Titration Calorimetry as a Tool to Determine Thermodynamic and Kinetic Parameters of Enzymes

Bianconi, M. Lucia

doi:10.1002/0470011122.ch9

Cited by 4 publications

(3 citation statements)

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“…The advantage with ITC is the possibility to perform continuous measurements of LPL activity in plasma, using changes in heat rate for determination of the reaction rate. There is, in general, a linear relationship between the reaction rate and the heat rate (19). However, in the case of lipolysis in plasma, the heat rate includes heat effects of at least four different processes: hydrolysis of ester bonds, structural changes of the lipoproteins due to removal of lipids, neutralization of the released conditions, a reduction of the reaction rate by apoC-III was observed at the start of the reaction and the reduction remained unchanged for more than 1 h (data not shown).…”

Section: Discussionmentioning

confidence: 99%

“…ITC provides a continuous assay using the observable heat rate that is directly proportional to the rate of the lipolysis (19). Raw data from ITC experiments are presented as thermograms in which the changes in the heat rate (also named heat flow, thermal power or heat flux) are monitored at a constant temperature.…”

Section: Itc Measurementsmentioning

confidence: 99%

“…4B). The slope of this relationship was used for calculation of the apparent enthalpy H, using the equation: Q = [P]VH, where Q is the total heat production, P is the concentration of released fatty acids, and V is the volume of the ITC cell (19). This calculation resulted in a H value equal to 38.8 kJ/mol.…”

Section: Itc Can Be Used To Record Complete Hydrolysis Of Plasma Lipimentioning

confidence: 99%

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Lipoprotein lipase activity and interactions studied in human plasma by isothermal titration calorimetry

Reimund

Kovrov

Olivecrona

et al. 2017

Journal of Lipid Research

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LPL is produced and secreted from parenchymal cells like adipocytes and myocytes for transport to the luminal side of the endothelium via interaction with the glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1) (8). Several plasma components have been shown to directly or indirectly modulate the activity of LPL. apoC-II and apoA-V increase the activity of LPL, while apoC-I, apoC-III, angiopoietin-like protein (ANGPTL)3, ANGPTL4, and ANGPTL8 decrease the activity (1, 9). The expression of each of these proteins depends on nutritional and hormonal factors, so that lipid uptake in tissues to a large extent is regulated by posttranslational effects on LPL (1, 9). It is possible that the macromolecular environment in plasma itself may be an influence on the interaction of LPL with its ligands. The protein concentration of plasma (80 g/l) has been shown to cause significant crowding effects (10). It is also possible that some plasma regulators of LPL activity have not been identified yet.LPL activity can be measured in vitro by using artificial, usually emulsified, systems of radiolabeled, fluorogenic, or chromogenic substrates, or isolated triglyceride-rich lipoproteins (TRLs). The reaction products are detected at certain time points by chemical quantification or by determination of radioactivity or fluorescence. These methods have been used to unravel important aspects of the action of LPL and also to quantitate the levels of LPL activity in cells and tissues. Only small amounts of LPL activity are normally present in the circulating blood (11). Therefore, intravenous injections of heparin are made to release LPL from its endothelial binding sites. Determination of LPL activity in postheparin plasma, using artificial substrate systems, is considered to give an estimation of the amount of active LPL at the vascular endothelium (12).Lack of a suitable technique for continuous monitoring of triglyceride hydrolysis in plasma has hampered the understanding of the action of LPL under physiological Abstract LPL hydrolyzes triglycerides in plasma lipoproteins. Due to the complex regulation mechanism, it has been difficult to mimic the physiological conditions under which LPL acts in vitro. We demonstrate that isothermal titration calorimetry (ITC), using human plasma as substrate, overcomes several limitations of previously used techniques. The high sensitivity of ITC allows continuous recording of the heat released during hydrolysis. Both initial rates and kinetics for complete hydrolysis of plasma lipids can be studied. The hydrolytic breakdown of plasma triglycerides by LPL at the capillary endothelium is a crucial event that contributes to control of the levels of triglycerides in plasma (1, 2). Many recent studies support the view that an elevated level of triglycerides in plasma is an independent risk factor for development of atherosclerosis (3-5). Therefore, the LPL system is considered to be an interesting target for drug design (6, 7). Education and Research Grant IUT 19-9,...

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Section: Discussionmentioning

confidence: 99%

Section: Itc Measurementsmentioning

confidence: 99%

Section: Itc Can Be Used To Record Complete Hydrolysis Of Plasma Lipimentioning

confidence: 99%

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