Purification and characterization of cytosolic phospholipase A2 activities from canine myocardium and sheep platelets

Stanley L. Hazen, Lori A. Loeb, Richard W. Gross

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

This chapter describes the assay method, purification procedures, and properties of the spleen phospholipases A2 enzymes. As an optimal molar ratio depends on the detergent-phospholipid combination, the ratio is determined for each set of detergent and phospholipid. The solvents are evaporated under nitrogen, and then the samples are further dried in vacuo for about 30 min. An appropriate amount of the buffer is added, and the tubes are vortexed for 2 min at about 37°. The key step of the purification procedures is reversed-phase high-performance liquid chromatography (HPLC). The PLA2 preparations (PLA2 S-1 and PLA2 M) purified from the two sources are homogeneous as judged by sodium dodecyl sulfate (SDS)–gel electrophoresis and analytical HPLC. Both enzymes migrates on SDS gels as a single protein band to the same position as rat pancreatic PLA2. The apparent molecular weight of both enzymes is estimated to be 13,600, in agreement with the weights calculated from the amino acid sequences. The amino acid compositions of PLA2 S-1 and pancreatic PLA2 are similar and the peptide maps and sequences of the amino-terminal 32 residues of the two enzymes are identical. PLA2 S-1 is immunochemically identical to rat pancreatic PLA2, whereas there is no immunochemical similarity between PLA2 M and pancreatic PLA2. An antipancreatic PLA2 antibody do not recognize PLA2 M, and conversely an anti-PLA2 M antibody do not recognize the pancreatic PLA2.

Original languageEnglish
Pages (from-to)400-411
Number of pages12
JournalMethods in enzymology
Volume197
Issue numberC
DOIs
StatePublished - Jan 1 1991

Fingerprint

Dive into the research topics of 'Purification and characterization of cytosolic phospholipase A2 activities from canine myocardium and sheep platelets'. Together they form a unique fingerprint.

Cite this