There are five lactate dehydrogenase (LDH) isoenzymes, composed of various combinations of two types of subunits. LDH-5, which contains only the LDH A subunit, is known to be present in both the cytoplasm and the nucleus, to act as a single-stranded DNA-binding protein possibly functioning in transcription and/or replication, and to undergo phosphorylation of tyrosine 238 in approximately 1% of the enzyme after cell transformation by certain tumor viruses. We have characterized LDH from wild-type PC12 pheochromocytoma cells and from a PC12 variant (MPT1) that exhibits altered lactate metabolism and altered expression of multiple genes. Wild-type and MPT1 cells contain different proportions of LDH isoenzymes, with LDH-5 being more predominant in wild-type cells than in the variant. A small fraction of LDH from PC12 cells contains phosphotyrosine. Approximately 99% of the total LDH activity is located in the cytoplasm, but all of the phosphotyrosine-containing LDH is located in the nucleus. Furthermore, essentially all of the nuclear LDH contains phosphotyrosine. These results suggest that tyrosine phosphorylation can affect its role in the nucleus.Lactate dehydrogenase (LDH, EC 1.1.1.27) catalyzes the reversible conversion of pyruvate to lactate. Five LDH isoenzymes exist because the enzyme is a tetramer that is composed of various combinations of two kinds of Mr 35,000 subunits named A and B (9). The isoenzymes and their respective structures are LDH-5 (A4), LDH-4 (A3B), LDH-3 (A2B2), LDH-2 (AB3), and LDH-1 (B4). The LDH isoenzyme composition varies from tissue to tissue.In addition to its role in the metabolism of lactate and pyruvate, one of the LDH isoenzymes, LDH-5, may function in the regulation of gene transcription and/or DNA replication, since LDH-5 is present in the nucleus (5) and it has recently been found to be a previously studied singlestranded DNA-binding protein (14,25,31). It had been shown earlier that antibody raised against this DNA-binding protein cross-reacts with single-stranded DNA-binding proteins from heterologous species including Drosophila spp. (22). Furthermore, studies of Drosophila polytene salivary chromosomes revealed that this protein is associated nonrandomly with the chromosomes and is usually concentrated in certain chromosome puffs that were active sites of transcription, e.g., heat shock puffs after heat shock treatment (22). A possible role in replication is suggested by the finding that LDH-5 stimulates the activity of DNA polymerase a in vitro (14).Another LDH variation arises from phosphorylation. A small percentage of chicken fibroblast LDH-5 is phosphorylated at seine residues; after transformation of these cells by Rous sarcoma virus, 0.5 to 1% of LDH-5 is also phosphorylated at a single tyrosine residue, tyrosine 238 (6, 7). It is not clear that this tyrosine phosphorylation affects glycolysis, because such a small fraction of the LDH in the transformed cells contains phosphotyrosine. The intracellular location of phosphotyrosine-containing LDH-5 has not previously...

We have cloned and characterized two variants of PC12 cells. MPT1 cells were selected by their resistance to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and variant 2068 was isolated nonselectively as a large, flat-cell variant commonly occurring in PC12 cultures. Variant 2068 cells also exhibit resistance to MPTP. Karyotype analysis revealed that these variants are true derivatives of wild-type PC12 cells; however, each variant is tetraploid, whereas the wild-type parent is diploid. The two variants contain an altered level and composition of lactate dehydrogenase isoenzymes, which could account for a previously described difference in lactate metabolism. Both variants exhibit a partial loss of transformed phenotype in culture in that they are nonrefractile, grow in monolayers, and fail to multiply in soft agar. We suggest that this alteration in transformed phenotype may result in altered mitochondria and lactate dehydrogenase and thus account for their resistance to MPTP. Compared with wild-type PC12 cells, MPT1 cells have a decreased level of fos mRNA and an increased level of myc mRNA; the latter results from an increased level of transcription of exon 1 of the myc gene. Studies with hybrid cells obtained by fusing MPT1 cells with wild-type-like cells show that most, but not all, of the parameters of the MPT1 phenotype predominate.

There are five lactate dehydrogenase (LDH) isoenzymes, composed of various combinations of two types of subunits. LDH-5, which contains only the LDH A subunit, is known to be present in both the cytoplasm and the nucleus, to act as a single-stranded DNA-binding protein possibly functioning in transcription and/or replication, and to undergo phosphorylation of tyrosine 238 in approximately 1% of the enzyme after cell transformation by certain tumor viruses. We have characterized LDH from wild-type PC12 pheochromocytoma cells and from a PC12 variant (MPT1) that exhibits altered lactate metabolism and altered expression of multiple genes. Wild-type and MPT1 cells contain different proportions of LDH isoenzymes, with LDH-5 being more predominant in wild-type cells than in the variant. A small fraction of LDH from PC12 cells contains phosphotyrosine. Approximately 99% of the total LDH activity is located in the cytoplasm, but all of the phosphotyrosine-containing LDH is located in the nucleus. Furthermore, essentially all of the nuclear LDH contains phosphotyrosine. These results suggest that tyrosine phosphorylation can affect its role in the nucleus.

L‐2‐aminobutyric acid (L‐2‐ABA) is a chiral precursor for the synthesis of anti‐epileptic drug levetiracetam and anti‐tuberculosis drug ethambutol. Asymmetric synthesis of L‐2‐ABA by leucine dehydrogenases has been widely developed. However, the limitations of natural enzymes, such as poor stability, low catalytic efficiency, and inhibition of high‐concentration substrates, limit large‐scale applications. Herein, by directed screening of a metagenomic library from unnatural amino acid‐enriched environments, a robust leucine dehydrogenase, TvLeuDH, was identified, which exhibited high substrate tolerance and excellent enzymatic activity towards 2‐oxobutyric acid. In addition, TvLeuDH has strong affinity for NADH. Subsequently, a three‐enzyme co‐expression system containing L‐threonine deaminase, TvLeuDH, and glucose dehydrogenase was established. By optimizing reaction conditions, 1.5 M L‐threonine could be converted to L‐2‐ABA with a 99% molar conversion rate and a space‐time yield of 51.5 g·L−1·h−1. In this process, no external coenzyme was added. The robustness of TvLeuDH allowed the reaction to be performed without the addition of extra salt as the buffer, demonstrating the simplest reaction system currently reported. These unique properties for the efficient and environmentally friendly production of chiral amino acids make TvLeuDH a particularly promising candidate for industrial applications, which reveals the great potential of directed metagenomics for industrial biotechnology.