Enzymatic and thermodynamic profiles of a heterotetramer lactate dehydrogenase isozyme in swine.
Lactate dehydrogenase (LDH) is a glycolytic enzyme that catalyzes the final step of glycolysis and produces NAD+. In somatic cells, LDH forms homotetramers and heterotetramers that are encoded by two different genes: LDHA (skeletal muscle type, M) and LDHB (heart type, H). Analysis of LDH isozymes is important for understanding the physiological role of homotetramers and heterotetramers and for optimizing inhibition of their enzymatic activity as it may result in distinct effects. Previously, we reported that hydroxychloroquine (HCQ) inhibited LDH activity, but we did not examine isozyme specificity. In the present study, we isolated heterotetrameric LDH (H2M2) from swine brain, determined its kinetic and thermodynamic properties, and examined the effect of HCQ on its activity compared to homotetrameric LDH isozymes. We show that: (1) the Kmvalues for H2M2-mediated catalysis of pyruvate or lactate were intermediate compared to those for the homotetrameric isozymes, M4and H4whereas the Vmaxvalues were similar; (2) the Kmand Vmaxvalues for H2M2-mediated catalysis of NADH were not significantly different among LDH isozymes; (3) the values for activation energy and van't Hoff enthalpy changes for pyruvate reduction of H2M2were intermediate compared to those for the homotetrameric isozymes; (4) the temperature for half residual activity of H2M2was closer to that for M4than for H4. We also show that HCQ had different affinities for various LDH isozymes.
Department of Life Science, Graduate School of Engineering Science, Akita University, Japan.
Tatsufumi
T
Goto
Department of Hematology, Nephrology, and Rheumatology, Master Course of Graduate School of Medicine, Akita University, Japan.
Kotomi
K
Sugawara
Department of Creative Engineering, Kitakyusyu National College of Technology, Japan.
Shigeyoshi
S
Nakamura
Department of Bioengineering, Graduate School of Engineering, Nagaôka University of Technology, Japan.
Shun-Ichi
SI
Kidokoro
Department of Life Science, Graduate School of Engineering Science, Akita University, Japan. Electronic address: wakui517@gipc.akita-u.ac.jp.
Hideki
H
Wakui
Department of Life Science, Graduate School of Engineering Science, Akita University, Japan; Research Center for Engineering Science, Graduate School of Engineering Science, Akita University, Japan. Electronic address: nunomura@gipc.akita-u.ac.jp.
Wataru
W
Nunomura
D000818:Animals; D001921:Brain / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006886:Hydroxychloroquine / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013552:Swine / Q000378:metabolism*; D013816:Thermodynamics
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D021122:Protein Subunits; D006886:Hydroxychloroquine; D007770:L-Lactate Dehydrogenase
H(2)M(2); HCQ; Heterotetrameric isozyme; Hydroxychloroquine; K(m); LDH; Lactate dehydrogenase; T(half); T(max); V(max); heterotetrameric lactate dehydrogenase isozyme consisting of two LDHA-encoded muscle type and two LDHB-encoded heart muscle type molecules; hydroxychloroquine; lactate dehydrogenase; optimum temperature for LDH pyruvate reduction activity; residual activity of LDH pyruvate reduction activity; van't Hoff enthalpy