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Localization of 5-lipoxygenase within human polymorphonuclear leukocytes
Abstract:
Human polymorphonuclear leukocytes (PMN) stimulated with the Ca-ionophore A23187 or opsonized zymosan not only release marker enzymes of specific granules but secrete 5-lipoxygenase activity as well. In the presence of BSA cells incubated with [14C]AA were able to synthetize 5-HPETE but failed to produce 5-HETE, LTB4, and its omega-oxidation metabolites. Subcellular fractionation studies by differential and isopycnic equilibrium density centrifugation demonstrated main lipoxygenase activity in particulate fractions consisting of specific granules, but not in cytosolic fractions. These results suggest the association of 5-lipoxygenase with specific granules. 5-lipoxygenase released from the cells upon appropriate stimulation reached its peak activity after 10 min and was then rapidly inactivated. It appears that the intermediate 5-HPETE may be generated extracellularly but has to re-enter the intracellular space for further metabolization.
Insights
Human polymorphonuclear leukocytes (PMN) secrete 5-lipoxygenase activity upon stimulation. This enzyme is primarily associated with specific granules, suggesting its role in inflammatory responses.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Human polymorphonuclear leukocytes (PMN) are key immune cells involved in inflammatory processes.
- The release of enzymes from granules is a critical mechanism in PMN function.
- Lipoxygenase activity plays a role in the synthesis of inflammatory mediators.
Purpose of the Study:
- To investigate the localization and activity of 5-lipoxygenase in human PMN.
- To determine the role of specific granules in 5-lipoxygenase secretion and activity.
- To elucidate the metabolic fate of arachidonic acid in stimulated PMN.
Main Methods:
- Human PMN were stimulated with Ca-ionophore A23187 or opsonized zymosan.
- Cells were incubated with [14C]arachidonic acid to track metabolite synthesis.
- Subcellular fractionation using differential and isopycnic equilibrium density centrifugation was performed.
- Lipoxygenase activity was measured in different cellular fractions.
Main Results:
- Stimulated PMN released 5-lipoxygenase activity along with granule marker enzymes.
- Synthesis of 5-HPETE was observed, but not 5-HETE or LTB4, in the presence of BSA.
- 5-lipoxygenase activity was predominantly found in particulate fractions, specifically associated with specific granules.
- Extracellularly generated 5-HPETE may require re-entry into the cell for further metabolism.
Conclusions:
- 5-lipoxygenase is associated with specific granules in human PMN.
- Secretion of 5-lipoxygenase occurs upon cellular stimulation.
- The localization suggests a role for specific granules in regulating inflammatory mediator production.