Partial purification and characterization of lactate dehydrogenase from Plasmodium falciparum

• Published in: Molecular and biochemical parasitology Vol. 4; no. 5; pp. 255 - 264
• Main Authors: Vander Jagt, David L., Hunsaker, Lucy A., Heidrich, John E.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.1981
• Subjects: Animals; Centrifugation; chromatography; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Deoxycholic Acid - pharmacology; enzymes; Enzymes, Host; Erythrocytes - parasitology; High-performance liquid chromatography; Kinetics; L-Lactate Dehydrogenase - isolation & purification; L-Lactate Dehydrogenase - metabolism; Lactate dehydrogenase; NAD - metabolism; Plasmodium falciparum; Plasmodium falciparum - enzymology; Protozoa; purification; Pyruvates - metabolism; Pyruvic Acid; Plasmodium falciparum; LDH; Lactate dehydrogenase; High-performance liquid chromatography; LDH-P; LDH-H 4 and LDH-M 4; HPLC
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/0166-6851(81)90058-X

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.