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Release and functional characterization of the leukotriene D4-metabolizing enzyme (dipeptidase) from human
Abstract:
Polymorphonuclear leucocytes released LTD4-dipeptidase activity in a time-, calcium-, and cell number-dependent fashion. The LTD4-dipeptidase released from polymorphonuclear leucocytes (PMN) by incubation with calcium (0.91 mM) was detectable up to a cell concentration of 1 X 10(6)/ml and increased with higher concentrations. Maximal LTD4-dipeptidase activity within the extracellular environment was detected after 15 min of incubation (2 X 10(7)/ml) in the presence of 2-4.5 mM calcium or after 30 min, when stimulation was carried out with 0.91 mM calcium. The activity of the released LTD4-dipeptidase was modulated by various metal ions and other compounds. The addition of Mn2+, Co2+, and Zn2+ (final concentration 1 mM) enhanced the LTD4-dipeptidase activity, while Cu2+ led to a complete inhibition. In the absence of exogenous calcium EDTA inhibited LTD4-dipeptidase. Calcium up to a concentration of 5 and 10 mM decreased the dipeptidase activity. The LTD4-dipeptidase is not affected by bestatin, leupeptin, or N-ethyl-maleinimide (NEM). The Km of LTD4-dipeptidase for LTD4 was 0.95 +/- 0.2 microM and Vmax was 737.5 +/- 112.5 pmol/min X mg protein (n = 3 +/- SEM). The highest LTD4-dipeptidase activity was obtained at physiological pH values. LTD4-dipeptidase activity can also be released from other cell types, but the enzyme activity from human PMN exceeded that of other cells (e.g. human lymphocytes/monocytes and basophils (LMB) and human lung cell suspension).
Insights
Polymorphonuclear leucocytes release LTD4-dipeptidase, an enzyme crucial for regulating inflammatory responses. This release is influenced by calcium levels and cell concentration, with specific metal ions modulating its activity.
Area of Science:
- Biochemistry
- Immunology
- Cell Biology
Background:
- Leukocytes, particularly polymorphonuclear leucocytes (PMN), play a critical role in inflammatory processes.
- Leukotriene D4 (LTD4) is a potent mediator of inflammation, and its metabolism is a target for therapeutic intervention.
- The enzymatic activity of LTD4-dipeptidase and its release from immune cells are not fully characterized.
Purpose of the Study:
- To investigate the release of LTD4-dipeptidase from human polymorphonuclear leucocytes (PMN).
- To determine the factors influencing the release and activity of PMN-derived LTD4-dipeptidase.
- To characterize the kinetic properties and modulators of this enzyme.
Main Methods:
- Human PMN were incubated under various conditions, including different calcium concentrations, incubation times, and cell densities.
- Extracellular LTD4-dipeptidase activity was measured.
- The effects of metal ions, chelating agents, and specific inhibitors were assessed.
- Enzyme kinetics (Km and Vmax) were determined using LTD4 as a substrate.
- pH dependence was evaluated.
Main Results:
- LTD4-dipeptidase was released from PMN in a time-, calcium-, and cell number-dependent manner.
- Maximal release occurred at specific calcium concentrations (2-4.5 mM) and incubation times (15-30 min).
- Mn2+, Co2+, and Zn2+ enhanced enzyme activity, while Cu2+ inhibited it; EDTA inhibited activity in the absence of calcium.
- The enzyme exhibited a Km of 0.95 +/- 0.2 microM for LTD4 and Vmax of 737.5 +/- 112.5 pmol/min x mg protein, with optimal activity at physiological pH.
- PMN-derived LTD4-dipeptidase activity exceeded that of other tested human cell types (lymphocytes, monocytes, basophils, lung cells).
Conclusions:
- Human PMN are a significant source of extracellular LTD4-dipeptidase.
- Calcium concentration and metal ions critically modulate the release and activity of this enzyme.
- The characterized kinetic properties and modulators provide insights into the enzyme's function in inflammatory pathways.
- PMN-derived LTD4-dipeptidase represents a potentially important factor in regulating LTD4 metabolism in inflammatory conditions.