A radioassay for phosphofructokinase-1 activity in cell extracts and purified enzyme

Sola‐Penna, Mauro; Santos, Ana Cristina dos; Alves, Gutemberg Gomes; El‐Bacha, Tatiana; Faber-Barata, Joana; Pereira, Monica Farah; Serejo, Fredson Costa; Poian, Andrea T. Da; Sorenson, Martha M.

doi:10.1016/s0165-022x(01)00180-4

Cited by 46 publications

(25 citation statements)

References 25 publications

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“…Radiometric Assay for PFK-1 Activity-PFK-1 activity was measured directly using a radiometric assay similar to that described previously (20) Identification of the Palmitoylation Sites of PFK-1 by Mass Spectrometry-Purified rabbit muscle PFK-1 (10 M) was incubated in the presence or absence of a stoichiometric amount of palmitoyl-CoA for 0 -60 min. PFK-1 treated with palmitoylCoA was then precipitated as described previously (22).…”

Section: Methodsmentioning

confidence: 99%

Reversible High Affinity Inhibition of Phosphofructokinase-1 by Acyl-CoA

Jenkins

Yang

Sims

et al. 2011

Journal of Biological Chemistry

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The enzyme phosphofructokinase-1 (PFK-1) catalyzes the first committed step of glycolysis and is regulated by a complex array of allosteric effectors that integrate glycolytic flux with cellular bioenergetics. Here, we demonstrate the direct, potent, and reversible inhibition of purified rabbit muscle PFK-1 by low micromolar concentrations of long chain fatty acyl-CoAs (apparent K i ϳ1 M). Collectively, these results demonstrate that fatty acyl-CoA modulates phosphofructokinase activity through both covalent and noncovalent interactions to regulate glycolytic flux and enzyme membrane localization via the branch point metabolic node that mediates lipid flux through anabolic and catabolic pathways.Glycolysis occupies a central role in eukaryotic energy metabolism through the production of NADH and ATP to meet immediate cellular energy demands, the generation of glycerol 3-phosphate for the de novo synthesis of phospholipids and triglycerides, and the production of pyruvate that is converted to acetyl-CoA for subsequent utilization in the TCA cycle or for de novo fatty acid biosynthesis. Thus, glycolysis and lipid metabolic flux are interwoven metabolic networks that must be coordinately regulated to maintain cellular bioenergetic homeostasis. Although the ability of fatty acids to suppress glycolytic flux has been known for 50 years (1-4), the mechanisms by which metabolically active tissues undergo a rapid shift from glucose utilization to fatty acid oxidation during metabolic transitions remain incompletely understood. The increasing prevalence of obesity, diabetes, and the metabolic syndrome, in which there is increased reliance on fatty acid substrate at the expense of glucose (5-8), has renewed interest in the chemical mechanisms regulating substrate utilization during metabolic transitions.Previous work has suggested that glycolysis and fatty acid ␤-oxidation are coordinately regulated by specific metabolites of each bioenergetic network (9 -11). A prevailing hypothesis to explain the regulation of glycolysis by fatty acids originally put forth by Randle and co-workers (3, 4) states that the availability and subsequent ␤-oxidation of fatty acids results in the production of tricarboxylic acid cycle intermediates that down-regulate glycolysis. Specifically, acetyl-CoA generated from fatty acid ␤-oxidation leads to the accumulation of citrate that is a potent inhibitor of phosphofructokinase, which catalyzes the rate-determining step in glycolysis. However, substantial experimental evidence suggests that the regulation of glycolytic flux involves the integration of complex networks that coordinately integrate metabolic transitions.Mammalian phosphofructokinase-1 (6-phosphofructo 1-kinase 3 ) catalyzes the first committed and rate-determining step of glycolysis and thus represents an essential metabolic control point or node for carbohydrate utilization. Accordingly, PFK-1 is subject to complex catalytic and allosteric regulation by multiple cellular metabolites, including AMP, ADP, ATP, fructose 2,6-bisphosph...

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Section: Methodsmentioning

confidence: 99%

Reversible High Affinity Inhibition of Phosphofructokinase-1 by Acyl-CoA

Jenkins

Yang

Sims

et al. 2011

Journal of Biological Chemistry

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“…Heart HK and PFK activities were measured as described previously (28) with modifications introduced in refs. 13, 29.…”

Section: Assay For Hk and Pfk Activitymentioning

confidence: 99%

Metformin reverses hexokinase and phosphofructokinase downregulation and intracellular distribution in the heart of diabetic mice

Silva¹,

Ausina²,

Alencar³

et al. 2012

IUBMB Life

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SummaryDiabetes mellitus is characterized by hyperglycemia and its associated complications, including cardiomyopathy. Metformin, in addition to lowering blood glucose levels, provides cardioprotection for diabetic subjects. Glycolysis is essential to cardiac metabolism and its reduction may contribute to diabetic cardiomyopathy. Hexokinase (HK) and phosphofructokinase (PFK), rate-limiting enzymes of glycolysis, are downregulated in cardiac muscle from diabetic subjects, playing a central role on the decreased glucose utilization in the heart of diabetic subjects. Thus, the aim of this study was to determine whether metformin modulates heart HK and PFK from diabetic mice. Diabetes was induced by streptozotocin injection on male Swiss mice, which were treated for three consecutive days with 250 mg/kg metformin before evaluating HK and PFK activity, expression, and intracellular distribution on the heart of these subjects. We show that metformin abrogates the downregulation of HK and PFK in the heart of streptozotocin-induced diabetic mice. This effect is not correlated to alteration on the enzymes' transcription and expression. However, the intracellular distribution of both enzymes is altered in diabetic hearts that show increased activity of the soluble fraction when compared to the particulate fraction. Moreover, this pattern is reversed upon the treatment with metformin, which is correlated with the effects of the drug on the enzymes activity. Altogether, our results support evidences that metformin alter the intracellular localization of HK and PFK augmenting glucose utilization by diabetic hearts and, thus, conferring cardiac protection to diabetic subjects.

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“…HK and PFK enzymatic activities were assessed by the radiometric method described by Sola-Penna et al [13] with the modifications proposed by Zancan and Sola-Penna [14,15]. This assay was performed at 37°C in a 0.4-ml reaction system containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl 2 and [c-32 P]ATP (4 lCi/lmol).…”

Section: Enzymatic Activity Assaysmentioning

confidence: 99%

Lactate downregulates the glycolytic enzymes hexokinase and phosphofructokinase in diverse tissues from mice

et al. 2010

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a b s t r a c tWe examined the effects of lactate on the enzymatic activity of hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) in various mouse tissues. Our results showed that lactate inhibited PFK activity in all the analyzed tissues. This inhibitory effect was observed in skeletal muscle even in the presence of insulin. Lactate directly inhibited the phosphorylation of PFK tyrosine residues in skeletal muscle, an important mechanism of the enzyme activation. Moreover, lactate indirectly inhibited HK activity, which resulted from its cellular redistribution, here attributed to alterations of HK structure. PK activity was not affected by lactate. The activity of HK and PFK is directly related to glucose metabolism. Thus, it is conceivable that lactate exposure can induce inhibition of glucose consumption in tissues.

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A radioassay for phosphofructokinase-1 activity in cell extracts and purified enzyme

Cited by 46 publications

References 25 publications

Reversible High Affinity Inhibition of Phosphofructokinase-1 by Acyl-CoA

Reversible High Affinity Inhibition of Phosphofructokinase-1 by Acyl-CoA

Metformin reverses hexokinase and phosphofructokinase downregulation and intracellular distribution in the heart of diabetic mice

Lactate downregulates the glycolytic enzymes hexokinase and phosphofructokinase in diverse tissues from mice

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