calpain and Favism

Favism is an acute hemolytic anemia triggered by ingestion of fava beans in genetically susceptible subjects with severe deficiency of glucose-6-phosphate dehydrogenase (G6PD) activity. Erythrocytes from 10 favic patients had constantly and markedly increased calcium levels, as compared with values detected in 4 asymptomatic G6PD-deficient controls. Correspondingly, the calcium permeability of erythrocytes, estimated as the fraction of intracellular calcium exchangeable with externally added 45Ca2+, was invariably enhanced in favism and returned to normal patterns after several months from the acute hemolytic crisis. In favic patients, the levels of erythrocyte calcium ATPase activities showed wide variability, ranging from 2.0-12.9 mumol Pi/ml RBC/h, while control values in asymptomatic G6PD-deficient subjects were 10.62 +/- 2.03 mumol Pi/ml RBC/h. Analysis of the calcium ATPase in situ in erythrocyte membranes from favic patients showed the same molecular mass of 134 kD as observed in the control subjects. Exposure of G6PD-deficient erythrocytes in vitro to autoxidizing divicine, a pyrimidine aglycone strongly implicated in the pathogenesis of favism which leads to late accumulation of intracellular calcium, caused: (i) a marked inactivation of calcium ATPase, without changes in the molecular mass of 134 kD; and (ii) the concomitant loss of spectrin, band 3 and band 4.1, all known substrates of the calcium activated procalpain-calpain proteolytic system. Thus, the increased intraerythrocytic calcium apparently results in the degradation of calcium ATPase observed in some favic patients. It is proposed that both enhanced calcium permeability and a calcium-stimulated degradation of the calcium pump are the mechanisms responsible for the perturbation of erythrocyte calcium homeostasis in favism.

Topics: Calcimycin; Calcium; Calcium-Transporting ATPases; Calpain; Erythrocytes; Favism; Glucosephosphate Dehydrogenase; Homeostasis; Humans; Male; Oxidation-Reduction; Pyrimidinones

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.

Topics: Calcium; Calpain; Enzyme Precursors; Erythrocytes; Favism; Glucosephosphate Dehydrogenase Deficiency; Heinz Bodies; Humans; Oxidants, Photochemical; Peptide Hydrolases

Damaged RBC drawn from favic patients during acute hemolysis showed marked alterations in their two major proteolytic systems. Cytosolic procalpain (i.e., the proenzyme species of Ca2+-activated neutral proteinase, or calpain) had considerably lower activity than in matched RBC from asymptomatic G6PD-deficient subjects. The total RBC activity of the three acid endopeptidases that are normally membrane-bound was not reduced in favism, but its subcellular distribution was mostly cytosolic, suggesting quantitative release from membranes. Changes in procalpain activity are the result of both autoxidation of divicine and of the intracellular elevation of Ca2+ that is found in favism. Changes in acid endopeptidase activity are the consequence of perturbed Ca2+ homeostasis. Overall, the picture shows a marked impairment of the RBC proteolytic machinery that in turn may worsen cellular damage.

Topics: Calcium; Calpain; Endopeptidases; Enzyme Precursors; Erythrocytes; Favism; Glucosephosphate Dehydrogenase Deficiency; Hemolysis; Humans; Male; Peptide Hydrolases; Pyrimidinones