Holt J C, Hatcher V B, Caulfield J B, Norton P, Umeda P K, Melendez J A, Martino L, Mudzinsky S P, Blumenstock F, Slayter H S, Margossian S S
Rhone-Poulenc Rorer Central Research, King of Prussia, PA, USA.
Mol Cell Biochem. 1998 Apr;181(1-2):125-35. doi: 10.1023/a:1006842332340.
A neutral protease with an estimated Mr of about 26 kD and responsible for cleavage ofmyosin LC2 was isolated from hamster skeletal muscle. Complementary DNAs were generated by RT-PCR using total hamster muscle RNA and degenerate oligonucleotide primers based on the sequences of two internal peptides. The nucleotide sequences of the resultant cDNAs were subsequently determined and the complete amino acid sequence of the protease deduced. Although the hamster protein shared 63-85% identity in nucleotide and amino acid sequences with rat and mouse mast cell proteases, it had a higher degree of specificity for myosin LC2 than mast cell proteases which also digested myosin LC1 and myosin heavy chains. As a result, the hamster protease was designated mekratin because of its unique enzymatic specificities to distinguish it from other mast cell proteases. A polyclonal antibody was raised specific to the hamster muscle and human cardiac muscle mekratins without apparent cross-reaction with rat mast cell proteases. We have earlier demonstrated the presence in excess of a neutral protease that specifically cleaves LC2 in human hearts obtained at end stage idiopathic dilated cardiomyopathy (IDC). Western analyses revealed that heart tissue from patients with IDC contained 5-10 fold more mekratin than control samples. Furthermore, the level of the protease in human IDC tissues was similar to that seen in myopathic hamster skeletal muscle. No bands were recognized by the antibody when IDC myofibrils were probed due to the removal of soluble proteins during sample preparation. Thus, these results strongly suggest that the anti-mekratin antibody will provide positive identification of IDC in many cases and diagnosis by exclusion may be replaced.
从仓鼠骨骼肌中分离出一种估计分子量约为26 kD、负责切割肌球蛋白轻链2（myosin LC2）的中性蛋白酶。利用仓鼠肌肉总RNA和基于两个内部肽段序列的简并寡核苷酸引物，通过逆转录聚合酶链反应（RT-PCR）生成互补DNA。随后测定所得cDNA的核苷酸序列，并推导蛋白酶的完整氨基酸序列。尽管仓鼠蛋白在核苷酸和氨基酸序列上与大鼠和小鼠肥大细胞蛋白酶有63%-85%的同一性，但它对肌球蛋白LC2的特异性高于肥大细胞蛋白酶，后者也能消化肌球蛋白轻链1（myosin LC1）和肌球蛋白重链。因此，由于其独特的酶特异性，仓鼠蛋白酶被命名为mekratin，以区别于其他肥大细胞蛋白酶。制备了一种对仓鼠肌肉和人心肌mekratin特异的多克隆抗体，与大鼠肥大细胞蛋白酶无明显交叉反应。我们早期已证明，在终末期特发性扩张型心肌病（IDC）患者的心脏中存在过量的特异性切割LC2的中性蛋白酶。蛋白质免疫印迹分析显示，IDC患者的心脏组织中mekratin含量比对照样品高5-10倍。此外，人IDC组织中该蛋白酶的水平与肌病仓鼠骨骼肌中的水平相似。由于样品制备过程中可溶性蛋白被去除，当检测IDC肌原纤维时，抗体未识别出条带。因此，这些结果强烈表明，抗mekratin抗体在许多情况下将为IDC提供阳性鉴定，且可取代排除性诊断。
