Testis-specific lactate dehydrogenase (LDH-C4) is among the lactate dehydrogenase (LDH) isozymes
March 28, 2017
Testis-specific lactate dehydrogenase (LDH-C4) is among the lactate dehydrogenase (LDH) isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. 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 analyzed by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (of the family is a small non-hibernating rodent that lives in remote mountain areas at an elevation of 3000-5000 m around the Qinghai-Tibet Plateau. The pika plays an important role in biodiversity of the ecosystem around the plateau and is regarded as a key species since ancient occasions [1 2 Fossil samples of pika that are nearly 37 million years old were found by archaeologistson TAK-901 the north edge of the Qinghai-Tibetan plateau . During long-term development the pikas developed a series of physiological adaptations that allow them to thrive in the harsh environment and become a highly advanced hypoxia-tolerant mammal. Specifically the pika attained air effectively in the hypoxic environment by bigger superficial pulmonary alveoli and higher capillary thickness  slim walled pulmonary arterioles and blunted hypoxic pulmonary vasoconstriction (HPV)  a rise in erythrocyte count number  decrease in the indicate corpuscular quantity  adjustments in hemoglobin (Hb)  and 2 3 concentrations  and a rise in the air affinity to Hb ; Second a pika includes a solid cardiac pumping function because of its bigger heart and smaller sized weights of right-to-left ventricular plus septum ; Finally a pika includes a high air utilization proportion by raising the densities of capillary and mitochondrial  and myoglobin focus in tissue [6 9 Furthermore to these physiological systems a pika decreases dependence on air by raising anaerobic glycolysis in its skeletal muscles  and gluconeogenesis in liver organ . The molecular systems of the adaptations in pika possess occurred because of some changes such as for example genetic progression the appearance TAK-901 of tissue-specific proteins and adjustments related to thin air TAK-901 including many useful cytokines as vascular endothelial development aspect (VEGF) [13 14 hemoglobin  HIF-1α [15 16 LDH-C4  pyruvate carboxylase  myoglobin  inducible nitric oxide synthase (iNOS)  leptin [19 20 and cytochromec oxidase . It really is popular that LDH family members enzymes catalyze the inter-conversion of pyruvate and lactate using the concomitant oxidation/decrease of the decreased type of nicotinamide adenine dinucleotide hydrogen (NADH) to nicotinamide adenine dinucleotide (NAD+) in the reactions TAK-901 . Different types of Lactate dehydrogenase (LDH) isozymes are comprised of expression products of three common genes: not only in testis and sperm but also in somatic cells of plateau pika . LDH-C4 has high thermostability  and its homolog from different species showed activity against longer carbon chain with α-hydroxy and α-keto acids than those of pyruvate and lactate [31 32 33 34 35 36 37 38 39 40 41 indicating that LDH-C4 has unique structural and functional properties [31 40 41 42 In the present study 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 Rabbit polyclonal to ISOC2. adaptation mechanism to the hypoxic environment of the Qinghai-Tibet Plateau. 2 Results 2.1 Plasmid Construction and Recombinant Protein Expression To study the enzyme TAK-901 kinetics of LDH-A4 LDH-B4 and LDH-C4 of plateau pika the expression plasmids pCold-SUMOand their correct expression Polymerase Chain Reaction (PCR) of the plasmids and sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) analysis of the expressed proteins were performed. Physique 1A-C show the agarose gel electrophoresis results of PCR products of (999 bp) (1005 bp) and (999 bp) as our previous study . By sequencing and alignment the sequences of and in the final plasmid constructs were decided.