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Fig. 3. Identification of PRL fragments generated by neutral proteases of chondrocytes. (A) Reducing western blot analysis of proteolytic products generated from human PRL by incubation with lysates from rat chondrocytes at pH 7 as revealed by the anti-human PRL antiserum (anti-PRL) or by monoclonal antibody INN-368 directed against the C-terminal region of PRL (C-Term). Black arrows indicate the PRL fragments detected by anti-PRL antiserum, and white arrows the PRL fragments detected by INN-368. (B) Western blot analysis under reducing (R) and non-reducing (NR) conditions illustrating the composition of proteolytic products generated from human PRL as revealed by their N-terminal sequencing. Under reducing conditions, the 16 and 14 kDa fragments gave the sequence LPICPGGA, corresponding to the N-terminus of undigested PRL, whereas the 5 kDa fragment had the N-terminal sequence LQMADEE starting at residue Leu156. Under non-reducing conditions, the 25 kDa PRL had both the N-terminal sequence of PRL and a second N-terminus starting at residue Leu156, indicating that it corresponds to a PRL cleaved between Ser155 and Leu156, which upon ß-mercaptoethanol reduction (ß-ME) yields the observed 16 and 5 kDa PRL products. Positions of the PRL isoforms of the indicated relative molecular mass are shown on the left of western blots. (C) Human and rat PRL amino acid sequences neighboring the chondrocyte cleavage site in the human (S155-L156) and the rat (S153-L154) hormones. The predicted chondrocyte cleavage site and its adjacent amino acids are shown for PRLs of different vertebrates.
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