Trypsins from the pyloric ceca of jacopever (Sebastes schlegeli), TR-J and elkhorn sculpin (Alcichthys alcicornis), TR-E, were purified by gel filtration on Sephacryl S-200 and Sephadex G-50. The molecular weights of the TR-J and TR-E were estimated to be 24,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The TR-J and TR-E revealed optimum temperatures of 60 o C and 50 o C, respectively, and showed the same optimum pH (pH 8.0) for hydrolysis of N α -p-tosyl-L-arginine methyl ester. The TR-J and TR-E were unstable at above 50 o C and 40 o C, respectively, and were more stable at alkaline pH than at acidic pH. Thermal stabilities of the TR-P and TR-E were highly calcium dependent. These purified trypsin enzymes were inhibited by serine protease inhibitors such as TLCK and Soybean trypsin inhibitor. The N-terminal amino acid sequences of the TR-J and TR-E were also investigated. The N-terminal amino acid sequences of the TR-J, IVGGYECKPYSQPHQVSLNS and TR-E, IVGGYECTPHSQAHQVSLNS were found, and these sequences showed highly homology to other fish trypsins.

Trypsin was purified from the pyloric ceca of spotted mackerel (Scomber australasicus) by gel filtration on Sephacryl S‐200 and Sephadex G‐50. The purification and yield were 20‐fold and 81%, respectively, as compared to those in the starting crude extract. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular weight of the enzyme was estimated to be 24,000 Da by SDS–PAGE. The trypsin was stable at pH 5–11 for 30 min at 30C, and its maximal activity against Nα‐p‐tosyl‐L‐arginine methyl ester was pH 8.0. Trypsin was heat‐stable up to about 50C for 15 min at pH 8.0. Optimum temperature of the trypsin enzyme was 60C. The enzyme was stabilized by calcium ion. The purified trypsin was strongly inhibited by serine protease inhibitors such as N‐p‐tosyl‐L‐lysine chloromethyl ketone and soybean trypsin inhibitor, suggesting that it is a trypsin‐like serine protease. N‐Terminal amino acid sequence of spotted mackerel trypsin was IVGGYECTAHSQPHQVSLNS.

ABSTRACT

BackgroundAlthough rare, cardiac sarcoidosis (CS) is potentially fatal. Early diagnosis and intervention are essential, but histopathologic diagnosis is limited. We aimed to detect Propionibacterium acnes, a commonly implicated etiologic agent of sarcoidosis, in myocardial tissues obtained from CS patients.Methods and resultsWe examined formalin-fixed paraffin-embedded myocardial tissues obtained by surgery or autopsy and endomyocardial biopsy from patients with CS (n = 26; CS-group), myocarditis (n = 15; M-group), or other cardiomyopathies (n = 39; CM-group) using immunohistochemistry (IHC) with a P. acnes-specific monoclonal antibody. We found granulomas in 16 (62%) CS-group samples. Massive (≥14 inflammatory cells) and minimal (<14 inflammatory cells) inflammatory foci, respectively, were detected in 16 (62%) and 11 (42%) of the CS-group samples, 10 (67%) and 10 (67%) of the M-group samples, and 1 (3%) and 18 (46%) of the CM-group samples. P. acnes-positive reactivity in granulomas, massive inflammatory foci, and minimal inflammatory foci were detected in 10 (63%), 10 (63%), and 8 (73%) of the CS-group samples, respectively, and in none of the M-group and CM-group samples.ConclusionsFrequent identification of P. acnes in sarcoid granulomas of originally aseptic myocardial tissues suggests that this indigenous bacterium causes granuloma in many CS patients. IHC detection of P. acnes in massive or minimal inflammatory foci of myocardial biopsy samples without granulomas may be useful for differentiating sarcoidosis from myocarditis or other cardiomyopathies.