ABSTRACT
Phosphofructokinase-M (PFKM) is a key enzyme in glycolysis. The expression and activity of PFKM is closely related to the occurrence and development of malignant tumors, but its role in the regulation of renal cell carcinoma (RCC) is still unknown. We found that the expression of PFKM was lower in RCC tumor tissue than in adjacent normal tissues, and that low expression of PFKM was related to the poor overall survival of RCC patients. In addition, our results showed that FOXO3 mediated PFKM inhibited the growth, migration and invasion of RCC cells, suggesting that PFKM is a protective factor for RCC.
Subject(s)
Carcinoma, Renal Cell/metabolism , Forkhead Box Protein O3/metabolism , Kidney Neoplasms/metabolism , Phosphofructokinase-1, Muscle Type/metabolism , Signal Transduction , Carcinoma, Renal Cell/diagnosis , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Forkhead Box Protein O3/analysis , Humans , Kidney Neoplasms/diagnosis , Kidney Neoplasms/pathology , Phosphofructokinase-1, Muscle Type/analysis , PrognosisABSTRACT
Longissimus muscle samples from the pig genotypes Duroc (Du), Pietrain (MHS homozygote negative (PiNN), positive (PiPP)) and a Duroc-Pietrain crossbreed (DuPi) were analyzed. The PiPP samples showed a faster pH drop and higher electrical conductivity, drip loss and lightness values. Before slaughter the concentrations of the adenine nucleotides were comparable between the genotypes, but 40 min after slaughter (p.m.) the ATP concentrations decreased and IMP increased, to a higher extent in the PiPP pigs. The nucleotide values of the 12 h p.m. samples were again comparable. Activities of glycogen phosporylase (GP), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) were nearly similar before slaughter. Forty minutes after slaughter the LDH activities increased in all pigs and the PFK activities in all genotypes but not in the PiPP. GP results were rather inconsistent indicating an earlier activation of this enzyme. The study showed that the reduced meat quality in the PiPP pigs is accompanied with rapid ATP degradation and accelerated enzyme activation.
Subject(s)
Adenine Nucleotides/analysis , Glycogen Phosphorylase/metabolism , L-Lactate Dehydrogenase/metabolism , Meat , Muscle, Skeletal/enzymology , Phosphofructokinase-1, Muscle Type/metabolism , Adenosine Triphosphate/metabolism , Animals , Electric Conductivity , Genotype , Glycogen Phosphorylase/analysis , L-Lactate Dehydrogenase/analysis , Mutation , Phosphofructokinase-1, Muscle Type/analysis , Ryanodine Receptor Calcium Release Channel/genetics , Ryanodine Receptor Calcium Release Channel/metabolism , Swine/classification , Swine/genetics , Swine/physiologyABSTRACT
Phosphofructokinase-1 plays a key role in the regulation of carbohydrate metabolism. Its activity can be used as an indicator of the glycolytic flux in a tissue sample. The method most commonly employed to determine phosphofructokinase-1 activity is based on oxidation of NADH by the use of aldolase, triosephosphate isomerase, and alpha-glycerophosphate dehydrogenase. This method suffers from several disadvantages, including interactions of the auxiliary enzymes with phosphofructokinase-1. Other methods that have been used also require auxiliary enzymes or are less sensitive than a coupled assay. Here, we propose a direct method to determine phosphofructokinase-1 activity, without the use of auxiliary enzymes. This method employs fructose-6-phosphate and ATP labeled with 32P in the gamma position ([gamma-32P]ATP), and leads to the formation of ADP and fructose-1,6-bisphosphate labeled with 32P ([1-32P]fructose-1,6-bisphosphate). Activated charcoal is used to adsorb unreacted [gamma-32P]ATP, and the radioactive product in the supernatant, [1-32P]fructose-1,6-bisphosphate, is analyzed on a liquid scintillation counter. The proposed method is precise and relatively inexpensive, and can be applied to determine phosphofructokinase-1 activity in cellular extracts as well as in the purified enzyme.
