Dalsgaard Trine Kastrup, Nielsen Jacob Holm, Larsen Lotte Bach
Department of Food Science, Research Centre Foulum, Faculty of Agricultural Sciences, University of Aarhus, Denmark.
Mol Nutr Food Res. 2007 Apr;51(4):404-14. doi: 10.1002/mnfr.200600112.
Five different milk proteins (alpha-casein, beta-casein, kappa-casein, beta-lactoglobulin, and lactoferrin) and a peptide substrate were applied as substrates for the investigation of how lactosylation affected proteolysis by different proteases. After a lactosylation period of 4 days in aqueous solution, at 65 degrees C and pH 6.8 in a protein: lactose ratio of 1000 the proteins were enzymatically hydrolyzed by the three milk relevant proteases plasmin, cathepsin D, and chymosin. Lactosylation of all substrates affected hydrolysis by plasmin negatively, with the largest effect on the globular proteins. This could be explained by modification of lysine residues, being the preferred cleavage site for plasmin, but also the residue generally preferred for lactosylation. Lactosylation of the caseins and of beta-lactoglobulin did not affect subsequent cleavage by cathepsin D and chymosin significantly, but for beta-lactoglobulin, both the secondary as well as the tertiary structure were affected by lactosylation. In contrast, decreased hydrolysis by cathepsin D and chymosin was observed for lactoferrin after lactosylation. Decreased hydrolysis may be caused by a more compact tertiary structure induced by lactosylation of lactoferrin, as indicated by fluorescence spectroscopy measurements.
选用五种不同的乳蛋白（α-酪蛋白、β-酪蛋白、κ-酪蛋白、β-乳球蛋白和乳铁蛋白）以及一种肽底物，用于研究乳糖基化如何影响不同蛋白酶的蛋白水解作用。在65℃、pH 6.8的水溶液中，按照蛋白质与乳糖1000的比例进行4天的乳糖基化反应后，用三种与牛奶相关的蛋白酶——纤溶酶、组织蛋白酶D和凝乳酶对这些蛋白质进行酶解。所有底物的乳糖基化均对纤溶酶的水解产生负面影响，对球状蛋白的影响最大。这可以通过赖氨酸残基的修饰来解释，赖氨酸残基是纤溶酶的首选切割位点，也是乳糖基化通常优先选择的残基。酪蛋白和β-乳球蛋白的乳糖基化对随后组织蛋白酶D和凝乳酶的切割没有显著影响，但对于β-乳球蛋白，其二级和三级结构均受到乳糖基化的影响。相比之下，乳糖基化后的乳铁蛋白经组织蛋白酶D和凝乳酶水解减少。水解减少可能是由于乳铁蛋白乳糖基化诱导形成了更紧密的三级结构，荧光光谱测量结果表明了这一点。
