Enzymatic Kinetic Properties of the Lactate Dehydrogenase Isoenzyme C4 of the Plateau Pika (Ochotona curzoniae)

Abstract

Testis-specific lactate dehydrogenase (LDH-C4) is one of the lactate dehydrogenase (LDH) isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C4 in mammals was previously thought to be expressed only in spermatozoa and testis and not in other tissues. Plateau pika (Ochotona curzoniae) belongs to the genus Ochotona of the Ochotonidea family. It is a hypoxia-tolerant species living in remote mountain areas at altitudes of 3000–5000 m above sea level on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also in somatic tissues of plateau pika. To shed light on the function of LDH-C4 in somatic cells, Ldh-a, Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes of Lactate Dehydrogenase A4 (LDH-A4), Lactate Dehydrogenase B4 (LDH-B4), and LDH-C4 were prepared by a series of expression and purification processes, and the three enzymes were identified by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (PAGE). The enzymatic kinetics properties of these enzymes were studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km) of LDH-C4 for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate 90 times higher affinity of LDH-C4 for pyruvate than for lactate. At relatively high concentrations of lactate, the inhibition constant (Ki) of the LDH isoenzymes varied: LDH-A4 (Ki = 26.900 mmol/L), LDH-B4 (Ki = 23.800 mmol/L), and LDH-C4 (Ki = 65.500 mmol/L). These data suggest that inhibition of lactate by LDH-A4 and LDH-B4 were stronger than LDH-C4. In light of the enzymatic kinetics properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C4.

Mechanisms driving the lactate switch in Chinese hamster ovary cells

“…However, the isozyme pattern within a cell would affect how quickly the reaction once again reaches equilibrium if a disturbance should occur (Quistorff & Grunnet, 2011b;Wang et al, 2016). Additionally, even if the reaction is considered close to dynamic equilibrium, the cellular environment is not a closed system.…”

The expression ofLdh-cin the skeletal muscle of plateau pika (Ochotona curzoniae) enhances adaptation to a hypoxic environment

“…These findings imply that LDH-C 4 has an affinity for pyruvate that is 60-fold higher than that for lactate, and suggest that pyruvate turnover to lactate may be high even at high concentrations of endogenous or extracellular lactate ( Clausen and Ovlisen, 1965 ; Wilkinson and Withycombe, 1965 ; LeVan and Goldberg, 1991 ; Wong et al, 1997 ). We also found that pika LDH-C 4 had a lower K m for pyruvate (∼0.052 mmol/l) and a higher K m for lactate (∼4.934 mmol/l), and that it was beneficial to catalyse the conversion of pyruvate to lactate even at high concentrations of lactate endogenously ( Wang et al, 2016 ). Those results suggest that the biochemical properties, which separated LDH-C 4 from the other LDH isoforms are conducive to the high glycolytic flux level.…”

“…LDH-C 4 is expressed in the spermatozoa and testis of birds and mammals ( Goldberg, 1964 , 1975 , 1984 ; Coonrod et al, 2006 ), and is one of the LDH isoenzymes ( Blanco and Zinkham, 1963 ; Goldberg, 1963 ). LDH-C 4 has higher affinity for pyruvate than lactate, and catalyses the conversion of pyruvate to lactate in the semen of humans and other species ( Clausen and Ovlisen, 1965 ; Wilkinson and Withycombe, 1965 ; Coronel et al, 1983 ; LeVan and Goldberg, 1991 ; Wong et al, 1997 ; Hereng et al, 2011 ; Wang et al, 2016 ), which is crucial for glycolysis to continue production of adenosine triphosphate (ATP) ( Coronel et al, 1983 ; LeVan and Goldberg, 1991 ; Odet et al, 2011 ). LDH-C 4 , is the key factor of sperm glycolysis, which has an important role in sperm motility to provide ATP ( Coronel et al, 1983 ; Williams and Ford, 2001 ; Mukai and Okuno, 2004 ; Odet et al, 2011 ).…”

“…LDH-C 4 , is the key factor of sperm glycolysis, which has an important role in sperm motility to provide ATP ( Coronel et al, 1983 ; Williams and Ford, 2001 ; Mukai and Okuno, 2004 ; Odet et al, 2011 ). In our previous studies, we found that the affinity of LDH-C 4 for pyruvate was ∼90-fold higher than that for lactate, and it was not easily inhibited by high lactate concentration ( Wang et al, 2016 ). LDH-C 4 was conducive to catalysis of the conversion of pyruvate to lactate.…”

Effect of Hypoxia on Ldh-c Expression in Somatic Cells of Plateau Pika

“…Compared with LDH-A 4 and LDH-B 4 , LDH-C 4 had a lower K m for pyruvate (~0.052 mmol/L) and a higher K m for lactate (~4.934 mmol/L); thus, LDH-C 4 was less sensitive to lactate inhibition than LDH-A 4 and LDH-B 4 [47]. These properties of pika LDH-C 4 were beneficial for catalyzing the conversion of pyruvate to lactate even at a high concentration of lactate.…”