calpain and divicine

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

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

Human red cells were treated with 100 microM Ca2+ and ionophore A 23187. This treatment induces remarkable changes in the activities of the two major proteolytic systems of red cells, i.e. Ca2+-dependent neutral proteinase and acid endopeptidases. Ca2+-dependent neutral proteinase undergoes intracellularly preliminary activation of the inactive proenzyme species, followed by eventual inactivation through self-proteolysis. Transient activation is shown by selective degradation of cytoskeletal proteins known to be targets of this enzyme system. Concomitantly, acid endopeptidase activity is substantially released from the membrane into the cytosol. Preliminary inactivation of the Ca2+-dependent neutral proteinase by exposure of Glucose 6-phosphate dehydrogenase-deficient red cells to auto-oxidizing divicine prevents alterations induced by Ca2+ loading on cytoskeletal membrane proteins, while leaving solubilization of acid endopeptidase activity unaffected. The two events, although dependent on Ca2+ loading, are therefore unrelated to each other.

Topics: Calcimycin; Calcium; Calpain; Cytoskeletal Proteins; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Enzyme Precursors; Erythrocytes; Glucosephosphate Dehydrogenase; Humans; Pyrimidinones; Solubility; Subcellular Fractions

Calpain, the micromolar Ca2+-requiring form of Ca2+-stimulated neutral proteinase purified from human red cells, is remarkably inactivated during autoxidation of divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism. Inactivation of purified calpain is produced, in decreasing order of efficiency, by transient, probably semiquinonic species arising from autoxidation of divicine, by the H2O2 that is formed upon autoxidation itself, and by quinonic divicine, respectively. Purified procalpain, the millimolar Ca2+-requiring form that can be converted to the fully active calpain form by a variety of mechanisms, is less susceptible than calpain itself to inactivation by the same by-products of divicine autoxidation. When intact red cells are exposed to autoxidizing divicine, procalpain undergoes a significant loss of activity. At 1 mM divicine, intracellular inactivation is observed with procalpain only, while the activity of a number of red cell enzymes is unaffected. Inactivation of procalpain is consistently greater in red cells from glucose-6-phosphate dehydrogenase-deficient subjects than in normal cells. Restoration of normal levels of glucose-6-phosphate dehydrogenase activity by means of entrapment of homogeneous human glucose-6-phosphate dehydrogenase in the deficient red cells results in normal stability of intracellular reduced glutathione; decreased susceptibility of procalpain to inactivation by autoxidizing divicine. These findings suggest that in the glucose-6-phosphate dehydrogenase-deficient red cells the procalpain-calpain system is a major target of divicine cytotoxicity.

Topics: Calpain; Enzyme Precursors; Erythrocytes; Glucosephosphate Dehydrogenase Deficiency; Glutathione; Humans; Oxidation-Reduction; Pyrimidinones