An ultramicro bioluminescence assay of enolase: Application to human cerebrospinal fluid

Viallard, J L; Murthy, M.R.V.; Dastugue, B.

doi:10.1007/bf00988598

Cited by 17 publications

(12 citation statements)

References 14 publications

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“…Enzyme activity was expressed as mIU, defined as the amount of enzyme that catalyzes the conversion of 1 nmol of substrate per minute. As previously described (Viallard et al, 1985), the lowest activity measured by this method is 0.4 ϫ 10 Ϫ6 IU of enolase. The assay is linear from 0.4 ϫ 10 Ϫ6 to 50 ϫ 10 Ϫ6 IU with a variation coefficient of 6.7% and 2.2% for the low and high values, respectively.…”

Section: Bioluminescence Determination Of Enolase Activitysupporting

confidence: 53%

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Enolase Isoforms Activities in Spermatozoa From Men With Normospermia and Abnormospermia

FORCE,

VIALLARD,

GRIZARD

et al. 2002

Journal of Andrology

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Total enolase as well as enolase‐αα (ENO‐aa, ubiquitous) and enolase‐S (ENO‐S, sperm‐specific) activities were measured in total and Percoll‐selected sperm from 30 normospermic fertile men and 20 abnormospermic infertile patients. The total enolase activity was significantly higher in total sperm from patients with abnormospermia compared with normospermic patients (11.1 ± 1.9 vs. 4.8 ± 0.5 mIU/107 sperm P < .05). ENO‐αα activity was significantly higher in total sperm from abnormospermic men than from normospermic men (P < .05). ENO‐αα activity in Percoll‐selected sperm was significantly lower compared with total sperm in both group of patients; however, for the same sperm fraction ENO‐αα activity did not differ between normospermic and abnormospermic men. ENO‐αα activity was related to the cell contamination ratio and to the percentage of spermatozoa with abnormal morphology. Furthermore, ENO‐αα was positively correlated with the percentage of immature sperm showing an excess of residual cytoplasm. ENO‐S activity was significantly higher in total sperm from normospermic patients than from abnormospermic patients (P < .05). ENO‐S activity in Percoll‐selected sperm was not significantly different compared with total sperm in both group of patients. However, this activity was significantly lower in Percoll‐selected sperm from abnormospermic men compared with normospermic men (P < .05). ENO‐S activity was not related to the cell contamination ratio but was significantly correlated with the percentage of spermatozoa with normal morphology. The 2 enolase isoforms seem to reflect 2 opposite aspects of sperm cells quality: ENO‐αα is associated with abnormal spermatozoa, immature spermatozoa, or both; and ENO‐S is associated with normal spermatozoa. As an additional index to distinguish normal from abnormal semen, the ENO‐S:ENO‐αα ratio was evaluated for total and Percoll‐selected sperm in both groups. This ratio seems to be a new, valuable marker of the global sperm quality in a given semen sample, and may represent a predictive index of sperm fertilizing potential.

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Section: Bioluminescence Determination Of Enolase Activitysupporting

confidence: 53%

“…We determined total enolase activity according to a method described by Viallard et al (1985). In this procedure the phosphoenolpyruvate formed from 2-phosphoglycerate (G2P) by enolase is transformed into adenosine triphosphate (ATP) in the presence of pyruvate kinase and adenosine diphosphate.…”

Section: Bioluminescence Determination Of Enolase Activitymentioning

confidence: 99%

Enolase Isoforms Activities in Spermatozoa From Men With Normospermia and Abnormospermia

FORCE,

VIALLARD,

GRIZARD

et al. 2002

Journal of Andrology

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“…Cr content was expressed in nanomoles per milligram of protein (nmol Cr/mg protein). Enolase activity was measured according to a previously described bioluminescence method [35] using an ATP determination kit (Invitrogen, Carlsbad, CA), with slight modifications to the enzyme buffer. For this analysis powdered LV tissue from 7 CrT-OE, 6 WT, 6 aortic banded, and 6 sham control hearts was made up to a concentration of 10 mg/ml in a buffer containing 50 mM Tris-acetate, 1mM EDTA, 1 mM EGTA, 3 mM potassium-acetate, 2 mM AMP, 2 mM DAPP, 400 units per ml of pyruvate kinase, 2 mM ADP, 5 mM potassium-phosphate, 5 mM MgCl 2 , 5 μM antimycin A, 100 μM KCN, 10 μM oligomycin-B, and ATP monitoring reagents (ATP determination kit, Invitrogen, Carlsbad, CA).…”

Section: Methodsmentioning

confidence: 99%

“…After obtaining a baseline measurement, 1 mM 2-phosphoglycerate was added and ATP production was measured over the course of 1 min. This assay takes advantage of coupled reactions in which ATP is produced as the final product by pyruvate kinase; under these reaction conditions the amount of ATP formed is directly proportional to enolase activity [35]. All assays were performed in quadruplicate.…”

Section: Methodsmentioning

confidence: 99%

Mice over-expressing the myocardial creatine transporter develop progressive heart failure and show decreased glycolytic capacity

Phillips¹,

Hove²,

Schneider³

et al. 2010

Journal of Molecular and Cellular Cardiology

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The metabolic phenotype of the failing heart includes a decrease in phosphocreatine and total creatine concentration [Cr], potentially contributing to contractile dysfunction. Surprisingly, in 32 week old mice over-expressing the myocardial creatine transporter (CrT-OE), we previously demonstrated that elevated [Cr] correlates with left ventricular (LV) hypertrophy and failure. The aim of this study was to determine the temporal relationship between elevated [Cr] and the onset of cardiac dysfunction and to screen for potential molecular mechanisms. CrT-OE mice were compared with wild-type (WT) littermate controls longitudinally using cine-MRI to measure cardiac function and single-voxel 1H-MRS to measure [Cr] in vivo at 6, 16, 32, and 52 weeks of age. CrT-OE mice had elevated [Cr] at 6 weeks (mean 1.9-fold), which remained constant throughout life. Despite this increased [Cr], LV dysfunction was not apparent until 16 weeks and became more pronounced with age. Additionally, LV tissue from 12 to 14 week old CrT-OE mice was compared to WT using 2D difference in-gel electrophoresis (DIGE). These analyses detected a majority of the heart’s metabolic enzymes and identified 7 proteins that were differentially expressed between groups. The most pronounced protein changes were related to energy metabolism: α- and β-enolase were selectively decreased (p<0.05), while the remaining enzymes of glycolysis were unchanged. Consistent with a decrease in enolase content, its activity was significantly lower in CrT-OE hearts (in WT, 0.59±0.02 μmol ATP produced/μg protein/min; CrT-OE, 0.31±0.06; p<0.01). Additionally, anaerobic lactate production was decreased in CrT-OE mice (in WT, 102±3 μmol/g wet myocardium; CrT-OE, 78±13; p=0.02), consistent with decreased glycolytic capacity. Finally, we found that enolase may be regulated by increased expression of the β-enolase repressor transcription factor, which was significantly increased in CrT-OE hearts. This study demonstrates that chronically increased myocardial [Cr] in the CrT-OE model leads to the development of progressive hypertrophy and heart failure, which may be mediated by a compromise in glycolytic capacity at the level of enolase.

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“…As an alternative to antibody capture, fluid phase enzymatic reactions have been used to detect plasma NSE by means of its enzymatic activity [ 13 ] [ 14 ]; however, in the 30 years since these assays were first described, to our knowledge, no related PoCT technologies have been reported. Semi-solid phase bioluminescence detection of NSE through its binding to immunobeads enabled high sensitivity detection [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection

et al. 2015

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BackgroundRapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT) for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs). As proof of principle, we use oriented immobilization of pyruvate kinase (PK) and luciferase (Luc) on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE), a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices.Methods and findingsWe performed in-vitro, animal model, and human subject studies. First, the efficiency of coupled enzyme activities when tethered to NPs versus when in solution was tested, demonstrating a highly sensitive and rapid detection of physiological and pathological concentrations of NSE. Next, in rat stroke models the enzyme-based assay was able in minutes to show a statistically significant increase in NSE levels in samples taken 1 hour before and 0, 1, 3 and 6 hours after occlusion of the distal middle cerebral artery. Finally, using the tethered enzyme assay for detection of NSE in samples from 20 geriatric human patients, we show that our data match well (r = 0.815) with the current gold standard for biomarker detection, ELISA—with a major difference being that we achieve detection in 10 minutes as opposed to the several hours required for traditional ELISA.ConclusionsOriented enzyme immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. Together, our findings provide proof of concept for using oriented immobilization of active enzymes on NPs as the basis for a highly rapid and sensitive biomarker detection platform. This addresses a key challenge in developing a PoCT platform for time sensitive and difficult to diagnose pathologies.

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An ultramicro bioluminescence assay of enolase: Application to human cerebrospinal fluid

Cited by 17 publications

References 14 publications

Enolase Isoforms Activities in Spermatozoa From Men With Normospermia and Abnormospermia

Enolase Isoforms Activities in Spermatozoa From Men With Normospermia and Abnormospermia

Mice over-expressing the myocardial creatine transporter develop progressive heart failure and show decreased glycolytic capacity

Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection

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