Enolase Activity Research Articles

Clinical value of an amplified electrophoretic determination of enolase isoenzymes in small cell lung carcinoma patients

Neurone specific enolase (NSE); alpha gamma and gamma gamma isoenzymes of the glycolytic enzyme enolase, is found in considerable quantity in serum of patients with small cell lung cancer (SCLC). The spectrophotometric measurement of serum enolase activity, plus the electrophoretic separation of isoenzymes (alpha alpha, alpha gamma and gamma gamma) using an amplification reaction constitute a simple method, the application of which has not yet been demonstrated. In patients, serum values of higher than 25 U/l of total enolase activity, with more than 10% NSE, were considered to be positive. Seventy patients diagnosed as SCLC were classified before treatment as having either limited (LD) or extensive (ED) disease, and after chemotherapy as being in complete (CR) or partial remission (PR), stable state (SS) or in relapse (R). The levels of enolase activity and NSE (M +/- SE) in these patients (enolase: 67 +/- 7 U/l, NSE 27 +/- 2%) were different from those in a control group of 19 patients with non-small cell lung cancer (enolase: 29 +/- 2 U/l, NSE: 7 +/- 1%) (p less than 0.001) at the time of diagnosis. Mean enolase and NSE levels in patients with SCLC were seen to differ significantly according to the clinical stage. The results of those patients with ED differed from those of patients with LD (p less than 0.001). The results of the group of patients that achieved remission differed from that of patients during relapse (p less than 0.0001). Serial measurements demonstrated a good correlation between enolase and NSE serum levels and the progression of the disease. The usefulness of this method in the early assessment of treatment was also demonstrated. The clinical usefulness of the dosage of NSE with that of two other tumour markers CKBB and mitochondrial CK was compared.

An immunobioluminescence assay for gamma-gamma enolase activity in human serum and cerebrospinal fluid

A solid phase immunobioluminescence assay for the measurement of gamma-gamma enolase activity in human serum and cerebrospinal fluid is described. The assay combines the specificity of the antiserum with the specificity of measuring enolase activity, thus making the assay highly specific for gamma-gamma enolase. The sensitivity of the method allows determinations in unconcentrated cerebrospinal fluid samples. The new immunobioluminescence assay has been compared with a radioimmunoassay method. The correlation is good both for serum and CSF samples. However, different correlation lines are found for serum and CSF.

Tau-crystallin/alpha-enolase: one gene encodes both an enzyme and a lens structural protein.

tau-Crystallin has been a major component of the cellular lenses of species throughout vertebrate evolution, from lamprey to birds. Immunofluorescence analysis of the embryonic turtle lens, using antiserum to lamprey tau-crystallin showed that the protein is expressed throughout embryogenesis and is present at high concentrations in all parts of the lens. Partial peptide sequence for the isolated turtle protein and deduced sequences for several lamprey peptides all revealed a close similarity to the glycolytic enzyme enolase (E.C. 4.2.1.11). A full-sized cDNA for putative duck tau-crystallin was obtained and sequenced, confirming the close relationship with alpha-enolase. Southern blot analysis showed that the duck genome contains a single alpha-enolase gene, while Northern blot analysis showed that the message for tau-crystallin/alpha-enolase is present in embryonic duck lens at 25 times the abundance found in liver. tau-Crystallin possesses enolase activity, but the activity is greatly reduced, probably because of age-related posttranslational modification. It thus appears that a highly conserved, important glycolytic enzyme has been used as a structural component of lens since the start of vertebrate evolution. Apparently the enzyme has not been recruited for its catalytic activity but for some distinct structural property. tau-Crystallin/alpha-enolase is an example of a multifunctional protein playing two very different roles in evolution but encoded by a single gene.

Metabolic changes in gladiolus due to airborne fluoride

Gladiolus plants were exposed to airborne fluoride in order to study effects at the metabolic level. Two varieties of gladiolus, one sensitive and the other resistant to fluoride, were grown on uniform soil at a clean-air site. One set of plants was then exposed to ambient air in the vicinity of an aluminum smelter plant. A second set of control plants was placed at a location free from fluoride-polluted air. At the end of the exposure period of two weeks, plants were harvested and analyzed. The fluoride content in exposed plants was about four times higher than that in control plants. Chlorophyll content remained unchanged, but the soluble protein content of exposed plants decreased by up to 50% relative to the control plants. Activities of glutamine synthetase, glutamate dehydrogenase, malate dehydrogenase and enolase in the tissue of exposed plants were reduced to 30-50% of the activities in control plants. These effects of fluoride were more pronounced in the sensitive variety than in the resistant one. The results demonstrate that gladiolus plants can be used as a sensitive bioindicator for fluoride pollution.

Effect of short-term exposure to hexachlorophene on rat brain cell specific marker enzymes

Seven cell specific marker enzymes in brain and optic nerve and morphological evaluation by light microscopy were used to characterize the neurotoxicity associated with exposure of rats to hexachlorophene (HCP; 40 mg/kg/day, po, for 9 days). In vitro exposure to HCP at concentrations up to 100 μ m had no direct inhibitory effect on the marker enzymes, validating their use in evaluating brain function in vivo. Rats exhibited a reduction in body weight gain, weakness, and ataxia of the hind limbs by the ninth day of HCP exposure. At 24 hr following the last day of exposure to HCP, the activities of the three neuron specific enzymes, glutamic acid decarboxylase, tyrosine hydroxylase, and choline acetyltransferase, in rat brain were unchanged from those of the vehicle-treated control group. Of the two astroglial enzyme markers measured, a small but significant increase was observed in the activity of nonneuronal enolase in the cerebellum and glutamine synthetase in the hippocampus of HCP-treated rats. The optic nerve appeared to be the most sensitive tissue in that the activity of both the astroglial marker, nonneuronal enolase, and the myelin marker, 2′,3′-cyclic nuceleotide phosphohydrolase, was significantly decreased following HCP exposure. This decrease in enzyme activity is consistent with the histological observations demonstrating extensive vacuolization and edema in the optic nerve after exposure to HCP.

Blood cell superoxide dismutase and enolase activities as markers of alcoholic and nonalcoholic liver diseases

Monitoring of chronic alcoholism would be facilitated by using sensitive biochemical markers in blood cells, mainly to detect differences between alcoholic subjects with or without liver injury. We propose two types of markers: the first one is superoxide dismutase (SOD) activity involved in the conversion of superoxide radicals (O 2 •- ) formed during acetaldehyde oxidation by xanthine oxidase after chronic alcohol consumption; the second one is enolase activity with both isoenzyme forms: nonneuronal enolase (NNE) and neuron specific enolase (NSE) which has been shown to be modified in many injuries related to the glycolytic pathways. For SOD activity we found a significant increase in alcoholic patients with liver injury and mainly in cirrhotic patients with ascitis. Both enolase activities were also found to be significantly increased in alcoholic patients with liver injury but NNE activity was also increased in alcoholics without apparent liver disease. Our results suggest that increased activity of SOD and NSE in blood cells may be related to liver injury mainly in alcoholism while increased NNE activity may also be a marker of alcohol abuse without liver injury.

Complex of brain d-phosphoglycerate mutase and γ enolase and its reactivation by d-glycerate 2,3-bisphosphate

The dissociabilities of dimeric γ enolase, α enolase, and phosphoglycerate mutase of brain origin were tested using fluorescein isothiocyanate attached covalently to these enzymes. The dissociation constant of dimeric γ enolase is lower ( K d = 0.03 μM) than that of the α enolase ( K d = 3 μM), while dimeric mutase seems to be nondissociable in the concentration range 0.1–10 μ m, at pH 7.3 in 50 m m imidazole buffer at 20 °C. Interaction of neuron-specific γ enolase with d-phosphoglycerate mutase was detected with the same fluorescence-labeling technique as well as by a kinetic analysis. The determined dissociation constant of the enolase-mutase complex was found to be in the range 5–40 μ m, independent of the technique used. A mixed type of inhibition in the binding of d-glycerate-2-P and mutase to the d-glycerate-2-P binding site on enolase was observed in the absence of d-glycerate-2,3-P 2. However, the inhibition of the enolase activity by brain d-phosphoglycerate mutase in the d-glycerate-2-P → phospho enolpyruvate transformation is almost fully reverted by d-glycerate-2,3-P 2, probably via the proper coordination of the active centers in the ternary complex of enolase, d-phosphoglycerate mutase, and their common intermediate, d-glycerate-2-P. The mechanism of intermediate transfer by consecutive enzyme pairs in a nondivergent metabolite flux (around the transformation of d-glycerate-2-P) is examined and conclusions of the present experiments are compared with the results of an extended analysis performed earlier with a divergent metabolite flux (around the transformation of multiusage triosephosphates, d-glyceraldehyde-3-P, and dihydroxyacetone phosphate).

Evidence that stimulation of gluconeogenesis by fatty acid is mediated through thermodynamic mechanisms

We have studied the stimulatory effects of palmitate on the rate of glucose synthesis from lactate in isolated hepatocytes. Control of the metabolic flow was achieved by modulating the activity of enolase using graded concentrations of fluoride. Unexpectedly, palmitate stimulated gluconeogenesis even when enolase was rate-limiting. This stimulation was also observed when the activities of phospho enolpyruvate carboxykinase and aspartate aminotransferase were modulated using graded concentrations of quinolinate and aminooxyacetate, respectively. Linear force-flow relationships were found between the rate of gluconeogenesis and indicators of cellular energy status (i.e. mitochondrial membrane and redox potentials and cellular phosphorylation potential). These findings suggest that the fatty acid stimulation of glucose synthesis is in part mediated through thermodynamic mechanisms.

Nutrition and fetal brain maturation: II. Impact of maternal starvation on changing levels of acetylcholinesterase and enolase in vitro

The impact of maternal starvation during Days 17–20 of gestation was examined in 20-day fetal rat brain tissue cultured for 6 days in MEM and 10% adult rat serum. Acetylcholinesterase (AChE) activities were consistently greater in fetal brain cell cultures from starved mothers. When fetal tissues from starved mothers were continuously exposed to 72-h fasted serum, AChE activities increased from 1.03±0.14 to 1.59±0.21 μmol/h/mg protein ( P<0.001). In fetal tissues from fed mothers, lower AChE activities were increased from 0.78±0.09 to 1.04±0.07 μmol/h/mg protein ( P<0.05) when 72-h fasted serum was used to replace the fed serum during incubation. When fetal brain cell cultures from fed mothers were exposed for 6 days to graded concentrations of fed serum (2.5–15%), the activities of AChE fell reciprocally from 1.34±0.10 to 0.82±0.12 μmol/h/mg protein ( P<0.05). The levels of AChE activity in tissues exposed to fasted serum were consistently greater, but fell similarly from 1.62±0.10 to 0.97±14 μmol/h/mg protein ( P<0.01), when serum concentrations were increased from 2.5 to 15%. AChE activities were 30% higher in tissues incubated with cycloheximide 10 −3 M ( P<0.02). Unlike AChE, fetal brain enolase activities were unaffected by maternal starvation. In fetal brain cell cultures from fed mothers, enolase fell from 1.85±0.10 to 1.37±0.12 μmol/min/mg protein following exposure to fasted instead of fed serum ( P<0.02). In fetal cultures from starved mothers, enolase activities were depressed similarly from 1.76±0.08 to 1.41±0.09 μmol/min/mg protein when fasted replaced fed serum ( P<0.02). Thus, the fetal brain cell cultures appear to maintain enzymatic realignments imposed by maternal starvation for at least 6 days. In addition, serum from fasted animals has significant growth inhibiting properties manifested by heightened activities of AChE and lower activities of enolase.

Peripheral small cell undifferentiated carcinoma of the lung. Clinicopathologic features of 17 cases.

Seventeen cases of resectable peripheral small cell undifferentiated carcinoma of the lung were studied (average size, 2.1 cm; range, 0.9-3.5 cm). The carcinomas exhibited predominantly mixed intermediate and fusiform/spindle subtypes; in seven carcinomas there were at least some foci of oat cell subtype. There was no histologic evidence of typical carcinoid tumor. Seven cases were fixed in solutions appropriate for immunohistologic study; these exhibited prominent neuron-specific enolase activity but less prominent and more variable staining for the carcinoembryonic antigen and cytokeratins. All 17 cases were deemed resectable and had no clinical or radiologic evidence of metastasis. Seven (41%) patients died with recurrent and/or metastatic carcinoma (average survival, 1.7 years); five of these patients had carcinomas with at least some foci of oat cell foci subtype.

The capacity of plastids from developing pea cotyledons to synthesise acetyl CoA

In order to determine whether the enzymes required to convert triose phosphate to acetyl CoA were present in pea (Pisum sativum L.) seed plastids, a rapid, mechanical technique was used to isolate plastids from developing cotyledons. The plastids were intact and the extraplastidial contamination was low. The following glycolytic enzymes, though predominantly cytosolic, were found to be present in plastids: glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), and pyruvate kinase(EC 2.7.1.40). Evidence is presented which indicates that plastids also contained low activities of enolase (EC 4.2.1.11) and phosphoglycerate mutase (EC 2.7.5.3). Pyruvate dehydrogenase, although predominantly mitochondrial, was also present in plastids. The plastidial activities of the above enzymes were high enough to account for the rate of lipid synthesis observed in vivo.

Neuron-specific enolase in comatose children.

Sequential examination of neuron-specific enolase in cerebrospinal fluid and serum was performed in 20 comatose children with acute encephalitis, acute encephalopathy, or Reye's syndrome. Neuron-specific enolase activities corresponded to the degree of brain damage. As neuron-specific enolase levels increased to greater than 80 ng/mL, patients had more severe impairment or died. Neuron-specific enolase may be a useful marker for evaluating the degree of neuronal damage and prognosis.

An enzymoimmunoassay for rat gamma enolase using two monoclonal antibodies

Monoclonal antibodies specific for rat ? enolase were prepared by fusion between ?? enolase immunized mouse spleen cells and mouse myeloma cells. Isolated hybrid cell lines were screened against both ?? and ?? enolase with an enzymoimmunoassay. Two types of antibodies were identified: one type, corresponding to 21% of the generated clones, recognized both ? and ? subunits, the other type, representing 3% of the clones, was specific for ? enolase. The specificity of antibodies was ascertained by immunofixation of ?? enolase activity. Two monoclonal antibodies (2H12 and 8A9) were used for quantitative determination of rat ? enolase in a "sandwich type" enzymoimmunoassay. The ? enolase has been shown to be a specific neuronal and neuroendocrine product. The development of a ? enolase assay will facilitate the study of the regulation of ? enolase expression.

Glycolytic enzyme activities in breast cancer metastases.

The activities of hexokinase, phosphofructokinase, aldolase, enolase and pyruvate kinase were studied in breast cancer metastases occurring at various sites and compared with the enzyme activities in a series of primary breast cancers. The activities of all enzymes studied were significantly higher in the metastases compared to the primary tumors (p less than or equal to 0.05). However, no changes in the isoenzyme patterns of enolase and pyruvate kinase were observed when the metastases were compared with primary breast cancers. Differences in location of the metastases did not lead to differences in enzyme activities. Our data suggest an association of an increasing rate of glycolysis with tumor progression.

Nutrition and fetal brain maturation: I. Responses in vitro and in vivo

The glycolytic enzyme enolase increases during the perinatal period of brain development and was utilized as a marker for examining the effect of culture environment on differentiation of cells from 20-day fetal rat brain. Enolase activity in cell cultures increased from 0.91 ± 0.03 (Day 0) to 2.11 ± 0.10 μmol/min/mg protein (Day 6). Comparable levels were not reached in vivo until neonatal pups were 15 days old. The in vitro increase was inhibited by both cycloheximide and actinomycin D. Enolase activity in the cells responded to alterations in both incubation media and homologous serum. After 6 days in culture, cells incubated in rat serum (10%) added to MEM or RPMI produced twice as much enolase activity as cells incubated similarly in Ham's medium, i.e., 1.96 ± 0.09 and 1.85 ± 0.21 vs 1.02± 0.09, P < 0.001. Results of a comparable magnitude were obtained when fetal calf serum replaced adult rat serum, but enolase production was somewhat lower when newborn calf serum replaced adult rat or fetal calf serum. When cells were incubated for 6 days with graded concentrations of adult rat serum (2.5–15%), enolase activity increased progressively. The pattern of enolase response suggests that the fetal rat brain cell model described herein will provide a sensitive probe with which to gain insight into nutrition and fetal brain development.

Isoenzyme pattern of enolase in the diagnosis of neuroendocrine bronchopulmonary tumors.

Electrophoretic separation of enolase isoenzymes and the measurement of enolase activity were performed in 25 lung tumor extracts. In 13 neuroendocrine (NE) tumors (nine small cell lung carcinoma [SCLC], three atypical NE tumors, and one carcinoid tumor), the NE differentiation was assessed by ultrastructural determination of neurosecretory granule (NSG) density. Twelve non-NE lung tumors also were studied (three adenocarcinomas, four epidermoid, two composite, two large cell undifferentiated carcinomas, and one lymphoma). Four normal lung tissues and 1 human brain were used as controls. The gamma gamma isoenzyme was present at a high level (mean +/- SE, 12 +/- 3%) in all NE carcinomas and consistently absent in all non-NE tumors as well as in normal lung. The alpha gamma isoenzyme was found in significantly higher proportion in NE carcinomas (mean +/- SE, 29 +/- 2%) than in non-NE tumors (mean +/- SE, 8 +/- 1%) (P less than 0.0001), despite an equally high level of total enolase activity in both groups of tumor. The separation of alpha gamma and gamma gamma isoenzymes of enolase allows for the accurate diagnosis of NE tumors and NE components of atypical NE carcinomas, and the gamma gamma isoenzyme, in contrast to gamma chain detection by immunoassay, can be considered to be a specific marker in itself of NE differentiation in lung neoplasms.

CHLOROPLAST AUTONOMY FOR THE BIOSYNTHESIS OF ISOPENTENYL DIPHOSPHATE IN GUAYULE (PARTHENIUM ARGENTATUM GRAY)

SUMMARYPurified chloroplasts (80% intact) with a photosynthetic activity of 195 μmol O2 mg chlorophyll−1 h−1 were prepared from the leaves of guayule. The activities of phosphoglyceromutase, enolase and pyruvate kinase were estimated in the purified chloroplasts and compared with those of Phaseolus radiatus Roxb. Activity of the pyruvate dehydrogenase complex (PDC) in the chloroplasts of guayule (448 nkat mg protein−1) was substantially higher than the PDC activity of Phaseolus (282 nkat mg protein−1). Incorporation of carbon from the added labelled carbon compounds, [14C]bicarbonate, [2‐14C]pyruvate and [U‐14C]‐3‐phosphoglycerate into isopentenyl diphosphate (IDP) was significant, indicating the ability of the chloroplasts to utilize Calvin cycle intermediates for IDP biosynthesis via acetyl CoA and mevalonate. DCMU inhibited the incorporation of 14CO2 and [14C]pyruvate into IDP. Acetoacetyl CoA thiolase, HMG‐CoA reductase, mevalonate kinase, phosphomevalonate kinase and pyrophosphomevalonate decarboxylase activity were localized in the isolated chloroplasts. It is believed that the chloroplasts in the leaf of guayule are autonomous for the biosynthesis of IDP and this is presumed to be dependent on photosynthetic energy and carbon flow.

Enolase Isozymes from Developing Oilseeds: A Survey

Summary Enolase activity was used to demonstrate the occurrence of both plastid and cytosolic glycolysisin the developing storage organs of several oil-rich seeds. Plastids isolated from these tissues and purified by rate-zonal sedimentation on discontinuous sucrose gradients contained substantial amounts of enolase activity. The enolase activity associated with isolated plastids could be separated from that found in the cytosol by ion-filtration and ion exchange chromatography and could be attributed to - a distinct isozyme. Hence, plastid glycolysis in developing oilseeds appears to be universal.

Studies of the in vitro effect of methylmercury chloride on rat brain neurotransmitter enzymes.

The in vitro effect of methylmercury (MM) on the enzymatic activities of brain cell specific marker enzymes, choline acetyltransferase (CAT), glutamic acid decarboxylase (GAD), 2',3'-cyclic nucleotide phosphohydrolase (CNP), glutamine synthetase (GS) and enolase was examined. The results demonstrate that at 100 microM MM, GS activity was not affected whereas a small decrease in the activity of both GAD (20%) and enolase (10%) was observed. CNP and CAT activity appeared to be more sensitive toward MM with 100 microM MM producing inhibition of 50% and 30%, respectively. The addition of sulfhydryl protecting reagents such as DTT or sodium thioglycolate can restore the enzyme activities to normal control levels despite prior exposure of the enzymes to MM.

Studies on the regulation of enolases and compartmentation of cytosolic enzymes in Saccharomyces cerevisiae.

Three enolase isoenzymes can be distinguished after electrophoresis of yeast crude extracts. After adding glucose to derepressed cells, there was a coordinated increase in the activity of enolase I and decrease in enolase II activity. Enolase I was found to be repressed and enolase II simultaneously induced by glucose. The third enolase activity remained unchanged and was identified as that of a hybrid enzyme. Enolase catalyses the first common step of glycolysis and gluconeogenesis. Gluconeogenic enolase I shows substrate inhibition for 2-phosphoglycerate (glycolytic substrate) and glycolytic enolase II is substrate-inhibited by phosphoenolpyruvate (gluconeogenic substrate). The gluconeogenic reaction was inhibited up to 45% by physiological concentrations of fructose 1,6-bisphosphate. To test for cytological compartmentation, a method was developed for isolating microsomes. Effective enrichment of rough and smooth endoplasmic reticulum was demonstrated by electron microscopy. No evidence was obtained for any compartmentation of either enolases or other glycolytic enzymes.