Enolase Activity Research Articles

Assignment of human genes: β-glucuronidase to chromosome 7, adenylate kinase-1 to 9, a second enzyme with enolase activity to 12, and mitochondrial IDH to 15

Assignment of human genes: β-glucuronidase to chromosome 7, adenylate kinase-1 to 9, a second enzyme with enolase activity to 12, and mitochondrial IDH to 15

Enolase Isoenzymes in Mammalian Tissues

The existence of isoenzymes of enolase (2-phospho-D-glycerate hydro-lyase, EC4.2.1.11) has been demonstrated electrophoretically, chromatographically and immunologically (Rider &Taylor, 1974). The three forms have been numbered on the basis of the electrophoretic migration of the rat enzymes, according to the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (1972). In the rat, enolase 1 is the predominant isoenzyme in liver, kidney, spleen, testis, adipose tissues and erythrocytes, whereas skeletal muscle contains only enolase 3. Both these isoenzymes exist in heart together with the hybrid form, enolase 2. In human tissues, the isoenzyme distribution is essentially similar, except that small amounts of enolase 2 also occur in liver, and the relative proportion of the isoenzymes in heart is altered, the human organ having less enolase 1. Molecular-weight studies, using Sephadex G-I50 chromatography and polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, have revealed that all three isoenzymes are dimers with a mol. wt. of around 90000. No significant difference in kinetic parameters has yet been found. Antisera raised in the chicken against the partially purified rat enolases 1 and 3 show that no immunological cross-reaction occurs between these two isoenzymes, whereas enolase 2 is susceptible to both antisera. It is concluded from these structural and immunological data that the observed heterogeneity of mammalian enolase is due to existence of two independent genetic loci, a and a. Thus enolase 1 is a dimer of two a subunits, enolase 3 is a dimer of two bsubunits, and enolase 2 is the aa dimer (Rider & Taylor, 1975). Developmental changes in the isoenzyme composition of rat heart and muscle were investigated by pooling tissues taken from litters whose ages had been determined by daily inspection of the cages. The enolase activity was determined and immunochemical titrations were carried out exactly as described previously (Rider & Taylor, 1974). The total enolase activity of foetal heart is the same as that of the adult tissue, 50 units/g wet wt., although there is a decrease to around 35 units/g between 10 and 30 days after birth. The developing muscle however, shows a continuous tenfold increase in enolase activity in the first 50 days of life from the foetal value of 20 units/g wet wt., a change similar to that previously reported by Hommes & Wilmink (1968). Both foetal heart and muscle contain enolase 1 as the predominant isoenzyme, and in both tissues the proportion of enolase 3 increases in the neonatal period. In muscle there is a rapid transition, for by the fifth day enolase 3 is the predominant form, whereas heart shows a more gradual and limited increase in the proportion of subunits, such that at 80 days of life enolase 1 still accounts for half the total tissue enolase activity. A further difference between the two tissues is that whereas the isoenzyme proportions in the heart approximate to a binomial distribution throughout development, this is not the case in the muscle. A phylogenetic study in which the muscle, heart and liver enolases from a wide range of species were tested against the two specific anti-(rat enolase) sera by the previously

Immunological determination of muscle-type enolase in the serum as a diagnostic test for myocardial infarction

An antiserum raised in the chicken against purified rat skeletal muscle enolase neutralized 86% of the enolase activity of human heart muscle and 8.7% ± 4.8% (mean ± S.D.) of the enolase activity in normal human serum. The fraction of enolase inhibited by the antiserum, designated as ‘immunologically reacting muscle enolase’, promptly rose after myocardial infarction in the serum of affected subjects, reaching peak values in excess of 40% in all but one of 23 subjects. Only one subject with ischaemic heart disease, but without infarction, and 2 with miscellaneous conditions, among 117 such patients tested, yielded comparable values. The test appears to be highly specific for myocardial infarction when muscle disease can be excluded and would not be difficult to introduce as a laboratory routine.

Demonstration of Enolase Activity Connected to the Brain‐Specific Protein 14‐3‐2

An enzyme activity, enolase (2‐phospho‐D‐glycerate hydro‐lyase, E.C. 4.2.1.11), was shown to be connected to the brain‐specific protein 14.3.2. Specific staining methods were used on immuno precipitates of the protein. The enzyme activity was strongly inhibited by the addition of fluoride in the presence of phosphate. Metal ion binding capacity of the protein was shown by means of an auto radiographic method.

The absence of 2,3-diphosphoglycerate in the erythrocytes of goldfish ( Carassius auratus) acclimated to high temperatures

1. 1. DPG Dg content was measured in erythrocytes of goldfish ( Carassius auratus) acclimated at 22, 31 and 35°C. 2. 2. Erythrocytes were incubated with NaF to inhibit enolase activity and thereby increase DPG levels. 3. 3. DPG was not detected in the erythrocytes of any goldfish, nor in erythrocytes incubated with NaF.

Studies on the re-citrate synthase reaction.

re‐Citrate synthase catalyzes the hydrolysis of R‐citryl‐CoA and R‐malyl‐CoA. The S diastereomers are not attacked. The stereospecificity of the hydrolase activity is an agreement with the stereochemical course of citrate synthesis as catalyzed by this enzyme. The cooperative action of 2‐oxoglutarate and metal ions (Co2+) induces maximum acetyl‐CoA enolase activity of re‐citrate synthase. R‐Malate is not effective in this reaction. Since S‐malate is the best inducer of the enolase activity of si‐citrate synthase this suggests differences in the architecture of the active sites of these enzymes.When re‐citrate synthase is incubated with citrate and coenzyme A in tritiated water, tritium is incorporated into citrate. This indicates that the re‐citrate synthase reaction is reversible.

Fluoride inhibition of enolase activity in vivo and its relationship to the inhibition of glucose-6-P formation in Streptococcus salivarius

The intracellular concentrations of glucose-6-P, 2-P-glycerate, P-enolpyruvate, and pyruvate were determined in cells of Streptococcus salivarius metabolizing glucose in the presence and absence of NaF in an attempt to elucidate the mechanism of action of this inhibitor. The addition of 2.4 mm NaF to metabolizing whole cells resulted in a cessation of glucose uptake and an immediate decline in the cellular glucose-6-P content. A comparison of the 2-P-glycerate and P-enolpyruvate levels in treated and untreated cells indicated that fluoride had also inhibited intracellular enolase (EC 4.2.1.11) activity. These two apparently separate fluoride effects could not be separated regardless of the pH of the incubation medium (pH 7.2, 8.0, and 5.8), or the concentration of NaF added to the cells (final concentration, 2.4, 0.36, and 0.12 mm). The addition of 2.4 mm NaF to cells metabolizing intracellular glycogen at pH 7.2 also resulted in the inhibition of enolase activity; however, in this case, inhibition of cellular glucose-6-P synthesis was not observed. These results indicated that fluoride was directly involved in the inhibition of glucose-6-P formation from exogenous glucose. The possible relationship between the two fluoride-sensitive sites was made clear by the demonstration that the P-enolpyruvate-phosphotransferase system was involved in the transport of glucose into cells of S. salivarius. The evidence strongly suggests that the fluoride inhibition of enolase results in the inhibition of glucose transport by reducing the amount of P-enolpyruvate available for phosphorylation.

Studies on Yeast Enolase: QUANTITATIVE END GROUP ANALYSES AND THE EFFECT OF EXOPEPTIDASE DIGESTION

Abstract The structural model of yeast enolase has undergone substantial modification in recent years, and the quantitative end group analysis of the enzyme was undertaken as a means of establishing the most recent two-subunit model by a chemical method. Carboxypeptidase digestion and hydrazinolysis gave 1.95 and 1.85 moles, respectively, of carboxyl-terminal leucine per mole of enzyme, and amino-terminal analysis by the cyanate method gave 1.8 moles of amino-terminal alanine per mole of enzyme, thus confirming the model of yeast enolase as an 88,000-dalton protein consisting of two 44,000-dalton subunits. Contrary to reports in the literature, pure enolase was found to be very resistant to endopeptidase-free exopeptidase digestion. The earlier findings that large segments of both the amino-terminal and carboxyl-terminal sequences can be removed by exopeptidases without loss of enolase activity can thus not be reproduced in this laboratory. Some possible reasons for this discrepancy are discussed.

Metabolic observations on platelets from patients with familial thrombopathic thrombocytopenia.

Summary. Glycolytic enzyme activities and adenine nucleotide content were studied in normal platelets and in platelets from patients with hereditary thrombopathic thrombocytopenia. This study revealed that most of the enzyme activities, the nucleotide content, and the platelet volumes of the patients were increased to two to three times the normal values. Taking into account the increased platelet volumes, the enzyme activities and nucleotide content were almost normal except for the activities of enolase, pyruvate kinase, and lactate dehydrogenase, which remained increased. Related to the platelet volume there was a normal lactate production and an impaired oxygen consumption. The existing platelet factor‐3 deficiency, changes in lipid composition, and absence of collagen‐induced platelet aggregation, together with the findings here described, are interpreted as manifestations of an underlying, genetically determined, disturbance in membrane structure.

Effect of fluoride on glycolytic enzymes. : (Report 3) Inhibition of enolase activity in muscle

Effect of fluoride on glycolytic enzymes. : (Report 3) Inhibition of enolase activity in muscle

Enzyme Activity in the Platelets of Newborn Infants

Platelet hexokinase, phosphofructokinase, phosphoglycerate kinase, enolase, pyruvate kinase, lactic dehydnogenase, glucose-6-phosphate, dehydrogenase, and glutamic-oxaloacetic transaminase activity was assayed in platelets from newborn infants and contrasted with those of adults. Platelet phosphofnuctokinase activity was decreased and platelet phosphoglycerate kinase and glutamic-oxaloacetic transaminase activity were elevated in the platelets of newborn infants. It would appear that certain biochemical properties of the erythrocytes of the newborn are shared by the platelets as well. The metabolic consequences of these differences remain to be explored.

The effect of fluoride on glycolytic enzyme system. 1. Effect on enolase

The effect of fluoride on purified enolase and the supernatant of mouse muscle homogenate, was studied in vitro and the change of enolase activity in mouse muscle was determined after feeding fluoride water of graded concentration for a week. The crude enolase in homogenate supernatant was more strongly inhibited than purified enolase, particularly when the activity was assayed by the sample mixed with fluoride in advance, namely as high as 50% inhibition in 0.5mM F. The author introduced a new hypothesis of incomplete inhibition (reversible) and complete inhibition (irreversible). In the latter case, it required the third factor, i. e. homogenate factor (H-factor), to be fully demonstrated. It was contained in the homogenate and probably comosed of phosphate, magnesium or other divalent metal and special protein. With the H-factor, the enolase became very sensitive to fluoride and was strongly and irreversibly inhibited. The inhibition went on to the greater extent with time.In vivo experiment, the slight inhibition of enolase activity was only observed at 100 and 400 ppm F feeding. The level was, however, so high that other enzyme systems might also be disturbed and the result might not be attributed solely to the effect of fluoride.

Kinetic studies on the activation of yeast enolase by divalent cations.

Kinetic studies on the activation of yeast enolase by divalent cations.

RED CELL METABOLISM IN THE NEWBORN INFANT. V. GLYCOLYTIC INTERMEDIATES AND GLYCOLYTIC ENZYMES

Previous studies demonstrated that the erythrocytes of the newborn infant have a relative phosphofructokinase deficiency and inappropriate glucose consumption for cell age. In view of these findings, an analysis of red cell glycolytic intermediates and all Embden-Meyerhof pathway enzyme activities was made in the red cells of infants, normal adults, and subjects with reticulocytosis to define further differences in metabolism and search for evidence of a metabolic blockade. The erythrocytes of subjects with reticulocytosis were found to possess increased quantities of glucose-6-phosphate, fructose-6-phosphate and 2,3-diphosphoglycerate. The elevation of the hexose phosphates was even greater in the red cells of the premature infants and was associated with a decrease in levels of 2,3-diphosphoglycerate and phosphoenolpyruvate. Measurement of enzyme activities indicated that, in addition to the phosphofructokinase deficiency, the cells of the newborn infant have markedly increased activities of enolase and phosphoglycerate kinase. It is suggested that the increase in the level of the hexose phosphates in the erythrocytes of the infants and subjects with reticulocytosis is a reflection of this relative phosphofructokinase deficiency, with this deficiency being greater in newborn infants. The differences in the enzymatic profile between the erythrocytes of the newborn and those of the normal adult is not solely a function of their younger age. Some of the differences are a reflection of the presence of a cell unique to this period of life.

Carbohydrate metabolism in normal and thyroidectomized Bufo bufo adult males

1. 1. In adult male specimens of Bufo bufo, thyroidectomized by means of 131I administration, the blood-glucose level dropped 55 per cent below that in the normal toad. 2. 2. In the liver the rate of synthesis of lactate from glucose and glycogen, under anaerobiosis, is slowed down; the same is true for the velocity of hexokinase and phosphorylase reactions. 3. 3. There is no change in the activity of phosphoglucomutase, aldolase, triose-phosphate isomerase, glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase, enolase, pyruvate kinase and lactate dehydrogenase. 4. 4. Increase in both the phosphofructokinase activity and glycogen storage are seen to occur. 5. 5. Among the enzymes involved in the gluconeogenesis pathway, hexosediphosphatase appears unaltered, whilst glucose-6-phosphatase is inhibited up to 50 per cent.

Effect of prolonged storage on erythrocyte enzymes.

After 19 weeks' storage in autologous plasma at 4 C, the enzymatic activities of erythrocytic enolase, 3‐phosphoglycerate kinase, pyruvate kinase and ATPase each were reduced by about one half, nucleoside diphosphokinase, glutathione reductase, and hexokinase were reduced by about one third, and glucose‐6‐phosphate dehydrogenase and lactate dehydrogenase were relatively unchanged in activity. After 19 weeks' storage at ‐120 C or ‐170 C in glycerol‐sucrose medium, the activity of these erythrocyte enzymes was essentially unchanged.

Steady state relaxation of enolase in vitro and metabolic throughput in vivo of red and white rabbit muscles.

The kinetics of rabbit muscle enolase in vitro are shown to follow a first order time law at substrate concentrations above as well as within the range of the Michaelis constants. The product (τssV1), where τss is the steady state relaxation time and V1 the maximum velocity in the forward direction, is found to be essentially a function of the total reactant concentration, and is almost independent of pH: , being the millimolar concentration of phosphoenolpyruvate at equilibrium. An expression for the relative steady metabolic throughput in vivo at the enolase step is derived. The deviation from apparent equilibrium and total reactant level are the important variables. For convenient use, a nomogram is given (see Fig.5). The thus developed expressions are then applied to red and white rabbit muscle at different states of activity. A calculation of the absolute metabolic throughput with available metabolite and enzyme level data correlates with the measured rate of lactate production. “Glycolytic potencies”, as deduced recently from enzyme patterns of the two muscles, prove to be proportional to the measured metabolic throughputs. The high cellular activity of enolase and other glycolytic enzymes is interpreted to serve mainly to minimize the deviation and the concomitant expenditure of free energy. The energy expended for the enolase step at maximal glycolytic flux is calculated to be 0.6 kcal per mole of lactate formed, in both types of muscle.

Modification of enzyme activities in dietary cirrhosis and fatty infiltration of rat liver.

Summary. The activities of enolase, phosphohexose isomerase and lactate dehydrogenase in cirrhotic and fatty livers of rats are increased when expressed on a cellular basis. Linear regression equations which permit the interconversion of enzyme activity expressed on the basis of supernatant protein to that of fresh weight, and freshweight corrected for lipid, collagen and particulate protein are presented.

Chandlerella hawkingi: Glucose utilization and glycolytic enzymes

Chandlerella hawkingi, the filarial parasite of the Indian jungle crow ( Corvus macrorhynchos Wagler) has been shown to utilize glucose to form mainly lactic acid. Eighty five to one hundred percent of glucose is aerobically converted into lactic acid and only 0.5% to pyruvic acid. The parasite possesses significant amounts of aldolase, triose phosphate dehydrogenase, enolase and lactate dehydrogenase activities. However, glucose-6-phosphate, 6-phosphogluconate and isocitrate dehydrogenases could not be detected in measurable amounts. From these results it is inferred that the parasite meets its energy requirement through the process of glycolysis.

Studies on the Mechanism of Substrate Protection of Enolase and Lactic Dehydrogenase against Ionizing Radiation

Beef heart lactic dehydrogenase (LDH) and rabbit muscle enolase were used to study the protective effect of substrates against ionizing radiation. The substrate of enolase, D-glyceric acid 2-phosphate, protected enolase activity against ionizing radiation, but the protection occurred in the absence of magnesium ions, which are required to bind the substrate molecule to the enzyme molecule. The substrate of enolase also protected LDH. Reduced nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide (NAD) protected LDH and also enolase. In both cases NADH gave better protection. NADH was oxidized to NAD by radiation both in the presence and in the absence of protein molecules. Cytidine-2′,3′-cylic phosphate, a substrate for ribonuclease, gave partial protection to both enolase and LDH. From the results of this study, it is concluded that specific "substrate protection" is not the primary mechanism involved. The data suggest that a "radical scavenger" mechanism would best account for th...