Yak Lactate Dehydgogenase A4: Purification, Properties, and cDNA Cloning

Abstract

Lactate dehydrogenase A4 (LDH-A4) was purified for yak skeletal muscle. Michaelis constant (Km) analysis showed that yak LDH-A4 for pyruvate was significantly higher than that of cattle. cDNA cloning of LDH-A revealed two amino acid substitutions between yak and cattle. We suggest that the higher Km of yak LDH-A4 might be a result of molecular adaptation to a hypoxic environment.Key words: yak; lactate dehydrogenase A; purification; molecular adaptationThe study of molecular adaptation has long been fraught with difficulties due to the numerous amino acid replacements accumulated over evolution, only a few of which are directly responsible for major adaptations. Though phylogenetic and phenotypic evidence is insufficient for an understanding of the molecular adaptation alone, 1) scientists must identify the replacements directly responsible for adaptive changes. Here we introduce a model to use in studying the molecular adaptation of the yak (Bos grunniens) to its living environment.The yak inhabits the steppes of the Himalayan highlands and was domesticated about 3,000 years ago.2) It has adapted to the high altitudes (2,000-5,000 m), hypoxia, and cold environment of QinghaiTibetan Plateau. It has high amino acid sequence similarities (> 98%) with domestic cattle (Bos taurus) in most proteins analyzed (our unpublished data), and their divergence time has been deduced to be about 1.2 to 2.2 million years ago. 3) Usually, there are only a few amino acid replacements when comparing the same protein of yak and cattle. Therefore, yak and cattle are model animals suited to the study of molecular adaptation to hypoxic ecological conditions by comparative methods.Lactate dehydrogenase (EC 1.1.1.27, LDH) is an enzyme that catalyzes the conversion of lactate and pyruvate in glycolysis. LDH in animals occurs in five common isoenzyme forms, each being one of the possible random tetrameric combinations of different subunits, designated A (M) and B (H). Here we chose LDH to study molecular adaptation for several reasons. First, LDH plays an important role in the anaerobic metabolism of glucose, and thus hypoxic conditions have the most probability to act on it during evolution selection. Second, it is a slowly evolving protein for which there are extensive data as to amino acid sequence 4-7) and three-dimensional structure. Third, structure-function analysis of the LDH of the bacterium Bacillus stearothermophilus has elucidated the roles of numerous amino acid residues in governing the enzyme's kinetic properties. [8][9][10][11][12] In this study, we purified and characterized LDH-A4 from skeletal muscle of yak and cattle, and found that yak LDH-A4 Km for pyruvate was much higher than that of cattle. Yak LDH-A cDNA was cloned and analyzed in order to elucidate the amino acid substitutions between yak and cattle.LDH-A4 was purified from skeletal muscles of yak and Chinese yellow cattle by modification of the methods reported by Dissing et al. (1998) and Burgos et al. (1995). 13,14) Skeletal muscle tissues were homogenized in ...

Cloning and expression profiles of yak lipoprotein lipase gene

“…This sequence encodes 478 amino acids fully identical to that of cattle. This result is consistent with many genes assayed in yaks (Zheng et al 2008), and also provides evidence for the close phylogenetic relationship between yak and cattle.…”

“…Yaks have lower intramuscular fat content than cattle (Cai and Wiener 1995). Because of the high similarity (98Á99%) in many protein sequences assayed between yak and cattle (Zheng et al 2008), yak is considered a suitable animal model to study the influences of hypoxia on production performance. Considering the functions of LPL in lipid metabolism and adipose tissue repartition, we thus cloned yak LPL gene and assayed the expression profiles of LPL mRNA and compared with cattle, in order to highlight the molecular basis of meat quality in yaks.…”

Comparison of enzyme activities and gene expression profiling between yak and bovine skeletal muscles

“…Jiulong yak is an important yak breed distributed in China and the biggest yak breed in bodyweight. Yaks share very high similarity (98% to 99%) with cattle in many protein sequences assayed such as LDH (Zheng et al, 2008), so it is an excellent animal model to study influences of environmental factors such as hypoxia on its performance. It is reported that permanent exposure or acclimation to severe hypoxia decreases the mitochondrial content of muscle fibers, and oxidative muscle metabolism is shifted towards a higher reliance on carbohydrates as fuel, and intramyocellular lipid stores are reduced (for review see Hoppeler et al, 2003).…”

Differences in mitochondrial gene expression profiles, enzyme activities and myosin heavy chain types in yak versus bovine skeletal muscles

“…Because of the high sequence similarity (98 to 99%) between yak and Yellow cattle in genes assayed (Zheng et al, 2008;Bai et al, 2010;Zhang et al, 2010), yak may act as a suitable animal model to study the mechanism of hypoxia adaptation. Due to the low oxygen environment, energy supply is an important aspect for studying adaptation in yaks.…”