Partial purification and characterization of lactate dehydrogenase from Plasmodium falciparum

Abstract

Lactate dehydrogenase (LDH) from Plasmodium falciparum was partially purified by two different procedures. In the first procedure, parasitized erythrocytes (80% parasitemia) were lysed, and the soluble fraction was purified on DEAE-Sephadex to separate the parasite LDH (LDH-P) from the LDH isozymes present in the human erythrocytes. LDH-P was then purified by high-performance liquid chromatography (HPLC) on a TSK-G-3000 SW protein column. This two-step procedure gave LDH-P with specific activity 85 μmol/min/mg protein; this represented a 700-fold increase in specific activity relative to the starting lysate. Alternatively, parasites of P. falciparum were isolated by mechanical rupture of infected erythrocytes followed by differential centrifugation. The 100 000 × g supernatant obtained after lysis of these parasites showed LDH-P specific activity 3.6 μmol/min/mg protein. This activity was free of contaminating erythrocyte LDH as determined by electrophoresis and specific staining for LDH. Further purification of LDH-P by HPLC, as before, gave material with specific activity 98 μmol/min/mg protein. Recoveries of activity on HPLC were 90%, demonstrating the usefulness of this procedure for the partial purification of small quantities of parasite protein. The kinetic properties of LDH-P were compared with those of two of the human isozymes, LDH-H 4 and LDH-M 4. LDH-P resembles LDH-H 4 in its kinetic properties: K M (NADH) is 7, 8.3 and 1.3 μM for LDH-P, LDH-H 4 and LDH-M 4, respectively; K M (pyruvate) is 30, 60 and 180 μM for LDH-P, LDH-H 4 and LDH-M 4. LDH-P differs significantly from LDH-H 4 and LDH-M 4 in that LDH-P is not sensitive to inhibition by high pyruvate nor sensitive to inhibition by the complex between NAD + and pyruvate. LDH-P is inactivated within seconds by sodium deoxycholate at concentrations that do not affect LDH-H 4 and only slowly inactivate LDH-M 4.