1. [Isolation of trypsin PC from the Kamchatka crab Paralithodes camtschatica and its properties]
G N Rudenskaia, V A Isaev, T S Kalebina, V M Stepanov, K V Mal'tsev, S V Shvets, N A Luk'ianova, Iu A Kislitsin, A I Miroshnikov Bioorg Khim. 1998 Feb;24(2):112-8.
Trypsin PC from the hepatopancreas of the king crab Paralithodes camtschatica was isolated and purified to apparent homogeneity by ion-exchange chromatography on Aminosilochrom and DEAE-Sephadex and affinity chromatography on arginine-agarose. The yield of the enzyme was 37.7%, and the purification degree was 21. Trypsin PC has a molecular mass of 29 kDa and pI < 2.5. It hydrolysis N-benzoyl-L-arginine p-nitroanilide at the optimum pH of 7.5-8.0 and at the temperature optimum of 55 degrees C (K(m) = 0.05 mM). Trypsin PC retained its activity within the pH range of 5.8-9.0 in the presence of Ca2+. The enzyme was inhibited by the specific inhibitors of serine proteases diisopropyl fluorophoshate and phenylmethylsulfonyl fluoride, by the trypsin inhibitor N-tosyl-L-lysylchloromethylketone, and by the trypsin inhibitors from soybean and potato. Trypsin PC was found to hydrolyze amide bonds formed by carboxylic groups of lysine and arginine in peptide substrates. The N-terminal sequence of this enzyme is IVGGTEVTPG.
2. Effects of exogenous protease effectors on beef tenderness development and myofibrillar degradation and solubility
L Uytterhaegen, E Claeys, D Demeyer J Anim Sci. 1994 May;72(5):1209-23. doi: 10.2527/1994.7251209x.
The effects of in situ postrigor injection (24 h postmortem) of exogenous aspartic, serine, and cysteine proteinase effectors into cylindrical beef longissimus samples on tenderness and myofibrillar protein degradation and integrity were studied. Injection of phenylmethanesulphonylfluoride (PMSF) and pepstatin did not influence shear force or protein degradation measured 8 d postmortem, confirming that neither serine nor aspartic proteinases affect tenderization. Injection of leupeptin, an epoxysuccinyl peptide (E-64), or N-acetyl-Leu-Leu-norleucinal (calpain inhibitor I) blocked tenderization completely, as observed by higher (P < .05) shear force values. A causal relationship between increased toughness and prevented action of the cysteine proteinases was suggested by a concomitant reduction of myofibrillar protein degradation, generally reflected in higher (P < .05) remaining troponin-T and titin amounts and lower (P < .05) levels of 30-kDa peptide, as evaluated by semiquantitative SDS-PAGE. Moreover, parallel to these changes, amounts of salt-soluble myofibrillar protein and semiquantitative concentrations of individual salt-soluble proteins (SDS-PAGE) were also reduced (P < .05). Injection of Triton-X-100 and Ca2+ increased (P < .05) tenderness, as well as myofibrillar protein degradation and solubility, and free Ca2+, whereas EDTA induced the opposite results, indicating an important role for calpains in tenderization. Because cathepsin B, D, H, and L inhibitors did not affect texture or proteolysis, our results suggest that calpains are the main proteases involved in beef tenderization.
3. High-molecular-weight serine proteinase from lobster muscle that degrades myofibrillar proteins
D L Mykles J Exp Zool. 1989 Jun;250(3):244-52. doi: 10.1002/jez.1402500303.
A latent alkaline serine proteinase (ASP) has been extracted from the soluble fraction of lobster claw and abdominal muscles. The enzyme, which was irreversibly activated 30- to 40-fold by brief (2-3 min) heating at 60 degrees C, had an optimal caseinolytic activity at pH 7.75. Its molecular weight was estimated to be 740,000 by gel filtration chromatography. Serine protease inhibitors (diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, benzamidine, and chloromethyl ketones) suppressed ASP activity 22 to 70%. In addition, sulfhydryl-blocking reagents and hemin inhibited activity 69 to 100%; leupeptin and E-64, however, did not. Pepstatin A, ethylenediaminetetraacetate, and adenosine triphosphate were without effect. These results suggest that the lobster ASP is a serine proteinase that contains one or more sulfhydryl groups essential for catalysis. ASP was stimulated by dithiothreitol and inhibited by CaCl2 and oleic and linoleic acids. The enzyme was partially activated by low concentrations of sodium dodecyl sulfate; 0.05% produced activities 13% of that of preparations heated at 60 degrees C. Neither poly-L-lysine, urea, dimethylsulfoxide, oleic acid, linoleic acid, nor N-ethylmaleimide activated the enzyme. The ASP degraded most myofibrillar proteins, but showed a preferential hydrolysis of paramyosin, troponin-I and -C, and myosin alpha light chain.
