Bioluminescent Assay of the Adenylate Energy Charge

Ford, Sharon R.; Leach, Franklin R.

doi:10.1385/0-89603-520-4:69

Cited by 14 publications

(17 citation statements)

References 23 publications

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“…We observed that phosphatase inhibition on ice as described by Guthrie et al (2011) in striped bass spermatozoa and the use of a Tricine boiling buffer (Ford and Leach, 1998) yielded highest ATP concentration regardless whether ATP was extracted from fresh or frozen samples. Therefore, ice conditions effectively prevented ATP degradation during phosphatase inhibitor treatment.…”

Section: Discussionmentioning

confidence: 79%

“…The energy charge was determined by modification of procedures described by Ford and Leach (1998). The modifications are described below.…”

Section: Methodsmentioning

confidence: 99%

“…The immediate use of a phosphatase inhibitor cocktail containing acidic and alkaline phosphatases as well as tyrosine protein phosphatases in aqueous solution prior to ATP release was found to increase the amount of detectable ATP in sperm samples of turkey, rooster and boar (Long and Guthrie, 2006). A simple method for extraction of ATP and other nucleotides is boiling of the sample in the presence or absence of deionized water or a boiling buffer (Ford and Leach, 1998; Yang et al, 2002; Yi et al, 2008). Other commonly used reagents for ATP extraction, e.g.…”

Section: Introductionmentioning

confidence: 99%

“…Protocol revision was based on the ATP and adenylate energy charge assay from Ford and Leach (1998) and key features of the ATP quantification assay described by Long and Guthrie (2006) for spermatozoa. The revised assay was applied to determine ATP content and energy charge in boar spermatozoa preserved at 17°C and 5°C with consideration of the nucleotide concentration in semen extender media.…”

Section: Introductionmentioning

confidence: 99%

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Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction

et al. 2016

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Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are regularly investigated in sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiencies in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to 20-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid-preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72 h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane-intact spermatozoa, supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa.

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Section: Discussionmentioning

confidence: 79%

“…The energy charge was determined by modification of procedures described by Ford and Leach (1998). The modifications are described below.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction

et al. 2016

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“…After centrifugation for 15 min at 20,000g, the supernatant was recovered and neutralized using a few drops of 2.5 M K 2 CO 3 . Adenylates were measured according to Ford and Leach (1998) using the ATP Bioluminescent Assay Kit (Sigma).…”

Section: Measurements Of Adenylate Concentrationsmentioning

confidence: 99%

Remodeled Respiration in ndufs4 with Low Phosphorylation Efficiency Suppresses Arabidopsis Germination and Growth and Alters Control of Metabolism at Night

et al. 2009

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Respiratory oxidative phosphorylation is a cornerstone of cellular metabolism in aerobic multicellular organisms. The efficiency of this process is generally assumed to be maximized, but the presence of dynamically regulated nonphosphorylating bypasses implies that plants can alter phosphorylation efficiency and can benefit from lowered energy generation during respiration under certain conditions. We characterized an Arabidopsis (Arabidopsis thaliana) mutant, ndufs4 (for NADH dehydrogenase [ubiquinone] fragment S subunit 4), lacking complex I of the respiratory chain, which has constitutively lowered phosphorylation efficiency. Through analysis of the changes to mitochondrial function as well as whole cell transcripts and metabolites, we provide insights into how cellular metabolism flexibly adapts to reduced phosphorylation efficiency and why this state may benefit the plant by providing moderate stress tolerance. We show that removal of the single protein subunit NDUFS4 prevents assembly of complex I and removes its function from mitochondria without pleiotropic effects on other respiratory components. However, the lack of complex I promotes broad changes in the nuclear transcriptome governing growth and photosynthetic function. We observed increases in organic acid and amino acid pools in the mutant, especially at night, concomitant with alteration of the adenylate content. While germination is delayed, this can be rescued by application of gibberellic acid, and root growth assays of seedlings show enhanced tolerance to cold, mild salt, and osmotic stress. We discuss these observations in the light of recent data on the knockout of nonphosphorylating respiratory bypass enzymes that show opposite changes in metabolites and stress sensitivity. Our data suggest that the absence of complex I alters the adenylate control of cellular metabolism.

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Halford

Samson

Kwan

et al. 2003

Molecular and Cellular Biochemistry

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Here, we report that the smooth muscle and endothelium of the pig coronary artery differ in the profiles of energy metabolism nucleotides. ATP levels in the freshly isolated smooth muscle (1490 +/- 93, all the values are in pmol/mg protein) were significantly greater than in the endothelium (418 +/- 68). In contrast, endothelium contained higher levels of NADH (328 +/- 21), NAD+ (1210 -/+ 28), NADPH (87 +/- 2), and NADP+ (77 +/- 4) than smooth muscle (17 +/- 2, 96 +/- 14, 7 +/- 1, and 8 +/- 1, respectively). However, smooth muscle and endothelium do not differ from each other in the ratios of NADH/NAD+ or NADPH/NADP+. Cells cultured from smooth muscle and endothelium contained less ATP (93 +/- 2, 141 +/- 6) and had lower ratios of NADH/ NAD+ than the freshly isolated tissues but the NADPH/NADP+ ratios remained similar. We conclude that (a) freshly isolated smooth muscle and endothelium differ in their profiles of the energy metabolism nucleotides, and (b) culturing the cells alters the profile.

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Bioluminescent Assay of the Adenylate Energy Charge

Cited by 14 publications

References 23 publications

Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction

Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction

Remodeled Respiration in ndufs4 with Low Phosphorylation Efficiency Suppresses Arabidopsis Germination and Growth and Alters Control of Metabolism at Night

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