CDP-diglyceride hydrolase from pig liver mitochondria.

Area of Science:

• Mitochondrial biochemistry
• Enzyme kinetics
• Lipid metabolism

Background:

• Mitochondria synthesize polyglycerophosphatides, essential components of mitochondrial membranes.
• The regulation of this synthesis pathway is not fully understood.
• CDP-diglyceride is a key precursor in phospholipid biosynthesis.

Purpose of the Study:

• To identify and characterize CDP-diglyceride hydrolase activity in pig liver mitochondria.
• To investigate the enzyme's properties, localization, and potential regulatory mechanisms.
• To explore its role in mitochondrial polyglycerophosphatide biosynthesis.

Main Methods:

• Synthesis of radiolabeled CDP-diglyceride ([3H]CMP).
• Enzyme assays measuring [3H]CMP release from CDP-diglyceride.
• Differential enrichment of enzyme activity in mitochondrial fractions and solubilized preparations.
• Kinetic analysis (Km, pH optimum) and substrate specificity determination.
• Investigation of inhibitor effects (divalent cations, nucleotides, phospholipids).

Main Results:

• CDP-diglyceride hydrolase activity was detected and enriched in pig liver mitochondrial membranes.
• The enzyme was solubilized using Triton X-100, with significant purification.
• Kinetic parameters (Km = 12.8 microM, pH optimum ~6.2) were determined.
• Dioleoyl CDP-diglyceride was the preferred substrate.
• Activity was modulated by divalent cations (inhibition in whole mitochondria, stimulation with phosphatidylglycerol), phospholipids, and cytidine nucleotides.

Conclusions:

• A novel CDP-diglyceride hydrolase activity exists in pig liver mitochondria.
• This enzyme is localized to mitochondrial membranes and can be solubilized.
• Its activity is influenced by various factors, suggesting a regulatory role.
• The enzyme is proposed to be involved in the biosynthesis of mitochondrial polyglycerophosphatides.