Purification and characterization of rat adipose tissue lipoprotein lipase

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Abstract
Lipoprotein lipase extracted from adipose tissue of glucose-fed rats with 5 mM-sodium barbital, pH 7.5, containing 20% (vol/vol) glycerol and 0.1% (vol/vol) Triton X-100, was partially purified by affinity chromatography on heparin linked to Sepharose 4B. Sodium dodecyl sulfate/polyacrylamide-gel electrophoresis [SDS-PAGE] of the partially purified enzyme preparation revealed the presence of 2 major Coomassie-staining bands (MW 62,000 and 56,000) as well as a number of minor bands. Treatment of partially purified enzyme with [1,3-3H]DFP resulted in the incorporation of radiolabel into the band of MW 56,000, but not into the band of MW 62,000. Both the amount of the MW 56,000 polypeptide and the incorporation of [1,3-3H]DFP into this band were greatly reduced in the enzyme preparations isolated from adipose tissue of 48 h-starved rats, whereas the amount of the MW 62,000 polypeptide was unaffected by starvation. Purification of lipoprotein lipase from adipose tissue of glucose-fed rats was also carried out using affinity chromatography on Sepharose 4B linked to heparin with low affinity for antithrombin-III. This procedure resulted in the presence of a single band of MW 56,000 on SDS-PAGE. The polypeptide of MW 56,000 probably corresponds to the subunit of lipoprotein lipase, whereas the MW 62,000 polypeptide probably represents antithrombin-III.