Key Regulatory Enzyme Of Glycolysis Research Articles

Contractile Activity Modifies Fru-2,6-P2 Metabolism in Rabbit Fast Twitch Skeletal Muscle

Modification of muscular contractile patterns by denervation and chronic low frequency stimulation induces structural, physiological, and biochemical alterations in fast twitch skeletal muscles. Fructose 2,6-bisphosphate is a potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. The concentration of Fru-2,6-P(2) depends on the activity of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which catalyzes the synthesis and degradation of this metabolite. This enzyme has several isoforms, the relative abundance of which depends on the tissue metabolic properties. Skeletal muscle expresses two of these isoforms; it mainly contains the muscle isozyme (M-type) and a small amount of the liver isozyme (L-type), whose expression is under hormonal control. Moreover, contractile activity regulates expression of muscular proteins related with glucose metabolism. Fast twitch rabbit skeletal muscle denervation or chronic low frequency stimulation can provide information about the regulation of this enzyme. Our results show an increase in Fru-2,6-P(2) concentration after 2 days of denervation or stimulation. In denervated muscle, this increase is mediated by a rise in liver PFK-2/FBPase-2 isozyme, while in stimulated muscle it is mediated by a rise in muscle PFK-2/FBPase-2 isozyme. In conclusion, our results show that contractile activity could alter the expression of PFK-2/FBPase-2.

Inhibition of platelet-derived growth factor BB-induced expression of glyceraldehyde-3-phosphate dehydrogenase by sodium butyrate in rat vascular smooth muscle cells.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key regulatory enzyme of glycolysis, which exists in nuclei and functions as a DNA-binding protein as well as a nuclear protein, appears to be modulated by cellular activities. Exposure of quiescent rat smooth muscle cells (SMCs) to platelet-derived growth factor BB (PDGF-BB), which stimulates SMCs proliferation, caused a time-dependent increase in mRNA for GAPDH and its catalytic activity. Treatment of quiescent SMCs with sodium butyrate (SB), which is shown to inhibit PDGF-BB-induced SMC proliferation, caused a time- and concentration-dependent decrease in PDGF-BB-induced GAPDH mRNA expression and its catalytic activity. Nuclear run-on studies revealed that the PDGF-BB-induced rate of GAPDH gene transcription was reduced by about 50% in the presence of 5 mmol/L SB. The protein synthesis inhibitor, cycloheximide, failed to abolish the SB-inhibited PDGF-BB-induced rate of transcription of GAPDH, suggesting that SB is not dependent on ongoing protein synthesis to exert its effects on PDGF-BB-induced GAPDH transcription. Furthermore, measurement of GAPDH mRNA stability at various times after the inhibition of transcription with actinomycin D indicated that 5 mmol/L SB has no significant effect on the half-life of PDGF-BB-induced mRNA. The reduction in PDGF-BB-induced GAPDH expression by SB is probably caused by a cycloheximide-insensitive transcriptional mechanism. Thus, the inhibition of PDGF-BB-induced expression of GAPDH by SB suggests a link between SMC proliferation, energy consumption, and GAPDH gene upregulation.

Muscle phosphofructokinase deficiency in two generations

Phosphofructokinase (PFK) is the key regulatory enzyme of glycolysis. Patients lacking the muscular isoform of PFK typically present with myopathy and compensated hemolysis (glycogenosis type VII or Tarui's disease). Since 1965 about 30 cases of muscular PFK deficiency have been reported. In most cases family history suggests a recessive inherited trait. We describe a family of Ashkenazi Jewish origin with two members in subsequent generations suffering from muscular PFK deficiency. The propositus, a 19-year-old male patient, presented with weakness, myalgias and exercise intolerance since early infancy. His father also had early fatigue on exercise with myalgias; the mother and a 12-year-old brother were asymptomatic. Muscle biopsy of both the propositus and his father showed increased glycogen storage and absent histochemical stain for PFK. Biochemical studies of muscle revealed a markedly decreased PFK activity and DNA analysis of the muscle PFK gene revealed compound heterozygosity in both cases. This is the first description of proven muscle PFK deficiency (glycogenosis type VII) in two subsequent generations.

6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog skeletal muscle: purification, kinetics and immunological properties.

Fructose 2,6-bisphosphate is the most potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. This study was prompted by the finding that the content of fructose 2,6-bisphosphate in frog skeletal muscle was dramatically increased at the initiation of exercise and was closely correlated with the glycolytic flux during exercise. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme system catalyzing the synthesis and degradation of fructose 2,6-bisphosphate, was purified from frog (Rana esculenta) skeletal muscle and its properties were compared with those of the rat muscle type enzyme expressed in Escherichia coli using recombinant DNA techniques. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was purified 5600-fold. 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities could not be separated, indicating that the frog muscle enzyme is bifunctional. The enzyme preparation from frog muscle showed two bands on sodium dodecylsulphate polyacrylamide gel electrophoresis. The minor band had a relative molecular mass of 55,800 and was identified as a liver (L-type) isoenzyme. It was recognized by an antiserum raised against a specific amino-terminal amino acid sequence of the L-type isoenzyme and was phosphorylated by the cyclic AMP-dependent protein kinase. The major band in the preparations from frog muscle (relative molecular mass = 53,900) was slightly larger than the recombinant rat muscle (M-type) isoenzyme (relative molecular mass = 53,300). The pH profiles of the frog muscle enzyme were similar to those of the rat M-type isoenzyme, 6-phosphofructo-2-kinase activity was optimal at pH 9.3, whereas fructose-2,6-bisphosphatase activity was optimal at pH 5.5. However, the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle differed from other M-type isoenzymes in that, at physiological pH, the maximum activity of 6-phosphofructo-2-kinase exceeded that of fructose-2,6-bisphosphatase, the activity ratio being 1.7 (at pH 7.2) compared to 0.2 in the rat M-type isoenzyme. 6-Phosphofructo-2-kinase activity from the frog and rat muscle enzymes was strongly inhibited by citrate and by phosphoenolpyruvate whereas glycerol 3-phosphate had no effect. Fructose-2,6-bisphosphatase activity from frog muscle was very sensitive to the non-competitive inhibitor fructose 6-phosphate (inhibitor concentration causing 50% decrease in activity = 2 mumol.l-1).(ABSTRACT TRUNCATED AT 400 WORDS)

A quantitative evaluation of the effect of enzyme complexes on the glycolytic rate in vivo: Mathematical modeling of the glycolytic complex

The cellular distribution of free and bound glycolytic enzymes in vivo was estimated by means of a model based on previously determined association constants for individual binding interactions and in vivo protein concentrations. The calculations revealed that a significant proportion of the enzymes would be either associated with F-actin, or bound in binary enzyme-enzyme complexes in vivo. An analysis of the relative concentration, and relative activity, of F-actin-bound enzymes suggested that a complete glycolytic complex, composed of all enzymatic steps from phosphofructokinase (PFK) to lactate dehydrogenase (LDH) does not exist. This was indicated by a very low concentration of F-actin-associated phosphoglycerate kinase (PGK) and by a very low activity of F-actin bound aldolase and PGK; this model showed that aldolase and PGK would be absent from any F-actin bound complex. An analysis of soluble enzyme-enzyme associations indicated that formation of binary enzyme complexes may lead to an increased overall flux through glyceraldehyde 3-phosphate dehydrogenase and LDH, but would serve to decrease flux through PFK and aldolase. A 1.4-fold activation of PFK, which occurs when the soluble enzyme binds to F-actin, suggested that reversible binding of PFK to F-actin may represent a novel cellular mechanism for controlling glycolytic flux during periods of increased metabolic demand by controlling the key regulatory enzyme of glycolysis.

Phosphofructokinase activity in the brain in Alzheimer's disease

The activity of phosphofructokinase, a key regulatory enzyme of glycolysis, was determined in neurosurgical samples of neocortex obtained from 6 patients with primary degenerative dementia, including 4 with Alzheimer's disease. No consistent reductions were seen compared with samples from nondemented controls. These results contrast with the greatly reduced activity reported previously for postmortem brain specimens from patients with Alzheimer's disease.

Regional assignment of human liver-type 6-phosphofructokinase to chromosome 21q22.3 by using somatic cell hybrids and a monoclonal anti-L antibody.

The three structural loci encoding human phosphofructokinase, a key regulatory enzyme of glycolysis, are located on separate chromosomes. The gene coding for the liver-type subunit PFKL has previously been assigned to chromosome 21. We have used a subunit- and human-specific monoclonal antibody to liver PFK to detect the expression of human PFKL in hamster X human hybrid cell lines. A cell line carrying an 8;21 translocation which contains all of chromosome 21 except the band 21q22.3 was negative for the expression of PFKL whereas cell lines carrying the reciprocal 8;21 translocation were positive. In addition, a cell line with a ring chromosome 21 containing a breakpoint which excluded the distal part of the q22.3 band was negative for expression of PFKL. These results indicate that human PFKL is located on chromosome 21q22.3.

Phosphoenolpyruvate and 2-phosphoglycerate: endogenous energy source(s) for sugar accumulation by starved cells of Streptococcus lactis.

In the absence of an exogenous energy source, galactose-grown cells of Streptococcus lactis ML3 rapidly accumulated thiomethyl-beta-D-galactopyranoside (TMG) and 2-deoxyglucose to intracellular concentrations of 40 to 50 mM. Starved cells maintained the capacity for TMG uptake for many hours, and accumulation of the beta-galactoside was insensitive to proton-conducting ionophores (tetrachlorosalicylanilide and carbonylcyanide-m-chlorophenyl hydrazone) and sulfydryl group reagents including iodoacetate and N-ethylmaleimide. Fluorimetric analysis of glycolytic intermediates in extracts prepared from starved cells revealed (a) high intracellular levels of phosphoenolpyruvate (13 mM; PEP) and 2-phosphoglycerate (approximately 39 mM; 2-PG), but an absence of other metabolites including glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, and triosephosphates. The following criteria showed PEP (and 2-PG) to be the endogenous energy source for TMG accumulation by the phosphotransferase system: the intracellular concentrations of PEP and 2-PG decreased with concomitant uptake of TMG, and a close correlation was observed between maximum accumulation of the beta-galactoside and the total available concentration of the two intermediates; TMG accumulated as an anionic derivative, which after extraction and incubation with alkaline phosphatase (EC 3.1.3.1) formed the original analogue; fluoride inhibition of 2-phospho-D-glycerate hydrolyase (EC 4.2.1.11) prevented the conversion of 2-PG to PEP, and uptake of TMG by the starved cells was reduced by 80%; and the stoichiometric ratio [TMG] accumulated/[PEP] consumed was almost unity (0.93). In cells metabolizing glucose, all intermediates listed in (a) and (b) were found. Upon exhaustion of glucose from the medium, the metabolites in (b) were not longer detectable, while the intracellular concentrations of PEP and 2-PG increased to the levels previously observed in starved cells. The glycolytic intermediates in (b) are all in vitro heterotropic effectors of pyruvate kinase (adenosine 5'-triphosphate:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from S. lactis ML3. It is suggested that the capacity of starved cells to maintain high intracellular concentrations of PEP and 2-PG is a consequence of decreased in vivo activity of this key regulatory enzyme of glycolysis.

Elevation of arterial phosphofructokinase activity associated with susceptibility to atherosclerosis in pigeons

The activity of phosphofructokinase (PFK, 2.7.1. 11) was measured in arteries of very young (5–8 week old) pigeons known to differ in susceptibility to atherosclerosis. The activity of the arterial enzyme was significantly higher in the atherosclerosis susceptible White Carneau (WC) pigeons than in the resistant Show Racers (SR). The difference was significant whether enzyme activity was calculated on the basis of extract protein, DNA content or fat-free dry weight. In the White Carneau arteries the activity of the enzyme was higher in the female than the male pigeons. PFK is a key regulatory enzyme of glycolysis and is subject to fine control adjustments. A low ATP/ADP ratio and a fall in citrate concentration, as, for example, induced by hypoxia, are mediators of a feedback mechanism leading to a rise in PFK activity and enhancement of glycolysis for energy production. This mechanism appears to be the cause of the higher PFK activity in the WC arteries, because related studies indicate impaired Krebs cycle activity in these vessels. It is suggested that the increased dependence of the WC arteries on glycolysis facilitates the development of atherosclerosis in this pigeon strain and that the mechanism is similar to the mechanism by which tissue hypoxia causes lipid accumulation and connective tissue alterations in the arterial wall.