Morelli A, Grasso M, Meloni T, Forteleoni G, Zocchi E, De Flora A
Blood. 1987 Jun;69(6):1753-8.
Red blood cells (RBC) from favic patients are characterized by (a) severe oxidative damage (contributed by autoxidation of divicine and isouramil, two pyrimidine aglycones present in fava beans) and (b) greatly increased calcium levels. In vitro, both autoxidation of divicine and calcium loading produced marked alterations of proteolytic systems in intact RBC. Specifically, autoxidizing divicine inactivated procalpain, the proenzyme species of calcium-activated cytosolic neutral proteinase, or calpain. Inactivation was much greater with glucose-6-phosphate dehydrogenase (G6PD)-deficient RBC than with normal RBC. On the other hand, loading of normal and G6PD-deficient RBC with calcium resulted in conversion of procalpain to calpain and eventual autoproteolytic inactivation of calpain itself, and extensive release of acid endopeptidase activity from the membranes into the cytosol. Damaged RBC from favic patients had significantly lowered procalpain activity and an abnormal subcellular distribution of acid proteinase activity that was found mostly in the cytosol. When purified calpain was incubated with membranes from acetylphenylhydrazine (APH)-treated RBC, significant proteolysis was observed affecting mostly band 3 and hemoglobin chains, ie, the two proteins involved in the onset of aggregation of Heinz bodies. Moreover, exposure of intact RBC to 20 mmol/L APH induced depletion of procalpain activity for which the time course was inversely related to formation of Heinz bodies. These findings support the role of procalpain in protecting G6PD-deficient RBC from oxidant-induced Heinz body formation and imply that exhaustion of the procalpain-calpain system is an important step in the mechanisms of RBC damage and destruction in favism.
(a) 严重的氧化损伤（由蚕豆中存在的两种嘧啶苷元——异胺基巴比妥酸和异尿嘧啶的自氧化作用导致）和 (b) 钙水平大幅升高。在体外，异胺基巴比妥酸的自氧化作用和钙负载都会使完整红细胞中的蛋白水解系统发生显著改变。具体而言，异胺基巴比妥酸的自氧化作用会使钙激活的胞质中性蛋白酶——钙蛋白酶原失活。与正常红细胞相比，葡萄糖 -6-磷酸脱氢酶（G6PD）缺乏的红细胞中这种失活作用更为明显。另一方面，正常红细胞和 G6PD 缺乏的红细胞在负载钙后，钙蛋白酶原会转化为钙蛋白酶，最终钙蛋白酶自身发生自蛋白水解失活，并且酸性内肽酶活性会从细胞膜大量释放到细胞质中。蚕豆病患者受损的红细胞中钙蛋白酶原活性显著降低，酸性蛋白酶活性的亚细胞分布异常，主要存在于细胞质中。当将纯化的钙蛋白酶与经乙酰苯肼（APH）处理的红细胞膜一起孵育时，会观察到显著的蛋白水解作用，主要影响带 3 蛋白和血红蛋白链，即参与亨氏小体聚集起始的两种蛋白质。此外，将完整红细胞暴露于 20 mmol/L 的 APH 会导致钙蛋白酶原活性耗尽，其时间进程与亨氏小体的形成呈负相关。这些发现支持了钙蛋白酶原在保护 G6PD 缺乏的红细胞免受氧化剂诱导的亨氏小体形成中的作用，并表明钙蛋白酶原 - 钙蛋白酶系统的耗尽是蚕豆病中红细胞损伤和破坏机制的一个重要步骤。
