Cathepsins B and L and cathepsin L-like proteinase degraded myosin heavy chain of actomyosin (AM) into several fragments with molecular masses (MW) of 154, 146, 138, 67, and 36 kDa; 164 and 155 kDa; and 135, 128, and 69 kDa, respectively. In the presence of 0.6 M NaCl, however, the MHC was degraded by cathepsin L into 164, 155, 41, and 37 kDa fragments. The hydrolytic rate of these three proteinases on AM was faster at pH 5.0 than at pH 6.0. Actin seemed to be resistant against hydrolysis by cathepsins B, L, and L-like in the absence of 0.6 M NaCl. According to SDS−PAGE analysis, cathepsin X, a novel cysteine proteinase, did not degrade AM. Keywords: Mackerel; cathepsins; actomyosin; proteolysis; seafood

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During surimi processing, cathepsins B and L activities in minced, leached and NaCl-ground meats were 6.02, 5.23, and 4.07 units/g, respectively. About 80% activity remained in surimi after 8 wk storage at Ϫ40ЊC suggesting that these proteinases were stable and difficult to remove. At 40Њϳ55ЊC, pH 6.5ϳ7.5, cathepsins B and L and purified cathepsin B had high hydrolytic activity on myosin heavy chain (MHC). The strength of surimi gel with cathepsins B and L or with purified B decreased (pϽ0.05) after 2 hr incubation at 55ЊC. This suggested that the residual cathepsins B and L had MHC-degrading activity and consequently caused gel softening.

In order to fast and economically purify MTGase from Streptoverticillium ladakanum, a stepwise elution method was developed and compared with linear gradient elution method. MTGase was purified to electrophoretical homogeneity by using CM Sepharose CL-6B and Blue Sepharose Fast Flow chromatographies by linear gradient or stepwise methods. The recovery of MTGase by linear gradient and stepwise methods were 68.4% and 81.0%, respectively. The optimal temperature and pH were 40 °C and 5.5, respectively. It was stable at pH 5.0 to 7.0 and had a rate constant (K D ) of 6.21 ∞ 10 -5 min -1 for thermal inactivation at 45 °C.After 4 d incubation of S. ladakanum, the culture fluid was filtered through 0.22 m filter paper. The production of MTGase

To investigate the properties of pig plasma factor XI11 (a zymogen of factor XIIIa, EC 2.3.2.13, TGase) and consequently to utilize the pig blood, factor XI11 was purified to electrophoretical homogeneity after DEAE-Sephacel chromatography. The molecular weights were 320 OOO estimated by Sepharose CL-6B and 75 OOO by SDS-PAGE, suggesting this zymogen contains four subunits with identical molecular weights of 75 OOO. This proenzyme was activated by thrombin and Ca2+. Accordingly, the purified proenzyme was identified as factor XIII. The optimal temperature for incorporating monodansylcadaverine to @-substrate was 55 "C. Factor XIIIa (activated factor XIII) was inhibited by N-ethylmaleimide, p-(chloromercuri)benzoate, and iodoacetic acid in the presence of Ca2+. The rate constants for thermal denaturation (&) at 55 OC of factor XI11 and XIIIa were 5.0 X lW5 and 0.6 X s-l, respectively. Factor XIIIa catalyzed the covalent cross-linking of myosin heavy chain. The addition of crude plasma factor XI11 (including thrombin) substantially increased the gel strength of minced mackerel.