Biosynthesis of prostaglandins

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Cyclooxygenase (COX) is a key enzyme in prostaglandin synthesis.
• The activity of COX can be modulated by various factors, including porphyrins.
• Understanding the structure and activation mechanism of COX is crucial for drug development.

Purpose of the Study:

• To investigate the stimulatory effect of protoporphyrin IX on purified sheep vesicular gland cyclooxygenase.
• To elucidate the role of heme and non-heme iron in the activation of cyclooxygenase.
• To determine the subunit composition and quaternary structure of the active cyclooxygenase holoenzyme.

Main Methods:

• Purification of cyclooxygenase from sheep vesicular glands.
• Enzyme activity assays in the presence of protoporphyrin IX.
• Determination of molecular weight and subunit composition using techniques like gel filtration and SDS-PAGE (implied).
• Analysis of heme and non-heme iron content.

Main Results:

• Highly purified cyclooxygenase is stimulated by protoporphyrin IX.
• Enzyme activation involves the binding of heme, with full activity achieved when half of the 70,000-dalton subunits bind heme.
• Approximately half of the subunits contain non-heme iron.
• The holoenzyme has an apparent molecular weight of ~300,000 daltons, suggesting a tetrameric complex (four 70,000-dalton subunits).
• Heme and non-heme iron appear to bind to distinct subunits.

Conclusions:

• Protoporphyrin IX acts as a cofactor, converting cyclooxygenase apoenzyme to its active holoenzyme form.
• The cyclooxygenase holoenzyme is a tetramer composed of four 70,000-dalton subunits.
• A proposed A2B2 arrangement suggests distinct subunits for heme and non-heme iron binding, contributing to enzyme activity.