Dependence on Ca2+ of lipoprotein lipase stability
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1Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel.
Abstract:
Macrophages continuously secrete lipoprotein lipase (LPL) into the culture medium. When LPL was collected from thioglycollate-elicited peritoneal macrophages (Tg-Mø) or J774.1 cells over a 4 h period in Ca2+ and Mg2(+)-free Dulbecco's modified Eagle's medium (d-DMEM) the activity in the collection medium was reduced by 40-62% and 23%, respectively, as compared to that expressed in full medium (DMEM). Ca2+ supplementation during the collection period in d-DMEM augmented LPL activity in the medium; about 1 mM Ca2+ was required for attainment of activity comparable to that expressed in DMEM. Addition of Ca2+ during the assay did not enhance LPL activity collected into d-DMEM. Addition of EGTA to the assay mixture reduced LPL activity by 34-60% and when present in the collection medium, EGTA led to a reduction in enzyme activity greater than 90%. A 4 h incubation of Tg-Mø in 3 mM EGTA led to an almost complete loss of intracellular Ca2+ (measured by efflux of 45Ca2+ from preloaded cells), yet there was no change in the overall synthesis and secretion of proteins and in the phagocytic capability of the cells. LPL activity in the enzyme collection medium after its removal from cell monolayers was stable at least up to 4 h at 0 degrees C and at 23 degrees C. Activity was progressively lost with increased temperatures: up to 40% loss at 37 degrees C in 4 h. Addition of EGTA to the above medium led to an enhanced rate of irreversible enzyme inactivation: 76-86% loss of activity in 4 h at 37 degrees C. No inactivation was observed at 0 degrees C and at 23 degrees C in the presence of EGTA. The results indicate a critical role for Ca2+ in enzyme stabilization.
Insights
Calcium ions (Ca2+) are crucial for stabilizing lipoprotein lipase (LPL) secreted by macrophages. Removing Ca2+ significantly reduces LPL activity, highlighting its essential role in enzyme stability.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Macrophages secrete lipoprotein lipase (LPL), an enzyme involved in lipid metabolism.
- The stability and activity of secreted LPL are critical for its biological function.
- Previous studies have not fully elucidated the factors influencing LPL stability in the extracellular environment.
Purpose of the Study:
- To investigate the role of calcium ions (Ca2+) in the stability and activity of macrophage-secreted LPL.
- To determine the effect of Ca2+ and chelating agents on LPL activity during collection and under different temperature conditions.
Main Methods:
- Lipoprotein lipase (LPL) was collected from thioglycollate-elicited peritoneal macrophages (Tg-Mø) and J774.1 cells in media with varying Ca2+ and Mg2+ concentrations.
- Ethylene glycol-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) was used to chelate Ca2+ during collection and enzyme assays.
- Intracellular Ca2+ levels were measured using 45Ca2+ efflux.
- LPL activity was assessed under different temperatures and in the presence of EGTA.
Main Results:
- LPL activity collected in Ca2+-free medium was significantly reduced compared to full medium.
- Ca2+ supplementation during collection restored LPL activity, with optimal levels around 1 mM.
- EGTA in the collection medium drastically reduced LPL activity (>90%).
- EGTA also accelerated LPL inactivation at 37°C, while Ca2+ stabilized the enzyme.
- Intracellular Ca2+ depletion did not affect protein synthesis or phagocytosis but was linked to reduced secreted LPL activity.
Conclusions:
- Calcium ions play a critical role in stabilizing secreted lipoprotein lipase (LPL) from macrophages.
- The presence of Ca2+ is essential for maintaining LPL activity in the extracellular medium.
- EGTA-induced Ca2+ depletion leads to significant irreversible inactivation of LPL, particularly at physiological temperatures.