Favism is an acute hemolytic anemia triggered by ingestion of fave 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 μmol Pi/ml RBC/h, while control values in asymptomatic G6PD-deficient subjects were 10.62 ± 2.03 μmol 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.

Membranes from human erythrocytes bind radioactive GTP and GTP analogs according to apparently homogeneous patterns. In spite of this uniform type of association, multiple guanine nucleotide binding proteins have been identified both by SDS-PAGE analysis of native and of variously ADP-ribosylated membrane preparations and by FPLC chromatography of solubilised erythrocyte membranes preliminarily incubated with [alpha-32P] GTP in the presence of 5 mM MgCl2. From eight to nine peak fractions of pronase-digestible GTP-binding activity were separated on a MA7Q anion exchange column, this pattern being highly reproducible with different membrane preparations. Prior incubation of membranes with [alpha-32P] GTP in the presence of excess unlabeled GDP resulted in displacement of bound labeled nucleotide from all FPLC fractions. The patterns of GTP binding were also markedly modified by preliminary treatment of membranes with N-ethylmaleimide. Detectable GTPase activity was present in each of the FPLC peak fractions. This wide heterogeneity of guanine nucleotide binding proteins raises so far unanswered questions as to their physiological significance in the mature erythrocyte.

Native membranes from human erythrocytes contain the following G proteins which are ADP-ribosylated by a number of bacterial toxins: Gi alpha and Go alpha (pertussis toxin), Gs alpha (cholera toxin), and three proteins of 27, 26 and 22 kDa (exoenzyme C3 from Clostridium botulinum). Three additional C3 substrates (18.5, 16.5 and 14.5 kDa) appeared in conditions of unrestrained proteolysis during hemolysis. SDS-PAGE separation of erythrocyte membrane proteins followed by electroblotting and incubation of nitrocellulose sheets with radiolabeled GTP revealed consistently four GTP-binding proteins with Mr values of 27, 26, 22 and 21 kDa. Although a 22 kDa protein was immunochemically identified as ras p21, the C3 substrate of 22 kDa is a different protein probably identifiable with a rho gene product. Accordingly, at least five distinct small molecular weight guanine nucleotide-binding proteins, whose functions are so far undetermined, are present in native human erythrocyte membranes.