Kinetic Properties of Three Isoforms of Trypsin Isolated from the Pyloric Caeca of Chum Salmon (Oncorhynchus keta)

Abstract

Trypsin, an important enzymes belonging to the large and diverse family of serine proteases, has been found in numerous higher mammals, fish, plants, insects and bacteria. Several studies on the isolation and characterization of trypsins from cold-adapted fishes, such as Atlantic salmon (Salmo salar), 1,2) chum salmon (Oncorhynchus keta), [3][4][5] Atlantic cod (Gadus morhua), 6) Greenland cod (Gadus ogac), 7,8) Antarctic fish (Paraotohhthenia magellanica), 9) Japanese anchovy (Engraulis japonicus), 10) and anchovy (Engraulis encrasicholus) 11) have been reported. These fish trypsins resemble mammalian trypsins in many respects, but are also different in some respects. For example, most of the fish trypsins studied are more unstable than those from mammals at acidic pH and high temperatures. 2,4,6,9,11) Moreover, trypsins from cold-adapted fish have higher catalytic efficiencies (measured by the k cat /K m ratio) than those from bovine and porcine. 2,[5][6][7][9][10][11] Furthermore multiple isoforms of trypsin have been found in many fish species, 2-6,10,11) while one cationic and one anionic form of trypsin have been found in humans, 12) bovines, 13) porcine, 14) and dogs. 15)Trypsin from chum salmon (Oncorhynchus keta) was first reported by Uchida et al. 3,4) Chum salmon trypsin is composed of seven anionic trypsins and one cationic trypsin, and they were separated by ion-exchange resin. Their functional properties were similar to those of bovine cationic trypsin; however, the detailed catalytic properties have not been reported. Thus, in our previous study, we isolated an anionic trypsin from chum salmon and measured the catalytic properties toward some substrates. The catalytic efficiency (k cat /K m ) of an anionic chum salmon trypsin for benzoyl-Larginine-p-nitroanilide (BAPA) hydrolysis is 22-fold higher than that of bovine trypsin at 5°C. 5) To improve our understanding of the higher level of catalytic efficiency at a lower temperature of enzymes from cold-adapted fish compared to mammalian enzymes, we also examined the X-ray crystallographic structure of an anionic chum salmon trypsin.16) The result suggested that a higher level of catalytic efficiency of anionic chum salmon trypsin might be achieved from the lower electrostatic potential of the S1-binding pocket. Studies on related enzymes from organisms adapted to living in a wide range of environments have revealed general patterns for how variations in sequence and structure allow enzymes to perform similar catalytic functions under different environmental conditions. These general patterns suggest mechanisms for how specific changes in structure may result in changes in enzymatic functions. Our studies are directed at the synthesis of non-covalent inhibitors of trypsin or trypsin-like enzymes (e.g. thrombin, kallikrein and urokinase) that may be used as scaffolds for the development of more specific drugs 17,18) ; therefore we are interested in the catalytic properties and three-dimensional structures of other isoforms of chum salmon trypsin. The...

A structural comparison of three isoforms of anionic trypsin from chum salmon (Oncorhynchus keta)

“…This may be the most significant factor in the higher catalytic efficiencies of CST-1 and CST-2 compared with CST-3. The catalytic efficiencies (K m /k cat ) of the chum salmon trypsin isoforms (CST-1, CST-2 and CST-3) in this study during BAPA hydrolysis were 20-fold more efficient than that of bovine trypsin (BT) at 308 K and their enzyme activity increased more at low temperatures (298-278 K;Toyota et al, 2007). The catalytic efficiency of anionic Atlantic salmon trypsin (AST) is also about 20-fold higher than BT at 310 K and 35-fold higher at 277 K (Quzen et al, 1996).…”

“…In our previous paper (Toyota et al, 2007), the K m values of CST-1 and CST-2 during BAPA hydrolysis were found to be slightly higher than that of CST-3, which is the main isoform. The k cat values for CST-1 and CST-2 were about twice as high as that of CST-3.…”

“…Salmon trypsin was isolated from the pyloric caeca of chum salmon according to a previously reported procedure . Three isoforms (CST-1, CST-2 and CST-3) were obtained by DEAE anion-exchange chromatography (Toyota et al, 2007). Their nucleotide sequences were determined using cDNA clones (Toyota et al, 2002).…”

“…The trypsins from chum salmon include seven anionic trypsins and one cationic trypsin. In a previous study (Toyota et al, 2007), we isolated two isoforms that are present in small amounts (CST-1 and CST-2) and the main isoform (CST-3) from the pyloric caeca of chum salmon. The isoelectric points of CST-1, CST-2 and CST-3 are 5.8, 5.4 and 5.6, respectively, and thus these isoforms are anionic trypsins.…”

Morphology and lectin‐binding sites of pyloric caeca epithelium in normal and GnRH‐treated Atlantic bluefin tuna (Thunnus thynnus) Linnaeus 1758

“…These appendages are present in only 60% of known fish species (Hossain and Dutta, 1996) and they differ in number, form, disposition and communication with the intestine (Khanna, 1961; Mohsin, 1962). The function of the pyloric caeca is to increase the surface area of the intestine (Buddington and Diamone, 1986) Pyloric caeca are involved in sugar, amino acid, peptide and lipid digestion and absorption (Buddington and Diamone, 1986; Horn, 1989; Lindsay and Harris, 1980; Martin andand Sandercock, 1967; Sastry, 1974; Toyota et al, 2007). Additionally, a secretory function of this organ and a role in salt and water balance (osmotic balance) have been postulated (Abaurrea‐Equisoah and Ostos‐Garrido, 1986; Anderson, 1986; Veillette et al, 2005).…”