Purification and Characterization of Lipoprotein Lipase from Pig Adipose Tissue

Abstract

Abstract Lipoprotein lipase was purified from acetone powders of pig adipose tissue. Extraction of acetone powders with 1.2 m NaCl in 0.005 m sodium-barbital buffer, pH 7.4, or heparin (200 units per ml) in distilled water, was 6 times as effective as extraction with 0.025 m NH4OH-NH4Cl buffer, pH 8.6, the commonly used extractant for lipoprotein lipase. At pH values below 7.5, over 85% of the activity extracted into 1.2 m NaCl could be recovered after 4 hours. The partially purified enzyme at later stages was stabilized by the inclusion of 20% glycerol in the buffers. Most of the purification was accomplished by affinity chromatography on Sepharose 4B columns containing covalently bound heparin. At this step, the preparation was purifed 600-fold. This purified enzyme binds reversibly to columns containing concanavalin A covalently bound to Sepharose. Lipolytic activity was eluted from concanavalin A-Sepharose column with 0.2 m α-methyl-d-mannoside, 1.0 m NaCl and 0.005 m sodium-barbital, pH 7.0. At this stage, the enzyme was purified 2100-fold. Isoelectric focusing yielded a single major peak of activity with an isoelectric point of 4.0. Minimum molecular weight determination by gel filtration in buffers containing 1.0 m NaCl and by disc gel electrophoresis in sodium dodecyl sulfate yielded values of 62,000 and 60,000, respectively. The crude enzyme, and that eluted from heparin-Sepharose columns, did not show stimulation by heparin, whereas that obtained after isoelectric focusing exhibited a 60 to 100% stimulation at 22 µg of heparin per ml. Activation by dialyzed serum was dependent on the stage of purification. The crude enzyme showed a 20-fold stimulation by serum but showed some activity in its absence; that purified by isoelectric focusing exhibited a complete dependence on the presence of serum for hydrolysis of triolein emulsions stabilized with gum arabic. Of the three very low density lipoprotein apoproteins studied, only apoLp-Glu could substitute for serum as an activator. In the presence of serum in the assay system, apoLp-Ser was as potent an inhibitor of lipoprotein lipase as apoLp-Ala.