Proteome analysis of rainbow trout (Oncorhynchus mykiss) liver proteins during short term starvation

摘要

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.