Interestingly, the enzyme activity profile in these two species was different during storage of fish fillets. was more randomly distributed within CT in salmon. In contrast, all these three MMPs were randomly distributed in CT in cod. In summary, our study reveals different MMP enzyme profiles in salmon and cod in the pin bone area, influenced by serine proteases, and suggests that MMPs and serine proteases must be taken in consideration when studying the conditions for early pin bone removal. in the two species is currently unknown. Degradation of the CT is enzymatic, involving numerous enzymes that can be regulated by various factors including pH, temperature and ion strength and processes that affect these factors could as such impact loosening of the pin bones (Larsen et al. 2008; Vargova et al. 2012). Proteases are central for CT degradation and are grouped based on their catalytic residues, matrix metalloproteases (MMPs), serine proteases, cysteine proteases, threonine proteases and aspartic proteases (Cawston and Wilson 2010). Rabbit polyclonal to PPA1 MMPs are the major group of proteases important for extracellular matrix (ECM) degradation. They are classified based on their substrate specificities and include collagenases (MMPs 1, 8, 13), gelatinases (MMPs 2, 9), matrilysins (MMPs 7, 11, 26) and stromelysins (MMPs 3, 10) (see (Pedersen et al. 2015) for review of MMPs in fish). The MMPs are normally secreted as zymogens, which are subsequently processed by proteolytic enzymes to generate the active forms (Okumura et al. 1997; Woessner 1991). Under normal physiological conditions, the proteolytic activity of the MMPs is controlled at any of the following three known stages: transcription, activation of the zymogens and inhibition of the active forms by various tissue inhibitors of MMPs (TIMPs) (Verma and Hansch 2007). Extracellular proteases influence and activate each other in a complex network, and often one protease pathway is combined with another (He et al. 1989; Shamamian et al. 2001; Zhu et al. 2001). In this study, we compared extracellular enzymes present in the attachment area of pin bones in salmon and cod during the period. The aim was to investigate the specific distribution of MMP activities in this specific area. Samples were harvested at different time points storage. Materials and methods Fish samples Tissues were obtained from L 888607 Racemate salmon (storage on ice (test In situ zymography visualizes the precise localization of the enzyme activities in the tissue. Our experiment with MMP substrate DQ-gelatin demonstrated the presence of MMP activity in the CT surrounding pin bones and in L 888607 Racemate the surrounding muscle and adipose tissue of salmon (Fig.?2, left panel). Using GM6001 and Pefabloc, the gelatinolytic activity in the CT was inhibited (Fig.?2, middle and right panel). Using different substrates, (gelatin, collagen and casein) we demonstrated MMP activity and serine protease activity in the CT close to the pin bones, summarized in Table?1. Inhibition of the enzyme activity in the CT close to pin bone was less visible in cod (Table?1), most likely reflecting less MMP activity present at the time point studied (6?h). Interestingly, although the enzyme activity was clearly inhibited in the CT, the activity in the surrounding tissue was not depressed by MMP or serine protease inhibitors, revealing a different enzyme profile in the CT close to pin bone compared to the CT in surrounding skeletal muscle and adipose tissue. MMPs exhibit a broad range of substrate specificities, including ECM proteins as well as non-ECM proteins. Collagen and gelatin are preferred substrates for the collagenase family and gelatinase family, respectively, although they can also be cleaved by other MMPs (Nagase 2001). Casein is a less common and preferred substrate for MMPs, but are frequently used in zymography for determining activity of MMP1 and MMP7 (Hu and Beeton 2010; Snoek-van Beurden and Von den Hoff 2005; Zeng et al. 2002). Casein is also a substrate for serine proteases. Under normal physiological processes, MMPs must be expressed to the exact extra- or peri-cellular location, at the right time and in the right amount. L 888607 Racemate Also, they must be activated or inhibited appropriately. Most MMPs are synthesized and secreted as inactive proenzymes, and plasmin has been described as a key activator of several MMPs (He et al. 1989; Murphy et al. 1999). However, many other serine proteases have also been shown to directly activate MMPs in vitro L 888607 Racemate or in vivo (Duncan et al. L 888607 Racemate 1998; Fang et al. 1997; Gruber et al. 1989;.