Pyruvate Kinase M1 Research Articles

Transcriptional stimulation by thyroid hormone of a cytosolic thyroid hormone binding protein which is homologous to a subunit of pyruvate kinase M1.

We have recently shown that the monomer of rat pituitary pyruvate kinase subtype M1 (p58-M1) is a cytosolic binding protein for 3,3',5-triiodo-L-thyronine (T3). To understand the role p58-M1 plays in thyroid hormone action, we examined the regulation of p58-M1 by T3 in GH3 cells. Expression of p58-M1 was evaluated by metabolically labeling GH3 cells cultured in regular medium, thyroid hormone depleted medium (Td medium), or Td medium supplemented with T3 (Td + T3 medium) followed by immunoprecipitation. T3 stimulates the expression of p58-M1 by 2-fold. Analysis by pulse-chase experiments indicates that the increased expression is not due to the increase of stability of p58-M1. Northern analysis of mRNA prepared from cells cultured in regular, Td, or Td + T3 medium demonstrates that T3 increases the accumulation of cytoplasmic mRNA by 2-fold. Nuclei from cells cultured in the three conditions were prepared, and the rates of synthesis of nascent nuclear RNA were compared by an in vitro transcription assay. Addition of T3 stimulates the rate of transcription by 2-fold. The parallel and identical magnitude in the increase of transcription rate and the accumulation of mRNA indicates that T3 stimulates the synthesis of p58-M1 by increasing the transcriptional activity of its gene.

Isolation and characterization of the human pyruvate kinase M gene

Genomic clones containing the human pyruvate kinase M (PKM) gene, which encodes the M1-type and M2-type isozymes, were isolated and their exon sequences were determined. The gene is approximately 32 kb and consists of 12 exons and 11 introns. Exons 9 and 10 contain sequences specific to the M1 and M2 types, respectively, indicating that the human isozymes are produced from the same gene by alternative splicing as in the case of the rat gene. The exon-intron structure of the human PKM gene is identical to that of the rat gene, and the introns of both genes interrupt the exons at the same points. Introns 6 and 7 begin with GC dinucleotide instead of the consensus GT, but the other exon-intron boundaries are consistent with the GT-AG rule. The gene is transcribed from multiple start sites. The 5'-flanking region of the gene contains putative Sp1-binding sites, but no TATA box or CAAT box, and shows high sequence similarity to that of the rat M gene. Bacterial chloramphenicol acetyltransferase assay revealed that the upstream region between positions -493 and -51 contained a cis-acting element(s) that was essential for expression of the M gene in HeLa cells. Long stretches of conserved regions were found in the introns around the M1-specific and M2-specific exons, suggesting that these regions may be involved in the alternative splicing machinery.

Purified pyruvate kinases type M2 from unfertilized hen's egg are substrates of protein kinase C.

To characterize pyruvate kinase isoenzymes from cells with the capability to proliferate, this enzyme was purified from yolk and vitelline membrane of unfertilized hen's egg. Pyruvate kinase type M2 from vitelline membrane was obtained in a homogeneous form after a 1150-fold purification to a specific enzymatic activity of 450 mumol X min-1 X mg-1. It was saturated half-maximally with phosphoenolpyruvate at KPPrv0.5 = 0.36 mM phosphoenolpyruvate and was activity by fructose 1,6-bisphosphate and L-serine at suboptimal substrate concentrations. After 11 000-fold purification to a specific enzymatic activity of 60 mumol X min-1 X mg-1, the pyruvate kinase isoenzymes type M2 (KPPrv0.5 = 0.32 mM) and M1 (KPPrv0.5 = 0.04 mM) were obtained from the yolk substance. Kinetic differences were noted between the pyruvate kinase type-M2 isoenzymes from vitelline membrane and yolk. A comparison of the amino acid composition of the purified pyruvate kinase isoenzymes from hen's egg revealed that all isoenzymes were related to pyruvate kinase type M1 from chicken breast muscle. The M2-type isoenzyme from vitelline membrane was related to the M2-type isoenzyme from chicken tumors, but was not related to the M2-type pyruvate kinase from chicken lung or liver. Protein kinase C from chicken oviduct phosphorylated in vitro both pyruvate kinase M2 isoenzymes from the unfertilized hen's egg preferably at serine and less at threonine residues. Pyruvate kinase type M1 from egg yolk was a weak substrate of protein kinase C. An activation of pyruvate kinase type M2 from vitelline membrane was observed at suboptimal concentrations of phosphoenolpyruvate under the conditions of phosphorylation, in the presence of phosphatidylserine.

Fluorometric assay of amino acids and amines by use of 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole(NBD-F) in high-performance liquid chromatography

The reaction of a newly developed fluoregenic reagent, 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole(NBD-F), with amino acids and biogenic amines was investigated. NBD-F was reactive to both primary and secondary amines including amino acids and biogenic amines such as catecholamines. The amino acids were reacted with the reagent, separated by high-performance liquid chromatography on μ-Bondapak C18 and detected at 10 to 100 fmol level. A few μg of protein hydrolysates, rabbit pyruvate kinase M1, rabbit aldolase A and papain, were adequate for the amino acids quantitation. An automatic amino acid analyzer with fluorometric detection by the post-column derivatization with NBD-F enabled the amino acid profile analysis in blood samples present in a paper disc of 3 mm diameter.

Immunohistochemical localization of pyruvate kinase isoenzymes in chicken tissues

A method for the localization of pyruvate kinase isoenzymes type L, M2 and M1 in tissue sections is described. Mono-specific antibodies directed against isoenzymes of pyruvate kinase from chicken and the peroxidase antiperoxidase method were used. The following preferential localizations of the isoenzymes in chicken tissues were observed: Pyruvate kinase M1 was found in skeletal muscle. The white muscle fibers were more intensely stained than the red. Some dark muscles (e.g., anterior latissimus dorsi) and the heart muscle showed no reaction with antiserum against pyruvate kinase M1. Pyruvate kinase type L was found in the hepatocytes and in kidney cortex. Pyruvate kinase type M2 was seen in the distal tubules of kidney, in hepatocytes and sinusoidal cells in liver, in lung, adipose tissue, and in the spleen mainly in the bursa dependent areas. Pyruvate kinase type M2 was detected in high concentrations in the granulation tissue of regenerating liver after partial hepatectomy. Liver sections of a hen bearing a pancreatic tumor showed an unusually high content of pyruvate kinase type M2 in some hepatocytes, which were each clustered to spots in the liver parenchyma. Thus, contrary to previous reports, the tissue distribution of isoenzymes in chicken is similar to that of other vertebrates.