Erythrocyte
AMP deaminase [
isoform E (AMPD3)] is activated in response to increased intracellular
calcium levels in
Tarui's disease, following exposure of
ionophore-treated cells to extracellular
calcium, and by the addition of
calcium to freshly prepared hemolysates. However, the assumption that Ca(2+) is a positive effector of
isoform E is inconsistent with the loss of sensitivity to this divalent
cation following dilution of erythrocyte lysates or
enzyme purification. Ca(2+) regulation of
isoform E was studied by examining in vitro effects of
calmodulin (CaM) on this
enzyme and by monitoring the influence of CaM antagonists on
purine catabolic flow in freshly prepared erythrocytes under various conditions of energy imbalance. Erythrocyte and recombinant
isoform E both adsorb to immobilized Ca(2+)-CaM, and relative adsorption across a series of N-truncated recombinant
enzymes localizes CaM binding determinants to within residues 65-89 of the AMPD3
polypeptide. Ca(2+)-CaM directly stimulates
isoform E catalytic activity through a K(mapp) effect and also antagonizes the
protein-
lipid interaction between this
enzyme and intracellular membranes that inhibits catalytic activity.
AMP is the predominant
purine catabolite in erythrocytes deprived of
glucose or exposed to
A23187 ionophore alone, whereas
IMP accumulates when Ca(2+) is included under the latter conditions and also during autoincubation at 37 degrees C. Preincubation with a CaM antagonist significantly slows the accumulation of erythrocyte
IMP under both conditions. The combined results reveal a
protein-
protein interaction between Ca(2+)-CaM and
isoform E and identify a mechanism that advances our understanding of erythrocyte
purine metabolism. Ca(2+)-CaM overcomes potent
isoform E inhibitory mechanisms that function to maintain the total
adenine nucleotide pool in mature erythrocytes, which are unable to synthesize
AMP from
IMP because of a developmental loss of
adenylosuccinate synthetase. This may also explain why
Tarui's disease erythrocytes exhibit accelerated
adenine nucleotide depletion in response to an increase in intracellular Ca(2+) concentration. This regulatory mechanism could also play an important role in
purine metabolism in other human tissues and cells where the AMPD3 gene is expressed.