Immunoaffinity purification, partial sequence, and subcellular localization of rat liver phospholipase A2

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Phospholipase A2 (PLA2) activity in rat liver occurs in various subcellular fractions.
• Little is known about the structural relationships and distribution of these enzymes.
• Calcium (Ca2+) and alkaline pH optimum are common characteristics.

Purpose of the Study:

• To purify and characterize rat liver mitochondrial phospholipase A2.
• To investigate the structural relationships and quantitative distribution of PLA2 enzymes in rat liver.
• To develop a rapid purification method using monoclonal antibodies.

Main Methods:

• Immunoaffinity chromatography using monoclonal antibodies against rat liver mitochondrial PLA2.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for purity assessment.
• N-terminal amino acid sequencing.
• Solubilization of PLA2 activity from homogenates and subcellular fractions.
• Gel filtration and immunological cross-reactivity studies.

Main Results:

• A rapid immunoaffinity method yielded a single-band purified mitochondrial PLA2.
• N-terminal sequencing revealed 96% identity to rat platelet/spleen PLA2, differing from pancreatic PLA2 (25% homology).
• PLA2 activity was primarily soluble or peripherally membrane-associated, solubilized by potassium chloride extraction.
• Membrane-derived PLA2 shared epitopes with the mitochondrial enzyme.
• Enzyme distribution correlated with mitochondrial marker cytochrome c oxidase.
• Rat liver cytosol contained additional Ca2+-dependent and -independent PLA2 activities.

Conclusions:

• Rat liver mitochondrial PLA2 is structurally related to membrane-associated forms found in platelets and spleen.
• Immunoaffinity chromatography is effective for purifying mitochondrial PLA2.
• Further characterization revealed distinct PLA2 activities in rat liver cytosol.