Leukotriene D4 and E4 formation by plasma membrane bound enzymes

Area of Science:

• Biochemistry
• Cell Biology
• Immunology

Background:

• Leukotrienes (LT) are inflammatory mediators derived from arachidonic acid.
• Rat basophilic leukemia cells synthesize both dihydroxy-leukotrienes and peptido-leukotrienes (LT C4, D4, E4).
• Enzymatic pathways for LT synthesis involve soluble and particulate fractions.

Purpose of the Study:

• To elucidate the subcellular localization of enzymes responsible for peptido-leukotriene metabolism.
• To identify the specific enzymes involved in the conversion of leukotriene C4 to D4 and E4.
• To determine if these enzymes are membrane-bound.

Main Methods:

• Differential centrifugation of rat basophilic leukemia cell homogenate.
• Sucrose gradient centrifugation to isolate membrane and pellet fractions.
• Incubation of fractions with leukotriene C4, followed by High-Performance Liquid Chromatography (HPLC) and UV spectroscopy for product identification.
• Enzyme inhibition studies using serine.borate and cysteine.

Main Results:

• The soluble fraction contained enzymes for LTA4 and LTB4 synthesis.
• Particulate fractions (10,000 X g pellet) contained enzymes for LTC4, LTD4, and LTE4 synthesis.
• Further fractionation identified gamma-glutamyl transpeptidase and amino peptidase in the membrane fraction, responsible for LTC4 to LTD4 and LTD4 to LTE4 conversion, respectively.
• Serine.borate inhibited LTD4 formation, and cysteine inhibited LTE4 formation.
• The sucrose pellet showed minimal conversion of LTC4 to LTD4.

Conclusions:

• Gamma-glutamyl transpeptidase and amino peptidase, crucial for LTD4 and LTE4 production, are localized to the plasma membrane.
• These findings clarify the cellular location of key enzymes in peptido-leukotriene metabolism.