scholarly journals Limitations of commonly used spectrophotometric assay methods for phosphoenolypyruvate carboxykinase activity in crude extracts of muscle

1982 ◽  
Vol 206 (1) ◽  
pp. 147-152 ◽  
Author(s):  
D A Duff ◽  
K Snell
Keyword(s):  
Enzyme Activity ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Lactate Production ◽  
Spectrophotometric Assay ◽  
Nadh Oxidation ◽  
Crude Extracts ◽  
Na Ions ◽  
Assay Methods ◽  
Lactate Formation ◽  
Assay Conditions

Phosphoenolpyruvate carboxykinase activity in crude extracts of muscle has frequently been determined by using a continuous spectrophotometric method, which is shown to grossly overestimate enzyme activity. NADH oxidation attributed to phosphoenolpyruvate carboxykinase activity in the assay is due to lactate production. Under the normal assay conditions. Na+ ions stimulate pyruvate kinase, providing pyruvate for lactate formation by lactate dehydrogenase and sufficiently to account for most of the observed NADH oxidation.

Separation of Pinusbanksiana Lamb. glyceraldehyde 3-phosphate (NAD) dehydrogenase from phenolic-based pigments with affinity chromatography

1982 ◽  
Vol 12 (4) ◽  
pp. 1035-1038
Author(s):  
Bruce E. Haissig
Keyword(s):  
Enzyme Activity ◽  
Affinity Chromatography ◽  
Electrophoretic Mobility ◽  
Spectrophotometric Assay ◽  
Disc Electrophoresis ◽  
Enzyme Complex ◽  
Crude Extracts ◽  
Ungerminated Seed ◽  
Catalytically Active ◽  
The Individual

Glyceraldehyde 3-phosphate (NAD) dehydrogenase was extracted from Pinusbanksiana Lamb, seed and from seedlings up to 12 days old. Enzyme activity in crude extracts of seedlings increased markedly from days 2–8 and then decreased slightly, as indicated by spectrophotometric assay. In contrast, staining of acrylamide gels after disc electrophoresis indicated that catalytically active isozymes were not present in crude extracts at and after day 6. Phenolics greatly increased in extracts from days 0–10 and polymerization of these phenolics apparently lead to binding of the resulting phenolic-based pigment to enzyme. The pigment–enzyme complex demonstrated greatly enhanced electrophoretic movement, in comparison with the individual isozymes, which caused frontal migration of the collective isozymes. Affinity chromatography restored electrophoretic mobility of isozymes equal to that obtained with enzyme from ungerminated seed. Enzyme was inseparable from pigment by several other methods.

Phosphoenolpyruvate Carboxykinase in C4 Plants: Its Role and Regulation

1997 ◽  
Vol 24 (4) ◽  
pp. 459 ◽  
Author(s):  
Robert P. Walker ◽  
Richard M. Acheson ◽  
László I. Técsi ◽  
Richard C. Leegood
Keyword(s):  
Molecular Mass ◽  
Malic Enzyme ◽  
Phosphoenolpyruvate Carboxykinase ◽  
C4 Plants ◽  
Panicum Maximum ◽  
C4 Plant ◽  
Cam Plants ◽  
Crude Extracts ◽  
High Concentrations ◽  
Regulatory Properties

Some of the recent findings which revise our view of the role and regulation of phosphoenolpyruvate carboxykinase (PEPCK) in C4 plants are discussed. Evidence is presented that PEPCK is present at appreciable activities in the bundle-sheath of some NADP-malic enzyme-type C4 plants, such as maize, but it was not detectable in NAD-malic enzyme-type C4 plants. PEPCK is rapidly inactivated in crude extracts of leaves of the C4 plant, Panicum maximum. This inactivation could be prevented by high concentrations of dithiothreitol or by the inclusion of ADP or ATP, suggesting the involvement of thiols at the active site. PEPCK is also subject to rapid proteolysis in crude extracts of a range of C4 plants, resulting in cleavage to a smaller (62 kDa) form. This can be reduced by extraction at high pH and by the inclusion of SDS, but it means that intact PEPCK has never been purified from a C4 plant. The molecular mass of PEPCK varies considerably in C4 plants, unlike C3 and CAM plants in which it is usually 74 kDa. PEPCK is phosphorylated during darkness (and reversed by light) in some C4 plants with PEPCK of a larger molecular mass, such as Panicum maximum (71 kDa), but it was not phosphorylated in the PEPCK-type C4 plant, Sporobolus pyramidalis (69 kDa). The known regulatory properties of PEPCK are discussed in relation to its role in C4 photosynthesis, in particular its sensitivity to regulation by adenylates and by Mn2+.

Carbohydrate metabolism by primary cultures of rabbit proximal tubules

1989 ◽  
Vol 256 (3) ◽  
pp. C532-C539 ◽  
Author(s):  
M. J. Tang ◽  
K. R. Suresh ◽  
R. L. Tannen
Keyword(s):  
Pyruvate Kinase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Lactate Production ◽  
Proximal Tubules ◽  
Glycolytic Metabolism ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular

Renal proximal tubular epithelia were used to assess the factors responsible for the induction of glycolysis in cultured cells. Primary cultures of rabbit proximal tubules, which achieved confluency at 6 days, exhibited hormonal responsiveness and brush-border characteristics typical of proximal tubular cells. Beginning at day 4, these cultured cells exhibited increased glycolytic metabolism reflected by enhanced glucose uptake and lactate production, along with parallel increases in activity of the glycolytic enzymes, pyruvate kinase and lactate dehydrogenase. The gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-bisphosphatase (FDP), were downregulated, and the cultured cells exhibited lower oxygen consumption rates than fresh tubules. Cells grown on a rocker, to mitigate hypoxia, exhibited a metabolic and enzymatic profile similar to cells grown under still conditions. ATP levels in cultured cells were higher than in fresh tubules. Furthermore, pyruvate kinase activity was higher in cells grown in media containing 0.5 as contrasted with 25 mM glucose. The enhanced glycolytic metabolism exhibited by cultured proximal tubular cells appears to be a characteristic of proliferation and is not a response to hypoxia, the Pasteur effect, or environmental glucose.

scholarly journals Sporting myths: the REAL role of lactate during exercise

2016 ◽  
Vol 19 (5) ◽  
Author(s):  
T Mann
Keyword(s):  
Lactate Concentration ◽  
Lactate Production ◽  
Blood Lactate Concentration ◽  
Endurance Performance ◽  
Post Exercise ◽  
Early Exercise ◽  
Important Intermediate ◽  
Lactate Formation ◽  
Exercise Muscle

Background. Lactate or, as it was customarily known, ‘lactic acid’ was one of the first molecules to attract the attention of early exercise scientists, mainly because blood lactate concentration could be measured and was shown to increase with increasing exercise intensity. This connection resulted in lactate being associated with numerous other events associated with high-intensity exercise including muscle cramps, fatigue, acidosis and post-exercise muscle soreness. Nobel prize-winning research by AV Hill and Otto Meyerhof provided a rational explanation linking lactate to anaerobiosis and acidosis, which resulted in this relationship being widely accepted as fact. It was only following isotopic tracer studies of George Brooks and others that the true role of lactate during rest and exercise was revealed. Conclusions. Lactate is now acknowledged as an important intermediate of carbohydrate metabolism, taken up from the blood by tissues such as skeletal and cardiac muscle as a substrate for oxidation. Furthermore, lactate formation consumes a proton, thereby buffering against muscle acidosis. For this reason, lactate production forms an essential aid to endurance performance rather than a hindrance.

scholarly journals Energy Requirement for Maintenance of Adenylate Energy Charge and Metabolic ATP in Platelets During Starvation

1977 ◽  
Author(s):  
J.W.N. Akkerman ◽  
G. Gorter ◽  
J.J. Sixma
Keyword(s):  
Steady State ◽  
Adenine Nucleotides ◽  
Energy Requirement ◽  
Energy Charge ◽  
Lactate Production ◽  
Stable Steady State ◽  
Adenylate Energy Charge ◽  
Consumption Energy ◽  
Steady State Levels ◽  
Lactate Formation

Energy requirements for maintenance of stable adenylate energy charge (AEC) and metabolic ATP(ATP-m)level were studied in gel filtered platelets at various degrees of starvation. Platelets gel filtered and subsequently incubated during 40 min.at 37°C with 1mM CN- and without glucose consumed their glycogen at a rate of 0.79 ± 0.23(± SD, n=6)/μmol glycosyl residues .min-1 10-11 cells. During this period AEC and ATP-m decreased linearly with time at rates of 5-6.10-3 and 0.75-1.05% of total radioactive adenine nucleotides .min-1.10-11 cells respectively. Addition of 25–1000μM glucose increased lactate production and decreased the fall of AEC and ATP-m proportional to the amounts of glucose added. Glycogenolysis remained active below 100μM glucose but ceased at higher glucose concentrations. From these data ATP-m production from glycogenolysis and glycolysis was calculated and compared with the decrease of steady state levels of AEC and ATP-m. A production of 3μmol ATP-m.min-1.10-11 cells was required to maintain initial AEC and ATP-m level. At lower rates of ATP-m production these values fell without reaching stable steady state levels in a lower range. After 40-50 min variations in AEC and ATP-m ceased and lactate formation stopped leaving the cells in a state of hybernation. Subsequent addition of glucoserestored lactate accumulation, AEC and ATP-m. On the basis of formation and steady state levels of ATP-m its consumption was calculated. A lowering production was not completely met by a lowering consumption. Energy consumption in resting platelets is therefore partly independent from energy production.

scholarly journals Hereditary Nonspherocytic Hemolysis With Erythrocyte Phosphofructokinase Deficiency

Blood ◽  
1972 ◽  
Vol 39 (3) ◽  
pp. 415-425 ◽  
Author(s):  
Larry Waterbury ◽  
Eugene P. Frenkel
Keyword(s):  
Enzyme Activity ◽  
Adenosine Triphosphate ◽  
Lactate Production ◽  
Kinetic Studies ◽  
Glycogen Storage ◽  
Muscle Disease ◽  
Phosphofructokinase Activity ◽  
Phosphofructokinase Deficiency

Abstract Hereditary nonspherocytic hemolysis associated with abnormal erythrocyte phosphofructokinase activity was demonstrated in a young man. Enzyme activity in the propositus, his mother, and maternal grandmother was approximately 60% of normal controls. There was markedly increased lability of enzyme activity on in vitro storage. Kinetic studies revealed increased sensitivity to adenosine triphosphate inhibition. Erythrocyte adenosine triphosphate levels were depressed. The absence of muscle disease and the presence of normal in vivo lactate production following ischemic exercise differentiated this kindred from those with Type VII glycogen storage disease.

Effect of Some Flavonic Compounds and Ascorbic Acid on Lactoferrin Stimulation of Erythrocyte Glycolysis and Na+/K+-ATPase Activity

2008 ◽  
Vol 63 (9-10) ◽  
pp. 773-779 ◽  
Author(s):  
Ana Maneva ◽  
Borislava Taleva
Keyword(s):  
Ascorbic Acid ◽  
Atpase Activity ◽  
Specific Binding ◽  
Lactate Production ◽  
Inhibitory Effect ◽  
High Concentration ◽  
Transport Chain ◽  
Strong Inhibitory Effect ◽  
Lactate Formation ◽  
Stimulation Of

The aim of the present study was to assess if some flavonic compounds (quercetin, piceatannol and apigenin) and ascorbic acid could interfere with the Lf stimulatory effect on the erythrocyte function. Quercetin (1.5 μm) and piceatannol (30 μm) showed an additive effect on Lf stimulation of Na+/K+-ATPase when used together with Lf. The enhancement of Lf stimulation on Na+/K+-ATPase in the presence of flavonoids was probably due to their antioxidative properties and/or to their involvement in the erythrocyte signaling. None of the estimated flavonoids showed an effect on Lf stimulation of the lactate production. Quercetin itself enhanced the ATPase activity but did not affect the lactate formation. Apigenin (1.5 μm) enhanced reliably the lactate generation, but it did not exert any effect on the ATPase activity. High concentration of ascorbic acid (60 mm) did not change the Lf stimulatory effect on Na+/K+-ATPase, but decreased the Lf-specific-binding. A significantly strong inhibitory effect on the Lf-specific binding exerted the electron acceptors NAD+ (2 mm) and FAD (2 mm). These effects concern most likely the competition with Lf for electron(s) which is (are) provided from the erythrocyte intercellular electron transport chain(s).

scholarly journals The effects of starvation and experimental diabetes on phosphoenol-pyruvate carboxykinase in the guinea pig

1977 ◽  
Vol 164 (2) ◽  
pp. 357-361 ◽  
Author(s):  
K R F Elliott ◽  
C I Pogson
Keyword(s):  
Enzyme Activity ◽  
Guinea Pig ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Blood Glucose Concentration ◽  
Mitochondrial Fraction ◽  
Kidney Cortex ◽  
Total Enzyme Activity ◽  
The Mean ◽  
Mean Blood Glucose ◽  
Total Enzyme

1. Approx. 85% of liver phosphoenolpyruvate carboxykinase is associated with the mitochondrial fraction in the fed guinea pig. Enzyme activity is unchanged in diabetes, but doubles during starvation. In contrast with earlier reports, both cytoplasmic and mitochondrial activities were found to be increased. 2. In kidney cortex, total enzyme activity is increased in both starved and diabetic animals. These changes are associated with increases in the cytoplasmic activity alone. 3. In diabetic animals the mean blood-glucose concentration was 23.1 mM. Other blood metabolites were lower than those in the rat, and the animals did not show significant ketosis. 4. Changes in the rates of gluconeogenesis from lactate and propionate paralleled those in phosphoenolpyruvate carboxykinase activity.

scholarly journals Evidence for a negative Pasteur effect in articular cartilage

1997 ◽  
Vol 321 (1) ◽  
pp. 95-102 ◽  
Author(s):  
Robert B. LEE ◽  
Jill P. G. URBAN
Keyword(s):  
Articular Cartilage ◽  
Glucose Uptake ◽  
Lactate Production ◽  
Pasteur Effect ◽  
Anoxic Conditions ◽  
Atp Production ◽  
Bovine Articular Cartilage ◽  
Atp Concentration ◽  
O2 Concentration ◽  
Lactate Formation

Uptake of external glucose and production of lactate were measured in freshly-excised bovine articular cartilage under O2 concentrations ranging from 21% (air) to zero (N2-bubbled). Anoxia (O2 concentration < 1% in the gas phase) severely inhibited both glucose uptake and lactate production. The decrease in lactate formation correlated closely with the decrease in glucose uptake, in a mole ratio of 2:1. This reduction in the rate of glycolysis in anoxic conditions is seen as evidence of a negative Pasteur effect in bovine articular cartilage. Anoxia also suppressed glycolysis in articular cartilage from horse, pig and sheep. Inhibitors acting on the glycolytic pathway (2-deoxy-d-glucose, iodoacetamide or fluoride) strongly decreased aerobic lactate production and ATP concentration, consistent with the belief that articular cartilage obtains its principal supply of ATP from substrate-level phosphorylation in glycolysis. Azide or cyanide lowered the ATP concentration in aerobic cartilage to approximately the same extent as did anoxia but, because glycolysis (lactate production) was also inhibited by these treatments, the importance of any mitochondrial ATP production could not be assessed. A negative Pasteur effect would make chondrocytes particularly liable to suffer a shortage of energy under anoxic conditions. Incorporation of [35S]sulphate into proteoglycan was severely curtailed by treatments, such as anoxia, which decreased the intracellular concentration of ATP.

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