Related Experiment Videos
Comparison of properties of membrane bound versus soluble forms of human leukocytic elastase and cathepsin G
1American Red Cross Holland Laboratory, Rockville, MD 20855.
Abstract:
Stimulation of human neutrophils with micromolar concentrations of N-formyl-methionyl-leucyl-phenyl-alanine (fMLP) or 4 beta-phorbol-12 beta-myristate-13 alpha-acetate (PMA), results in their degranulation and/or lysis with a concomitant release of Human Leucocyte Elastase (HLE; EC 3.4.21.37), Cathepsin G (cat G; EC 3.4.21.20) and Myeloperoxidase (MPO; EC 1.11.1.7) into the surrounding medium, some of which re-bind in an active form to neutrophil plasma membranes or membrane fragments. Histones, when present in the medium, prevent this association indicating that it is largely charge dependent. Bound proteinases have an increased resistance to inhibition by protein proteinase inhibitors, while bound MPO retains its ability to oxidatively inactivate alpha-1-proteinase inhibitor (alpha-1-PI). The attachment of oxidases and proteinases to plasma membrane or its fragments may allow them to remain active in an environment replete with proteinase inhibitors and, therefore, may be responsible for neutrophil or neutrophil-debris mediated tissue damage.
Insights
Stimulating human neutrophils releases active enzymes like Human Leucocyte Elastase and Myeloperoxidase. These enzymes bind to cell membranes, resisting inhibitors and potentially causing tissue damage.
Area of Science:
- Immunology
- Biochemistry
- Cell Biology
Background:
- Human neutrophils release enzymes such as Human Leucocyte Elastase (HLE), Cathepsin G (cat G), and Myeloperoxidase (MPO) upon stimulation.
- These released enzymes can re-bind to neutrophil membranes or fragments in an active form.
- Histones can prevent this re-binding, suggesting a charge-dependent interaction.
Purpose of the Study:
- To investigate the binding of neutrophil-released enzymes to plasma membranes.
- To understand the functional consequences of this enzyme-membrane association.
- To explore the role of this phenomenon in neutrophil-mediated tissue damage.
Main Methods:
- Stimulation of human neutrophils with N-formyl-methionyl-leucyl-phenyl-alanine (fMLP) or phorbol ester (PMA).
- Analysis of enzyme release (HLE, cat G, MPO) into the medium.
- Detection of enzyme re-binding to neutrophil membranes or membrane fragments.
- Assessment of enzyme activity and resistance to inhibitors after binding.
- Investigation of histone effects on enzyme-membrane association.
Main Results:
- Neutrophil stimulation with fMLP or PMA led to degranulation/lysis and release of HLE, cat G, and MPO.
- A portion of these released enzymes re-bound to neutrophil plasma membranes or fragments in an active state.
- Histones inhibited this enzyme-membrane association, indicating its charge-dependent nature.
- Bound proteinases exhibited increased resistance to proteinase inhibitors.
- Bound MPO retained its capacity to inactivate alpha-1-proteinase inhibitor (alpha-1-PI).
Conclusions:
- Enzyme attachment to neutrophil membranes enhances their stability against inhibitors.
- This membrane association may allow enzymes to remain active in inhibitor-rich environments.
- Neutrophil- or neutrophil-debris-mediated tissue damage could result from these active, membrane-bound enzymes.