Pressure-induced structural changes of pig heart lactic dehydrogenase

Area of Science:

• Biochemistry
• Protein Structure and Dynamics
• High-Pressure Science

Background:

• Lactic dehydrogenase (LDH) is a crucial enzyme in cellular metabolism.
• Understanding the effects of extreme conditions like high hydrostatic pressure on enzyme structure and function is vital for various scientific fields.
• Pig heart LDH isoenzyme exhibits unique quaternary structure stability characteristics.

Purpose of the Study:

• To investigate the reversible dissociation and denaturation of pig heart lactic dehydrogenase under high hydrostatic pressure.
• To characterize the structural and functional changes of LDH monomers at elevated pressures.
• To determine the role of the coenzyme NAD+ in stabilizing the enzyme structure.

Main Methods:

• High hydrostatic pressure application (above 1000 bar).
• Fluorescence spectroscopy to monitor protein unfolding and dissociation.
• Enzyme activity assays to measure deactivation and reconstitution.
• Hybridization experiments to identify dissociation products.

Main Results:

• Pig heart LDH reversibly dissociates into homogeneous monomers above 1000 bar, at lower pressures than the skeletal muscle isoenzyme.
• High pressure causes decreased fluorescence intensity without red shift, indicating monomer formation without significant unfolding.
• Dissociation, denaturation, and deactivation are reversible below a limiting pressure; irreversible denaturation and aggregation occur above it.
• The coenzyme NAD+ stabilizes the native tetramer, shifting the dissociation equilibrium to higher pressures.

Conclusions:

• High hydrostatic pressure induces reversible dissociation of pig heart LDH into monomers, a process distinct from unfolding.
• The enzyme's stability and reversibility are pressure-dependent, with a critical threshold for irreversible denaturation.
• NAD+ plays a significant role in maintaining the quaternary structure of LDH under pressure.