Characterization of adenosine deaminase from the malarial parasite, Plasmodium lophurae, and its host cell, the duckling erythrocyte

Abstract

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) was purified and characterized from the malarial parasite, Plasmodium lophurae, and its host cell, the duck ( Anas domesticus) erythrocyte, using chromatofocusing (Pharmacia) and adenosine affinity columns. Gel filtration of the enzymes gave molecular weights of 33,800 ( P. lophurae) and 36,500 (duck erythrocyte); both enzymes had broad pH optima (pH 6.8 to 8.0), similar stabilities when stored as crude lysates, and like K m values with adenosine: 2.74 ± 0.88 × 10 −5 M (parasite) and 1.74 ± 0.27 × 10 −5 M (erythrocyte). The P. lophurae adenosine deaminase had a pI of 5.37 ± 0.09, and the duck erythrocyte enzyme had a pI of 4.72 ± 0.09, as determined by chromatofocusing. The parasite enzyme exhibited a specific activity in the crude lysate that was an average 60-fold higher than that of the erythrocyte enzyme. The pattern of elution from the adenosine affinity column, as well as kinetic studies with three adenosine analogs, revealed distinct differences in the binding characteristics of the two enzymes. The P. lophurae adenosine deaminase was weakly retarded by the affinity column, whereas the duck erythrocyte enzyme was strongly retarded. With 9-β- d-arabinosyladenine as substrate, the K m values were similar (2.29 ± 0.98 × 10 −4 M for P. lophurae and 1.10 ± 0.21 × 10 −4 M for the duck erythrocyte). Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was a potent inhibitor of the duck erythrocyte enzyme with 100% inhibition at 1.3 μM, whereas the parasite adenosine deaminase was not inhibited at 422 μM even when incubated for 24 hr. Inhibitor studies with coformycin, a tight-binding inhibitor, resulted in K i values of 7.14 × 10 −11 M for P. lophurae and 1.86 × 10 −10 M for the duck erythrocyte. The molar equivalencies, E t , and catalytic numbers, k 3, were slightly different for both enzymes. The E t values were 2.80 × 10 −10 M ( P. lophurae) and 3.13 × 10 −10 M (duck erythrocyte); the k 3 values were 5.18 × 10 3 min −1 and 4.36 × 10 3 min −1 respectively.