Pyruvate kinase in pig liver mitochondria

Area of Science:

• Biochemistry
• Mitochondrial Metabolism
• Enzymology

Background:

• Pyruvate kinase (PK) is a key enzyme in glycolysis, typically found in the cytosol.
• The presence and function of PK within mitochondria, particularly in fatty acid synthesis, remain less understood.

Purpose of the Study:

• To investigate the existence and characteristics of pyruvate kinase (PK) in pig liver mitochondria.
• To explore the potential role of mitochondrial PK in metabolic pathways, including fatty acid synthesis.

Main Methods:

• Photometric monitoring of PK activity using phosphoenolpyruvate (PEP) or ADP as substrates in solubilized mitochondria.
• Immunological analysis to confirm the presence of mitochondrial PK.
• Digitonin titration to determine the localization of mitochondrial PK.
• Assessment of intramitochondrial NAD(P)+ levels and extracellular citrate/oxaloacetate following PEP addition.

Main Results:

• Pyruvate kinase (PK) activity was detected in pig liver mitochondria, distinct from the cytosolic isoenzyme.
• Mitochondrial PK exhibited sigmoidal kinetics with respect to PEP and ADP concentrations.
• Immunological assays confirmed the presence of mitochondrial PK, which was localized to the mitochondrial matrix.
• PEP addition to mitochondria led to reduced intramitochondrial NAD(P)+ and increased extracellular citrate/oxaloacetate.

Conclusions:

• Pig liver mitochondria contain a unique pyruvate kinase (PK) isoenzyme localized to the matrix.
• Phosphoenolpyruvate (PEP) may trigger fatty acid synthesis through its metabolism within mitochondria, involving mitochondrial PK.