Partial purification and characterization of lactate dehydrogenase from Plasmodium falciparum.
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 isoenzymes 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 micromol/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 X g supernatant obtained after lysis of these parasites showed LDH-P specific activity 3.6 micromol/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 micromol/min/mg protein. Recoveries of activity on HPLC were more than 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-H4 and LDH-M4 . LDH-P resembles LDH-H4 in its kinetic properties: KM (NADH) is 7, 8.3 and 1.3 microM for LDH-P, LDH-H4 and LDH-M4, respectively; KM (pyruvate) is 30, 60 and 180 microM for LDH-P, LDH-H4 and LDH-M4. LDH-P differs significantly from LDH-H4 and LDH-M4 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-H4 and slowly inactivate LDH-M4.
D000818:Animals; D002498:Centrifugation; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003840:Deoxycholic Acid / Q000494:pharmacology; D004912:Erythrocytes / Q000469:parasitology; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology*; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid
D011773:Pyruvates; D003840:Deoxycholic Acid; D009243:NAD; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase