Interaction between NADPH-cytochrome P-450 reductase and hepatic microsomes

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Biology

Background:

• NADPH-cytochrome P-450 reductase is crucial for microsomal enzyme activity.
• Microsomal enzyme activity can be modulated by reductase levels.

Purpose of the Study:

• To investigate the incorporation of purified NADPH-cytochrome P-450 reductase into microsomal membranes.
• To determine the functional consequences of reductase incorporation on enzyme activity.
• To explore the organization of reductase and cytochrome P-450 in the membrane.

Main Methods:

• Purification of NADPH-cytochrome P-450 reductase from rat liver microsomes.
• Assaying reductase incorporation by measuring ethoxycoumarin O-dealkylase activity enhancement.
• Separating incorporated reductase from unbound enzyme using Sepharose 4B gel filtration.
• Studying the reductase-hydroxylase activity relationship in trypsin-treated microsomes.

Main Results:

• Solubilized NADPH-cytochrome P-450 reductase enhanced various microsomal monoxygenase activities.
• Reductase incorporation into microsomes was temperature-dependent, with maximal incorporation at 37°C after 30 min.
• Trypsin treatment of microsomes removed reductase, decreasing hydroxylase activity, which was restored by adding purified reductase.

Conclusions:

• Purified NADPH-cytochrome P-450 reductase can be incorporated into the microsomal membrane.
• Incorporated reductase interacts with cytochrome P-450, restoring enzyme activity.
• Findings support a non-rigid model for the organization of P-450 and reductase in the microsomal membrane.