Clinical and Enzymological Studies on Human Lactate Dehydrogenase:I. Two Km Values of Serum LDH for Lactate

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The authors studied to find optimal conditions for determining Km values of human serum LDH (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) for lactate in order to screen "abnormal" LDH. LDH activity was measured at 25°C in four buffer systems. 1) The activity and its pH dependence differed slightly but clearly depending on the buffers used: glycine, pyrophosphate, Veronal, and Tris systems. 2) The pH-activity curves were essentially different when 1mM and 50 mM lactate were used. 3) LDH showed two different Km values for NAD+ in two ranges of its concentration: 79×10-6M (Veronal) and 44-57×10-6M (others) in 0.05-0.25mM, and 133×10-6M (Veronal) and 95-105×10-6M (others) in 0.25-3.5 mM. 4) Two values of Km were found also for lactate with a critical concentration around 2 mM: 1.2mM (Veronal) and 0.5mM (others) in the lower concentrations, and approximately 2mM (Veronal and Tris) and 1mM (others) in the higher ones. 5) The sera from patients, which had predominantly H or M type isoenzyme showed also two Km values with a critical concentration similar to that observed in sera with normal isoenzyme patterns. Therefore, this phenomenon may reflect a particular intrinsic property of the tetrameric enzyme: for example, subunit-subunit interaction.
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