Glycation and phosphorylation of α-lactalbumin by dry heating: Effect on protein structure and physiological functions
Glycation and phosphorylation of α-lactalbumin by dry heating: Effect on protein structure and physiological functions
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DOI：
10.3168/jds.2009-2014
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发表时间：
2009-07-01
影响因子：
3.5
通讯作者：
Aoki, T.
中科院分区：
文献类型：
--
作者：
Enomoto, H.;Hayashi, Y.;Aoki, T.
α-乳清蛋白（α-LA）与麦芽五糖（MP）通过美拉德反应（MP-α-LA）进行糖基化，然后在焦磷酸存在下通过干热进行磷酸化，以研究其结构和生理功能。糖基化有效地发生，并且通过美拉德反应，α-LA的糖含量增加了约22.3%。在焦磷酸盐存在下，随着干热时间的延长（1 ~ 5d），MP-α-LA的磷酸化程度增加，在pH4.0、85 ℃下干热5d，MP-α-LA的磷含量增加约1.01%。α-LA的电泳迁移率随着磷酸化水平的增加而增加。圆二色谱表明，糖化和随后的磷酸化的α-LA分子的二级结构的变化是轻微的。然而，糖基化后的磷酸化的色氨酸荧光强度增加。此外，α-LA的差示扫描量热热分析表明，磷酸化降低了MP-α-LA的变性温度。这些结果表明，熔融（部分未折叠）构象的α-LA形成的干热焦磷酸盐的存在下，糖化后。抗-α-LA抗体应答被糖化和随后的磷酸化显著降低。α-LA对脂多糖刺激后THP-1细胞产生促炎细胞因子如IL-6和肿瘤坏死因子-α的抑制作用通过MP的糖基化而显著增强，并通过糖基化后的磷酸化而进一步增强。磷酸化增强了α-LA溶解磷酸钙的能力。糖化和随后的磷酸化降低了α-LA的凋亡活性。这些结果表明，在通过美拉德反应与MP糖化后，在焦磷酸盐存在下通过干热进行磷酸化是改善α-LA生理功能的有用方法。
alpha-Lactalbumin (alpha-LA) was glycated with maltopentaose (MP) through the Maillard reaction (MP-alpha-LA) and subsequently phosphorylated by dry heating in the presence of pyrophosphate to investigate its structure and physiological functions. Glycation occurred effectively, and the sugar content of alpha-LA increased by approximately 22.3% through the Maillard reaction. The phosphorylation of MP-alpha-LA was enhanced with an increase in the dry-heating time from 1 to 5 d, and the phosphorous content of MP-alpha-LA increased by approximately 1.01% by dry heating at pH 4.0 and 85 C for 5 d in the presence of pyrophosphate. The electrophoretic mobility of alpha-LA increased with an increase in the phosphorylation level. The circular dichroism spectra showed that the change in the secondary structure of the alpha-LA molecule by glycation and subsequent phosphorylation was slight. However, the Trp fluorescence intensity was increased by phosphorylation after glycation. In addition, the differential scanning calorimetry thermograms of alpha-LA showed that the denaturation temperature of MP-alpha-LA was decreased by phosphorylation. These results indicated that molten (partially unfolded) conformations of alpha-LA were formed by dry heating in the presence of pyrophosphate after glycation. The anti-alpha-LA antibody response was significantly reduced by glycation and subsequent phosphorylation. The suppressive effect of alpha-LA on the production of proinflammatory cytokines such as IL-6 and tumor necrosis factor-alpha from THP-1 cells after stimulation with lipopolysaccharide was significantly enhanced by glycation with MP and was further enhanced by phosphorylation after glycation. The Ca phosphate-solubilizing ability of alpha-LA was enhanced by phosphorylation. The apoptotic activity of alpha-LA was reduced by glycation and subsequent phosphorylation. These results suggest that phosphorylation by dry heating in the presence of pyrophosphate after glycation with MP through the Maillard reaction is a useful method for improvement of the physiological functions of alpha-LA.