Enzyme Activity Assays Research Papers (original) (raw)

Global climate change is expected to intensify hypoxic conditions during coastal upwelling events along the west coast of North America, but the potential effects on nearshore fishes are not well understood. Viviparous rockfishes (genus Sebastes) that gestate and give birth during the upwelling season, such as gopher rockfish (S. carnatus), may be among the most affected. We tested the transgenerational effects of future hypoxic conditions on the metabolic status of gopher rockfish larvae. Four pregnant females were held during gestation in either ambient conditions (DO~8 mg/L) or a hypoxic treatment (DO~4 mg/L). A subset of larvae was sampled immediately post-parturition and remaining larvae were then split into two groups and placed in either control or hypoxic conditions for five days and sampled again. Citrate synthase (CS) and lactate dehydrogenase (LDH), proxies for aerobic and anaerobic metabolism, respectively, were assayed in pooled larval samples from all treatments. We expected that larvae would exhibit relatively greater reliance on aerobic metabolism (e.g., higher CS/LDH ratios) if exposure to hypoxia during pregnancy enhances larval performance to hypoxia post-parturition, compared with larvae from control mothers. We also expected that larvae from control mothers would exhibit relatively greater reliance on anaerobic metabolism if exposed to hypoxic post-parturition. Lastly, we hypothesized that larvae from hypoxic mothers would rely more on anaerobic metabolism immediately post-parturition. At birth, we found larvae from hypoxic mothers were more anaerobic compared to those from control mothers, but after five days we found trends that larvae from hypoxic mothers that continued developing in hypoxic conditions were more aerobic compared to those from control mothers. Our findings suggest the potential for larval gopher rockfish to exhibit a more aerobic phenotype when gestated during hypoxic conditions and placed in similar conditions post-parturition.

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- Hypoxia, Anaerobic Metabolism, Rockfish, Larval fish biology
- by John D Ferchak
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- Cellulase, Enzyme Activity Assays

Colorimetric methods are convenient for the determination of inorganic phosphate. However, the acidic conditions required can complicate measurement of ATPase through non-enzymatic ATP hydrolysis. Here we present an optimized antimony-phosphomolybdate microassay for the simple and rapid detection of ATPase activity, with micromolar sensitivity. The low acidity of the color reagent results in no interference for samples containing up to 0.5-5 mM ATP, dependent on the sample volume. The assay is compatible with common assay conditions and was similar in accuracy to an established continuous method. The simplicity of this method makes it ideal for medium to high throughput applications.

Gynura procumbens has been well known for its various medicinal properties such as being an antioxidant. It would be benecial to be able to dry and preserve the leaves of G. procumbens for commercial utilisation. However, enzymes such as polyphenol oxidases (PPO) and peroxidases (POD) may degrade the antioxidants present. It was found previously that air-drying at ambient temperatures, where PPO and POD activity should be at its optimal temperature, did not signicantly reduce the antioxidant activities in G. procumbens. However, oven-drying at 50°C and 100°C reduced the antioxidant properties signicantly. Therefore, this study aims to determine the effect of different methods of drying leaves on the enzyme activity of PPO and POD, such as open ambient air-drying, drying in a desiccator at 25% relative humidity, oven-drying at 50°C and 100°C. Results show that all methods of drying that were used fully diminished the enzyme activity of PPO. However, for POD, only oven-drying at 100°C and blanching were able to fully diminish the enzyme activity. Additionally, the desiccator shortened the drying time of open-air-drying by 2–5 days. For commercial utilisation, drying the leaves in a lowered relative humidity would be the best choice.

- by Jia Jun Fung and +1
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- Peroxidase, Gynura procumbens, Polyphenol Oxidase, Total Phenolic Content
- by taryn bodill
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- Organic Chemistry, Computer Simulation, Oxidoreductases, Drug Design

Context: Mara¸sMara¸s powder (MP), a different type of smokeless tobacco (ST) prepared from a tobacco called Nicotiana rustica Linn, is widely used in the Southern Turkey. Smoking and ST cause oxidative stress (OS) in the human body. Paraoxonase (PON) and arylesterase (ARE) are antioxidant enzymes. Objective: To investigate the effects of MP on activities of PON, ARE, and malondialdehyde (MDA) levels in plasma and to compare these parameters in smokers and MP users (MPU). Materials and methods: The study consisted of smokers, MPU, and control group (CG) neither smoking nor using MP healthy subjects. PON and ARE activities were measured spectrophoto-metrically using paraoxon and phenylacetate substrates, respectively. Results: PON and ARE activities were decreased whereas MDA levels were increased in tobacco groups compared to the CG. The differences of ARE and MDA values between the tobacco groups and CG were found statistically significant (p50.01). But no significant differences were detected between the groups in the activity of PON (p40.05). However, the lowest activities of the enzymes were obtained in MPU. Conclusion: Our results can help to evaluate harmful effects of cigarette and ST as MP. These effects can be attributed to increased OS. Increased plasma MDA levels and decreased ARE activities may be important in assessing oxidant/antioxidant imbalance in MPU as well as smokers. Also, using of MP has harmful effects at least cigarette smoking.

- by Naciye Kurtul and +1
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- Maras Powder, Smokers, Enzyme Activity Assays

High levels of recombinant protein expression can lead to the formation of insoluble inclusion bodies.These complex aggregates are commonly solubilized in strong denaturants, such as 6–8 M urea, although, if possible, solubilization under milder conditions could facilitate subsequent refolding and purification of bioactive proteins. Commercially available GST-tag assays are designed for quantitative measurement of GST activity under native conditions. GST fusion proteins accumulated in inclusion bodies are considered to be undetectable by such assays. In this work, solubilization of recombinantly produced proteins was performed in 4 M urea. The activity of rGST was assayed in 2 M urea and it was shown that rGST preserves 85% of its activity under such denaturing conditions. A colorimetric GST activity assay with 1-chloro-2, 4- dinitrobenzene (CDNB) was examined for use in rapid detection of expression targeted to inclusion bodies and for the identification of inclusion body proteins which can be solubilized in low concentrations of chaotropic agents. Applicability of the assay was evaluated by tracking protein expression of two GST fused allergens of biopharmaceutical value in E. coli, GST-Der p 2 and GST-Mus a 5, both targeted to inclusion bodies.

- by Milica Grozdanovic
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- Enzymology, Protein Purification, Protein Folding, Protein Engineering