The genotoxic carcinogen aflatoxin B1 (AFB1) inhibited the calmodulin-stimulated membrane-bound (Ca2+Mg2+)-ATPase. Using the purified enzyme, 12 nmoles per ml of AFB1 caused maximum inhibition of 28% and 50%, of the acidic phospholipid-stimulated and calmodulin-activated Ca2+-ATPase activity respectively. Treatment of red cell ghosts with increasing concentrations of Triton X-100, a non-ionic detergent caused a progressive loss of both the basal and calmodulin-stimulated Ca2+-ATPase activity. The activity of the phospholipid-free, detergent-solubilized enzyme was almost fully restored by phosphatidyl serine (PS) and its sensitivity to calmodulin was restored in the presence of phosphatidyl choline (PC). Analysis of the results obtained using varying concentrations of ATP shows that AFB1 did not affect the Km and Vmax of the unstimulated enzyme whereas these parameters were reduced by about 75% and 50%, respectively, in the presence of calmodulin. Using the product of limited proteolysis by trypsin i.e. the 90 kDa fragment which still retains its calmodulin binding-domain and the 76 kDa fragment which has lost this domain, kinetic studies on the enzyme activity revealed that AFB1 inhibited the calmodulin-activated 90 kDa fragment by about 50% while the 76 kDa was not affected at all by the toxin and calmodulin. The toxin had no significant affect on the basal activity of the 90 kDa limited proteolysis fragment of the enzyme. These observations suggest that AFB1 inhibits the activated Ca2+-ATPase by binding to an important site in the calmodulin-binding domain of the enzyme. It seems likely that the toxin binds to tryptophan in the calmodulin-binding domain, thus causing a reduction in the rate at which this domain can interact with Ca2+-calmodulin or acidic phospholipids. The implication of these observations is that Ca2+-extrusion and other calmodulin-activated enzymes and processes may be slowed down during prolonged exposure to AFB1 because of its anticalmodulin effect.
Abbreviations ATP, adenosine 5′-triphosphate; EGTA, ethylenglycolbis (β-aminoethylether) N,N′-tetraacetic acid; Hepes, 4-(2 hydroxyethyl)-1-piperazine ethanesulphonic acid; AFB1, aflatoxin B1; PMSF, phenylmethylsulfonylfluoride; TLCK, N-α-p-tosyl-L-lysine chloromethyl ketone; PC, phosphatidycholine; PS, phosphatidylserine; PI, phosphatidyl inositol; DPG, diphosphatidyl glycerol; SDS, sodium dodecyl sulphate; Tris-HCl, Tris (hydroxymethyl)aminomethane hydrochloride;