The Partial Purification of Human Erythrocyte Adenylate Kinase and the Effect of Some Common Antimalarial Drugs on this Enzyme

Abstract

The enzyme adenylate kinase (AK) was partially purified from human erythrocytes. The purification process involved the separation of the erythrocytes from whole human blood, rinsing of the cells in saline solution and finally lysing them in two volumes of water containing dithiothreitol. The crude haemolysate was dialysed against O.OlM sodium citrate buffer pH 5.0 and then treated with a suspension of CM-sephadex C-50, previously equilibrated in O.OlM sodium citrate buffer pH 5.0 containing 0.25M sodium chloride to reduce amount of haemoglobin present to effect better separation on column. After centrifuging at 30,000g the supernatant solution from this treatment was further subjected to sephadex G-100 column chromatography to obtain a partially purified enzyme preparation. The activity of adenylate kinase was measured using the coupled assay system by Oliver (1957), which is AK 2ADP ------- >AMP + ATP + hexokinase + Glucose + Mg2+ Glucose-6-phosphate + G6PD + NADP+ 6-phospho-gluconate + NADPH + H+ Absorbance at 340nm was measured for the NADPH produced over a period of 6 minutes using an SP 500 spectrophotometer with a recorder attached to it. This corresponded to ATP produced over the same period. It was found that AK was unstable at 70°C and optimum activity was observed around 37°- 40°C. Dilute solutions of AK were found to be unstable, but the enzyme was stable at 4°C in a concentrated form. Adenylate kinase activity was also found to be inhibited at high substrate (ADP) concentration, and it is known that AMP, one of the products of the reaction inhibits AK activity, competitively. From substrate concentration of 3-10mM ADP, the activity decreased. Optimum activity was obtained at substrate concentration of l-2mM ADP. The effect of some common antimalarial drugs on the enzyme activity was also looked at. The antimalarial drugs used included chloroquine diphosphate, chloroquine sulphate, quinine hydrochloride, quinine sulphate, proguanil hydrochloride, primaquine phosphate and mepacrine hydrochloride. All the drugs at a concentration of 10“^M inhibited the enzyme activity to various extent. The degree of inhibition depended on the incubation period of the enzyme with the particular drug. Quinine was found to inhibit the enzyme activity most. Inhibition of up to 62.0% was observed with quinine after 3 hours incubation period. For the same incubation period, primaquine phosphate inhibited the enzyme activity up to 42.0%, mepacrine up to 40.7%, chloroquine up to 30,3% and proguanil hydrochloride up to 28.0%. The type of inhibition, observed for chloroquine diphosphate, the most common antimalarial drug in the tropics, was noncompetitive inhibition.