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.
Highlights
The Qinghai-Tibet Plateau, with an average elevation of over 3000 m above sea level, is the highest and largest plateau on earth and is characterized by extremely harsh environmental conditions such as hypoxia, strong ultraviolet radiation, and cold temperature which expectedly have profound effects on human and animal survival
In order to verify that the vectors contained the sequences of Ldh-a, Ldh-b, and Ldh-c and their correct expression, Polymerase Chain Reaction (PCR) of the plasmids and sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-polyacrylamide gel electrophoresis (PAGE)) analysis of the expressed proteins were performed
The results showed that the Ki of lactate for lactate dehydrogenase (LDH)-C4 (Ki = 65.500 mmol/L) was almost three times higher than that for Lactate Dehydrogenase A4 (LDH-A4) (Ki = 26.900 mmol/L) and Lactate Dehydrogenase B4 (LDH-B4) (Ki = 23.800 mmol/L)
Summary
The Qinghai-Tibet Plateau, with an average elevation of over 3000 m above sea level, is the highest and largest plateau on earth and is characterized by extremely harsh environmental conditions such as hypoxia, strong ultraviolet radiation, and cold temperature which expectedly have profound effects on human and animal survival. The pika obtained oxygen effectively from the hypoxic environment by larger superficial pulmonary alveoli and higher capillary density [4], thin walled pulmonary arterioles and blunted hypoxic pulmonary vasoconstriction (HPV) [5], an increase in erythrocyte count [6], reduction in the mean corpuscular volume [7], changes in hemoglobin (Hb) [8], and 2,3-diphosphoglycerate concentrations [5], and an increase in the oxygen affinity to Hb [8]; Secondly, a pika has a strong cardiac pumping function due to its larger heart and smaller weights of right-to-left ventricular plus septum [9]; Thirdly, a pika has a high oxygen utilization ratio by increasing the densities of capillary and mitochondrial [10], and myoglobin concentration in tissues [6,9] In addition to these physiological mechanisms, a pika reduces dependence on oxygen by increasing anaerobic glycolysis in its skeletal muscle [11] and gluconeogenesis in liver [12]. We compared the enzymatic kinetic characteristics among LDH-A4, LDH-B4, and LDH-C4 of plateau pika with the aim of exploring the pika’s adaptation mechanism to the hypoxic environment of the Qinghai-Tibet Plateau
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