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Detection of Functional Matrix Metalloproteinases by Zymography
Published on: November 8, 2010
Detection of TIMP-2-like protein in Atlantic cod (Gadus morhua) muscle using two-dimensional real-time reverse
Jørgen B Lødemel1, Wolfgang Egge-Jacobsen, Ragnar L Olsen
1Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsø, Breivika, N-9037 Tromsø, Norway. jbl@nfh.uit.no
Abstract:
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.
Insights
Researchers identified tissue inhibitors of metalloproteinases (TIMPs) in Atlantic cod muscle, crucial for regulating matrix metalloproteinases (MMPs) and understanding fish muscle degradation during storage.
Area of Science:
- Biochemistry
- Marine Biology
- Proteomics
Background:
- Matrix metalloproteinases (MMPs) are implicated in postmortem fish muscle degradation.
- Tissue inhibitors of metalloproteinases (TIMPs) regulate MMPs in mammals, but their presence in fish is poorly understood.
Purpose of the Study:
- To investigate the presence and characteristics of TIMPs in Atlantic cod (Gadus morhua) muscle.
- To identify potential inhibitors of MMPs involved in fish muscle tissue breakdown.
Main Methods:
- Gelatin affinity chromatography
- Real-time reverse zymography (RTRZ), including a novel 2D RTRZ method
- Mass spectrometry (MS) for de novo peptide sequencing
Main Results:
- An inhibitor with a molecular weight similar to TIMP-2 was detected in cod muscle.
- 2D RTRZ confirmed the existence of a single inhibitory form.
- Peptide sequencing revealed high homology to TIMP-2 from humans and other fish.
Conclusions:
- Atlantic cod muscle contains a TIMP-2 homologue.
- This finding contributes to understanding the regulation of MMPs in fish muscle.
- The identified TIMP-2 may play a role in preventing postmortem muscle degradation.
