100mmol / L potassium cyanate in water incubator at 37 ℃ for 7d and 12d respectively. Let the transparent lens as the normal control group. All lenses were incubated in 50 mmol / L LDMEM solution, and then each lens capsule was removed and homogenized to extract water-soluble protein. The activity of GAPDH and TTase was measured separately with a spectrophotometer. Results: After incubation with potassium cyanate, the activity of GAPDH was significantly lower than that of the normal control group (P <0.01). The activity of GAPDH in 12d group was significantly lower than that of the 7d group. Compared with the control group, the activity of TTase was significantly different from that of the control group at 7d (P <0.05), but there was no significant difference between the 7d group and the 12d group Learning difference (P = 0.19296). Conclusion: The results showed that GAPDH and TTase activities in carbamylated group were significantly different from those in control group after being incubated with 100mmol / L potassium cyanate for 7d and 12d respectively, confirming that carbamylation can induce GAPDH and TTase Enzyme inactivation provides a new idea for the further study of TTase and GAPDH in the pathogenesis of cataract.