Actions of cathepsins on troponin T during postmortem aging of porcine muscle

In this study, we examined the contribution of the cathepsins (cathepsin D and crude cathepsins containing cathepsins B and L) to troponin T degradation during postmortem aging. The action of cathepsin D on troponin T was examined at various pHs (pH 4.0–6.5). The degradation of intact troponin T was observed at pH 4.0, but not observed at pH 5.5 and 6.5. As a result of the degradation of troponin T, the 30 kDa fragment was not generated in any pH condition. The action of the crude cathepsins on troponin T was also examined at various pHs (pH 4.0–6.5). The intact troponin T was degraded at pH 4.0 and the 30 kDa fragments were observed. These 30 kDa fragments disappeared during further incubation. On the other hand, at pH 5.5 and 6.5, the intact troponin T was degraded and the 30 kDa fragment was accumulated. These results suggested that the cathepsin D scarcely contributed to the degradation of troponin T during postmortem aging, but crude cathepsins containing cathepsins B and L were partially involved in the degradation of troponin T and the generation of 30 kDa fragments.     

应用重组日本血吸虫组织蛋白酶L诊断日本血吸虫病的研究

在大肠杆菌BL21中表达日本血吸虫组织蛋白酶L基因，经镍离子亲和层析柱纯化得到重组日本血吸虫组织蛋白酶L；以间接ELISA法，检测血吸虫病因牛血清104份，阳性检出率为67．31％；以血吸虫感染小鼠、兔、绵羊血清做阻断试验，得到的OD值与阴性血清相当。结果表明，以重组日本血吸虫组织蛋白酶L作为诊断抗原检测日本血吸虫病牛感染情况，有一定的应用前景。     

组织蛋白酶L生物学功能的研究进展

组织蛋白酶L(cathepsin L, CTSL)隶属于组织蛋白酶类,是一种木瓜蛋白酶家族中的半胱氨酸蛋白水解酶,以酶原形式广泛存在于动物各种组织细胞的溶酶体内,其相对分子量为30 000,在维持动物正常生命活动中扮演着重要角色。CTSL参与激素原的激活、体内抗原呈递、组织器官发育等多个生理过程,对于指导蛋白质的合成与分解、T细胞分选等具有重要的生物学意义。在病理状态下,由于炎症刺激、免疫损伤等多种原因均能造成细胞的损伤,导致CTSL大量释放到组织间隙或胞质中并被激活,使得细胞成分或细胞基质成分降解,常与肿瘤的浸润和转移、毛囊的形态发育及心肌病有关。目前,我国针对CTSL的相关生理特性研究相对较少,且有关CTSL研究方向较为单一。本文对CTSL的结构与功能,CTSL的生物合成与体内转运,及其在毛囊发育、激素合成、肿瘤标志物、维持心肌稳态、寄生虫防治和抗原递呈方面的典型生物学效应进行综述,并对其研究前景进行展望,以期对CTSL的深入研究提供理论依据,为CTSL研究提供方向。     

Occurrence of two distinct molecular species of cathepsin B in carp Cyprinus carpio

ABSTRACT:   We purified cathepsins B₁ and B₂ from the ordinary muscle of carp Cyprinus carpio . The N-terminal amino acid sequences (12 residues) of 29 kDa bands of cathepsins B₁ and B₂ are the same and showed high homology of 75% and 83%, respectively, with the heavy chain of rat and human cathepsins B. Based on conserved sequences of other cathepsins B and the N-terminal amino acid sequences of 29 kDa bands, we cloned carp cathepsin B cDNA. The nucleotide sequence of carp cathepsin B cDNA consists of 1470 bp including a 993 bp open reading frame, encoding a deduced protein of 330 amino acids. The deduced amino acid sequence of carp cathepsin B has similarity of 80% to rainbow trout cathepsin B and of 76–78% to other vertebrate cathepsins B. The sequence of its isoform was also determined during molecular cloning, which has 94.8% similarity with first cloned cathepsin B. They are completely same in N-terminal amino acid sequence of heavy chain, active site and potential N-glycosylation site. This indicates there are at least two kinds of cathepsin B functioning in vivo in carp.     

鲢鱼背肌肌原纤维蛋白自溶与内源组织蛋白酶B,L,H的关系

鲢鱼鱼糜制品在加工过程中容易发生热诱导 (5 0～ 70℃ )的鱼糜凝胶软化 (modori) ,导致鱼糜制品品质下降。为探明原因 ,本研究比较了在加工过程中漂洗前后鲢鱼鱼肉糜中组织蛋白酶B ,L ,H活性的变化 ,结果表明漂洗后仍残留了组织蛋白酶B ,L ,H活性 ,其中组织蛋白酶L活性的残留率最高 ,为 2 5 6 1%± 0 82 %。用SDS PAGE分析了有无E 6 4存在下 ,鲢鱼背肌肌原纤维蛋白自溶过程中 (pH 6 5 ,5 0℃ ) ,肌球蛋白重链相对含量的差异 ,结果显示肌球蛋白重链相对含量的下降趋势与残留的组织蛋白酶活性变化趋势吻合 ;而且E 6 4部分抑制了肌球蛋白重链的自溶。上述结果表明溶酶体半胱氨酸组织蛋白酶在鲢鱼鱼糜凝胶软化中起一定作用 ,而且组织蛋白酶L可能是重要的蛋白水解酶。     

南蓝鳕鱼糜中L型组织蛋白酶的研究

从南蓝鳕鱼糜中提取肌动球蛋白,对肌动球蛋白进行凝胶层析,测定各收集组分中L型组织蛋白酶的活性。结果表明,组织蛋白酶L依然存留于肌动球蛋白样品中,多次漂洗以及稀释-沉淀处理亦不能有效将其除去。凝胶层析图谱分析表明,该酶可能是肌动球蛋白非结合型酶。对凝胶层析所得粗酶液进行酶学性质分析,结果得知该酶最适温度为45℃,正好落在鱼糜凝胶劣化发生范围之内。专一性底物及酶激活剂、抑制剂影响研究表明,该酶为内含巯基的半胱氨酸型组织蛋白酶。该酶最适pH为5.5,在近中性pH范围内依然有较高的残留活性,表明该酶具有潜在凝胶劣化能力。