Hemocyanin with phenoloxidase activity in the chitin matrix of the crayfish gastrolith

L. Glazer, M. Tom, S. Weil, Z. Roth, I. Khalaila, B. Mittelman, A. Sagi
2013 Journal of Experimental Biology  
3 Gastroliths are transient extracellular calcium deposits formed by the crayfish 3 4 Cherax quadricarinatus von Martens on both sides of the stomach wall during pre-molt. 5 Gastroliths are made of a rigid chitinous organic matrix, constructed as sclerotized chitin-3 6 protein microfibrils within which calcium carbonate is deposited. Although gastroliths share 3 7 many characteristics with the exoskeleton, they are simpler in structure and relatively 3 8 homogenous in composition, making them
more » ... excellent cuticle-like model for the study of 3 9 cuticular proteins. In searching for molt-related proteins involved in gastrolith formation, two 4 0 integrated approaches were employed, namely the isolation and mass spectrometric analysis 4 1 of proteins from the gastrolith matrix, and 454-sequencing of mRNAs from both the 4 2 gastrolith-forming and sub-cuticular epithelia. SDS-PAGE separation of gastrolith proteins 4 3 revealed a set of bands at apparent molecular weights of 75-85 kDa, of which peptide 4 4 sequencing following mass spectrometry matched the deduced amino acid sequences of seven 4 5 hemocyanin transcripts. This assignment was then examined by immunoblot analysis using 4 6 anti-hemocyanin antibodies, also used to determine the spatial distribution of the proteins in 4 7 situ. Apart from contributing to oxygen transport, crustacean hemocyanins were previously 4 8
doi:10.1242/jeb.080945 pmid:23393281 fatcat:rozvtknd5bc3jip343sybwdt5q