1. Purification and characterization of a sarcoplasmic serine proteinase from threadfin bream Nemipterus virgatus muscle
Jin-Yang Liu, Asami Yoshida, Yi-Li Gao, Kazuya Shirota, Yasuhiko Shiina, Erika Noguchi, Koichi Kuwahara, Kiyoshi Osatomi Food Chem. 2019 Jun 30;284:198-204. doi: 10.1016/j.foodchem.2019.01.024. Epub 2019 Jan 15.
A sarcoplasmic serine proteinase (SSP) was purified from threadfin bream (Nemipterus virgatus) belly muscle by ammonium sulfate precipitation and a series of chromatographies including Q-Sepharose, Phenyl Sepharose and Superdex 200. The SSP was purified 1967 folds with a yield of 4.8%. The molecular weight of the SSP was estimated to be 43.5 kDa and 22.5 kDa on SDS-PAGE under non-reducing and reducing conditions, respectively. The N-terminal amino acid sequence of the two protein bands were determined as IVGGYEXQPYSQAHQVSLNSGY and corresponded. It is suggested that the SSP exists as a homodimer. Optimum pH and temperature were 9.5 and 50 °C, using Boc-Val-Pro-Arg-MCA as a substrate. Substrate specificity and effects of inhibitors indicated that the SSP was a trypsin-like serine proteinase. The SSP was responsible for hydrolyzing myosin heavy chain (MHC) and inducing modori phenomenon in the threadfin bream surimi gel. Thus, the SSP was considered as a modori-inducing proteinase.
2. A novel type of myofibril-bound serine protease from white croaker (Argyrosomus argentatus)
Makoto Ohkubo, Kiyoshi Osatomi, Kenji Hara, Tadashi Ishihara, Futoshi Aranishi Comp Biochem Physiol B Biochem Mol Biol. 2005 Jun;141(2):231-6. doi: 10.1016/j.cbpc.2005.03.005.
Myofibril-bound serine protease (MBSP) was purified from the myofibril fraction of white croaker (Argyrosomus argentatus) muscle and its enzymatic properties were compared with other fish MBSPs. White croaker MBSP was extracted by the heat treatment of myofibrils and then purified by a series of column chromatographies on Q-Sepharose, Sephacryl S-300, hydroxyapatite and Benzamidine Sepharose. The purified MBSP migrated as a single protein band at 67 kDa in SDS-PAGE under both reducing and non-reducing conditions. It was inhibited by Pefabloc SC, soybean trypsin inhibitor (STI), aprotinin and benzamidine, and was not affected by E-64, pepstatin A and EDTA. The enzyme was most active against Boc-Phe-Ser-Arg-MCA at pH 7.0 and 50 degrees C, and preferentially hydrolyzed Boc-Val-Pro-Arg-MCA and Boc-Asp-Pro-Arg-MCA. Unlike other marine fish MBSPs, white croaker MBSP considerably hydrolyzed Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA. Some enzymatic characteristics including the molecular structure and the substrate specificity for a lysine residue at the P(1) position are quite different not only from other fish MBSPs but also from soluble serine protease obtained from white croaker muscle (MSSP). White croaker MBSP could be therefore classified into a novel type of fish muscle MBSP.
3. New fluorogenic substrates for alpha-thrombin, factor Xa, kallikreins, and urokinase
T Morita, H Kato, S Iwanaga, K Takada, T Kimura J Biochem. 1977 Nov;82(5):1495-8. doi: 10.1093/oxfordjournals.jbchem.a131840.
Twenty peptide-4-methylcoumarin amides (MCA) were newly synthesized and tested as possible substrates for alpha-thrombin, factor Xa, kallikreins, urokinase, and plasmin. These fluorogenic peptides contained arginine-MCA as the carboxyl-terminus. Release of 7-amino-4-methylcoumarin was determined fluorometrically. Of these peptides, the following were found to be specific substrates for individual enzymes: Boc-Val-Pro-Arg-MCA for alpha-thrombin, Boc-Ile-Glu-Gly-Arg-MCA, and Boc-Ser-Gly-Arg-MCA for factor Xa, Z-Phe-Arg-MCA for plasma kallikrein, Pro-Phe-Arg-MCA for pancreatic and urinary kallikreins, and glutaryl-Gly-Arg-MCA for urokinase. Moreover, these peptide-MCA substrates were resistant to plasmin.
