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This study investigates the molecular mechanism by which yaks (Bos grunniens) adapt to hypoxia based on lactate dehydrogenase (LDH). Three LDH1 variants of the yak were revealed in tissue extracts by electrophoresis, including LDH1-F, LDH1-M, and LDH1-S. Kinetic analysis using purified LDH1 variants showed that the yak LDH1-M variant exhibited a similar K (m) (NADH) and the same mobility on a gel as bovine LDH1, and the LDH1-F variant showed significant differences in K (m) values for NADH or pyruvate from the other two variants of yak LDH1 and bovine LDH1. Among the three muscles assayed, yak longissimus dorsi showed the highest LDH activity and the lowest malate dehydrogenase (MDH) activity; heart muscle was exactly the opposite. Our results suggest that the three LDH1 variants might play an important role in the adaptation to hypoxia.

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 98%) with domestic cattle (Bos taurus) in most proteins analyzed (our unpublished data), and their divergence time has been deduced to be about 1.2 to 2.2 million years ago. 3) Usually, there are only a few amino acid replacements when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 ...

Native polyacrylamide gel electrophoresis showed that two types of lactate dehydrogenase (LDH) existed in yaks. Based on the electrophoresis characteristics of LDH isoenzymes, yak LDH variants were speculated to be the gene mutation on H subunit encoded by B gene. According to the mobility in electrophoresis, the fast-band LDH type was named LDH-Hf and the slow-band LDH type LDH-Hs. In order to reveal the gene alteration in yak LDH variants, total RNA was extracted from heart tissues of yaks with different LDH variants, and cDNAs of the two variants were reverse transcripted. Two variants of B genes were cloned by RT-PCR. Sequence analysis revealed that four nucleotides differed between LDH-Bf and LDH-Bs, which resulted in two amino acids alteration. By Deepview software analysis of the conformation of yak LDH1 variants and H subunit, these four nucleotides altered two amino acids that generated new hydrogen bonds to change the hydrogen bonds network, and further caused subtle conformational changes between the two LDH variants.

The objective of the present study was to confirm the widespread existence of alternative splicing of lactate dehydrogenase c (ldhc) gene in mammals. RT-PCR was employed to amplify cDNAs of ldhc from testes of mammals including pig, dog, rabbit, cat, rat, and mouse, as well as pigeon. Two to six kinds of splice variants of ldhc were observed in the seven species as a result of deletion of one or more exons or insertion of partial sequence of an intron in the mature mRNA. The deleted exons occur mostly in exons 5, 4, 6, and 3. The insertion of a partial sequence of introns, which resulted in an abnormal stop codon in the inserted intron sequence, was observed only in dog and rat. The deletion of exons also resulted in a reading frame shift and formation of a stop codon in some variants. No alternative splicing was observed for ldha and ldhb genes in testis of yak. Native polyacrylamide gel electrophoresis and Western blot analysis revealed no obvious LDH-C4 activity derived from expressed ldhc variants. Our results demonstrated the widespread and unique existence of alternative splicing of ldhc genes in mammals.

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The genotypes and protein polymorphisms of milk epithelial mucin (MUC1) were analyzed by touch-down PCR and SDS-PAGE respectively using blood samples and milk collected from 50 lactating yaks. A total of seven alleles were revealed, namely A, B, C, D, E, F, and G, and the corresponding number of repetitive units of variable number tandem repeat (VNTR) in MUC1 gene were 21, 20, 19, 18, 17, 16, and 15, respectively. Fifteen genotypes of MUC1 were observed in yaks. The genotypes of MUC1 gene matched completely to the phenotypes of milk MUC1 in each individual. This study demonstrated that the yak MUC1 exhibits abundant polymorphisms in both its gene and protein, and the polymorphisms are due to the expression of VNTR in MUC1 gene. The possible cluster of the VNTR was also discussed in different ruminants.