[44] Lysophospholipases from bovine liver

Abstract

This chapter focuses on the purification and brief characterization process of a membrane-bound and a soluble lysophospholipase from bovine liver. The incubation mixtures consist of varying amounts of enzyme protein in a total volume of 0.5 ml of 20 mM potassium phosphate buffer. The assay mixture for lysophospholipase I contains in addition 2 mM 2-mercaptoethanol. Fresh bovine liver is homogenized in two liters of 20 mM Tris-HCl containing 0.15 M NaCl during two min in a Waring blendor and filtered through two layers of cheesecloth to give a homogenate. Solubilization of lysophospholipase activity is achieved after delipidation of the homogenate by extraction with n -butanol. Purified lysophospholipase I is unstable and loses activity on repeated freezing and thawing. Lysophospholipase II is stable after lyophilization and can be stored in this form for years without loss of activity. Both enzymes give a single band in nondenaturating and in sodium dodecyl sulfate (SDS)–polyacrylamid gels. Molecular weights determined by SDS–PAGE and Sephadex G-100 gel filtration amount to 24,000 and 26,000, respectively, for lysophospholipase I. The corresponding values for lysophospholipase II are 63,000 and 57,000, respectively. These results indicate that both enzymes consist of single polypeptide chains.