Validation of methodologies for the study of horm one-sensitive lipase in lactating cows adipose tissue

Abstract

The objectives of this study were: a) to validate techniques to measure HSL in adipose tissue from lactating cows: and b) to measure activation of HSL by second messengers responsible for HSL phosphorylation. Radioactive labelled trioleine (³H-trioleina) was used as a substrate. The emulsion was prepared by sonication in a buffer solution containing 0.1M phosphate, pH= 7.0, which minimized interference of lipoprotein lipase in the assay. Utilization of a mixture of 25mg/ml de phosphatidil-choline and 15mg)ml phosphatidil-ethanolamine as emulsifiers produced the higher HSL activities. Enzyme was obtained by homogeneization, centrifugation at 90,000xg (50 min) and precipitation at pH= 5.2. Linearity of the assay was demonstrated for changes in amount of enzyme and reaction time. HSL was activated in vitro with cyclic dibutiril AMP (dbcAMP), ATP, protein kinase A and MgCl₂, and these are the first published results in the literature for ruminants. Concentrations of 2mM ATP and 3 μM dbcAMP were necessary to maximize activation of HSL. Kinectic studies of HSL demonstrated that the K_m for the bovine enzyme was 0.3mM of trioleina. Activation of HSL by dbcAMP, determined in various concentrations of substrate, was proportionally higher in concentrations near K_m, suggesting that activation alters the interface enzyme-lipid droplet, and not the turnover number. In conclusion, the methodology allows the determination of HSL activity and the activation of the enzyme appears to be the result of increased affinity for the substrate.