Techniques to measure lipase and esterase activity in vitro

Gilham, Dean; Lehner, Richard

doi:10.1016/j.ymeth.2004.11.003

Cited by 178 publications

(133 citation statements)

References 72 publications

Supporting

Mentioning

128

Contrasting

Unclassified

Order By: Relevance

“…Cells were then washed with Tris-buffered saline and harvested in an ice-cold homogenization buffer, and cytosolic and membrane fractions were prepared as described above. Lipolytic activities in cytosols or membranes were assessed by mixing 20 l of 250 M 4-methylumbelliferyl heptanoate in reaction buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 300 M taurodeoxycholate), 20 l of enzyme preparation (cytosol or membranes), and 60 l of the reaction buffer in 96-well black plates and incubating at room temperature with shaking (24). The fluorescence of the reaction product was continuously recorded over a 5-min period using FluoroScan Ascent FL (Thermo Labsystems) at 355 and 460 nm as excitation and emission wavelengths, respectively.…”

Section: Methodsmentioning

confidence: 99%

Attenuation of Adipocyte Triacylglycerol Hydrolase Activity Decreases Basal Fatty Acid Efflux

Wei¹,

Gao²,

Lehner

2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Fatty acids released from adipose triacylglycerol stores by lipolysis provide vertebrates with an important source of energy. We investigated the role of microsomal triacylglycerol hydrolase (TGH) in the mobilization of adipocyte triacylglycerols through inactivation of the TGH activity by RNA interference or chemical inhibition. Attenuation of TGH activity resulted in decreased basal but not isoproterenol-stimulated efflux of fatty acids from 3T3-L1 adipocytes. Lack of TGH activity was accompanied by accumulation of cellular triacylglycerols and cholesteryl esters without any changes in the expression of enzymes catalyzing triacylglycerol synthesis (diacylglycerol acyltransferases 1 and 2) or degradation (adipose triglyceride lipase and hormone-sensitive lipase). Inhibition of TGH-mediated lipolysis also did not affect insulin-stimulated Glut4 translocation from intracellular compartments to the plasma membrane or glucose uptake into adipocytes. These data suggest that TGH plays a role in adipose tissue triacylglycerol metabolism and may be a suitable pharmacological target for lowering fatty acid efflux from adipose tissue without altering glucose import.

show abstract

Section: Methodsmentioning

confidence: 99%

Attenuation of Adipocyte Triacylglycerol Hydrolase Activity Decreases Basal Fatty Acid Efflux

Wei¹,

Gao²,

Lehner

2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, that may affect enzyme stability because of toxicity and water immiscibility (Lehner and Verger 1997). Other dispersing systems have been used, such as detergents, lipids, bile acids, etc., that denatured the enzyme, which resulted in loss of activity (Gilham and Lehner 2005).…”

Section: Co-solvent For Promoting Substrate Solubilitymentioning

confidence: 99%

“…Various methods for esterase activity determination have been reported, such as inter alia, titration (Gilham and Lehner 2005;Schmidt and Bornscheuer 2005), high performance liquid chromatography (HPLC) (Faulds and Williamson 1995;O'Neill et al 1996;Barbe and Dubourdieu 1998;Yue et al 2009), and gas chromatography (GC) (Borneman et al 1990). All ascertain enzyme activity through the determination of acid or other hydrolytes after enzyme action.…”

Section: Introductionmentioning

confidence: 99%

Accurately Determining Esterase Activity via the Isosbestic Point of p-Nitrophenol

Peng¹,

Fu²,

Líu³

et al. 2016

BioResources

View full text Add to dashboard Cite

Esterase is an important enzyme for ester hydrolysis or synthesis. Its activity, however, has not been accurately ascertained due to a lack of accurate protocols. In this study, the isosbestic point of p-nitrophenol was found and used as the marker for its activity. The methodology avoided decomposition of the substrate, chromophore agents, and pH changes. The esterase activity was determined accurately and rapidly in a complex solution. In this protocol system, organic solvents were used for dissolving substrates, which influenced activity determination to some extent. Among the solvents tested, methanol exerted the least inhibitory influence. The results indicated that this modified method has potential to be applied for esterase activity determination on a large scale and in real time.

show abstract

“…Besides, lipases work as well in the presence of water. In fact, they need a certain hydric activity to maintain their tridimensional structure, so the presence of water is not a problem with this kind of catalyst -although excessive hydric activity affects the transesterification reaction because the substrates are water insoluble (Jaeger & Eggert, 2002;Shah et al, 2004;Gilham & Lehner, 2005;Fjerbaek et al, 2009). Lipases can operate at low or relatively low temperatures in the range of 20 to 70ºC, and at even lower temperatures if the enzyme has been obtained from psycrophilic microorganisms (Dabkowska & Szewczyk, 2009).…”

Section: Ph Temperature and Hydric Activitymentioning

confidence: 99%