Proteome analysis of rainbow trout (Oncorhynchus mykiss) liver proteins during short term starvation
Proteome analysis of rainbow trout (Oncorhynchus mykiss) liver proteins during short term starvation
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DOI：
10.1023/a:1014015530045
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发表时间：
2001-04-01
影响因子：
2.9
通讯作者：
Houlihan, DF
中科院分区：
文献类型：
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作者：
Martin, SAM;Cash, P;Houlihan, DF
喂食和饥饿的鲑鱼肝脏中蛋白质合成的基本速率是相似的；在饥饿期间，随着动物体重的减轻和肝脏的缩小，蛋白质的降解必然会增加。在不同的生理环境下，鱼体内的蛋白质降解是如何被控制的，人们知之甚少。在这项研究中，蛋白质组分析已经被用来鉴定丰度变化的蛋白质，这些蛋白质可能与虹鱼在14天不进食后肝脏蛋白质降解增加有关。全肝蛋白质提取液在高分辨双向凝胶上进行分析。每条鱼的蛋白质图谱被数字化，并使用计算机软件构建复合参考凝胶。总共确定了780个蛋白质点，并监测了喂食和饥饿的鱼群的蛋白质丰度。根据等电点(PI)、相对分子质量和丰度记录所有蛋白质斑点。发现24种蛋白质在两组之间的丰度存在差异，其中8种蛋白质在投喂鱼中增加，16种蛋白质的丰度因停食而增加。从凝胶中取出22个蛋白点，用胰酶消化，然后用MALDI-TOF光谱分析进行多肽分离。利用多肽质量检索GenBank数据库进行蛋白质鉴定。根据它们的肽谱与现有蛋白质序列的同源性，鉴定了其中12种蛋白质。一种在饥饿条件下含量增加的蛋白质被鉴定为组织蛋白酶D，这是一种参与蛋白质降解的溶酶体内肽酶。Northern印迹分析表明，组织蛋白酶D的表达增加，反映饥饿鱼肝脏组织蛋白D的转录增强或稳定，支持蛋白质组学证据。因此，在饥饿的鲑鱼中，溶酶体蛋白水解酶的增加可能在蛋白质的损失中起到一定作用。
Basal rates of protein synthesis in the liver are similar in fed and starved trout; during starvation protein degradation must increase as the animal loses weight and the liver decreases in size. Little is known about how protein degradation is controlled in fish under various physiological circumstances. In this study proteome analysis has been used to identify proteins that are changed in abundance that may be involved in increased protein degradation in the liver of rainbow trout following a period of 14 days without food. Protein extracts from whole liver were analysed on high resolution two dimensional gels. The protein profiles from individual fish were digitised and computer software used to construct a composite reference gel. In total 780 protein spots were identified and their abundance monitored for fed and starved groups of fish. All protein spots were recorded in terms of their isolelectric point (pI), molecular weight and abundance. Twenty four proteins were found to have differences in abundance between the two groups, 8 were increased in fed fish with 16 increased in abundance as a result of food withdrawal. Twenty two protein spots were excised from gels and subjected to trypsin digestion followed by peptide separation by MALDI-TOF spectrometry. Peptide masses were used to search the GenBank data base for protein identification. Twelve of the proteins were identified on the basis of the homology of their peptide profiles to existing protein sequences. One protein, which increased in abundance under starvation conditions, was identified as cathepsin D, a lysosomal endopeptidase involved in protein degradation. Northern blot analysis of RNA isolated from liver of rainbow trout showed an increase in expression of cathepsin D reflecting either increased transcription or stability of the mRNA in starved fish, supporting the proteome evidence. Thus in starved trout an increase in lysosomal proteases may play a part in the loss of proteins.