Detection of TIMP-2-like protein in Atlantic cod (Gadus morhua) muscle using two-dimensional real-time reverse zymography.
Matrix metalloproteinases (MMPs) have been proposed to participate in postmortem degradation of fish muscle connective tissues during storage. In the extracellular matrix (ECM) of mammals, a group of specific tissue inhibitors of metalloproteinases (TIMPs) contributes in regulating the MMPs present. However, little information exists on the presence of TIMPs in fish. In this paper, the presence of TIMPs in the muscle of Atlantic cod (Gadus morhua) was investigated using gelatin affinity chromatography, real-time reverse zymography (RTRZ) and mass spectrometry (MS). Using RTRZ inhibitory action against cod muscle, proteinases binding to gelatin were detected in the muscle. The inhibitor had similar molecular weight (21 kDa) as a human recombinant TIMP-2 used as a reference sample. Because isoforms of TIMP-2 homologues with similar molecular weight have been suggested in fish, a two-dimensional RTRZ (2D RTRZ) method was designed. The new method showed the existence of only one form with inhibitory action against cod muscle proteinases. Finally, de novo sequencing of two peptides derived from the cod muscle inhibitor showed high homology to TIMP-2s both from human and other teleosts.
Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsø, Breivika, N-9037 Tromsø, Norway. jbl@nfh.uit.no
Jørgen B
JB
Lødemel
Wolfgang
W
Egge-Jacobsen
Ragnar L
RL
Olsen
D000595:Amino Acid Sequence; D000818:Animals; D001329:Autolysis; D048389:Gadus morhua; D018093:Gelatinases / Q000037:antagonists & inhibitors; D006801:Humans; D009132:Muscles / Q000737:chemistry*; D010446:Peptide Fragments; D016415:Sequence Alignment; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000032:analysis / Q000302:isolation & purification* / Q000494:pharmacology
D010446:Peptide Fragments; D019716:Tissue Inhibitor of Metalloproteinase-2; D018093:Gelatinases