Hormone-sensitive lipase from bovine adipose tissue.
Hormone-sensitive lipase has been purified to near homogeneity from bovine perirenal adipose tissue. The purification method involves isoelectric precipitation at pH 5.0, followed by partial solubilisation in Triton N-101 and ion-exchange chromatography on DE-52. After additional solubilisation, the enzyme is further purified by chromatography on phenyl-Sepharose and heparin-Sepharose. This procedure can be completed within three working days and yields approx. 30 units of enzyme with a specific activity of 30 U/mg. The enzyme has been identified as a polypeptide of Mr 84 000 by affinity labelling with [3H]diisopropyl fluorophosphate. This polypeptide comprises approx. 60-80% of the protein in the final preparation, as judged by scanning densitometry of SDS-polyacrylamide gels stained with silver or with Coomassie blue R. The polypeptide of Mr 84 000 serves as a substrate for cyclic AMP-dependent protein kinase, phosphorylation correlating with activation of the lipase. Polyclonal antibody to the lipase has been raised in a rabbit and shown to specifically cross-react with the Mr 84 000 subunit.
S R
SR
Cordle
R J
RJ
Colbran
S J
SJ
Yeaman
D000255:Adenosine Triphosphate / Q000378:metabolism; D000273:Adipose Tissue / Q000201:enzymology*; D000345:Affinity Labels; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006728:Hormones / Q000494:pharmacology*; D007531:Isoflurophate / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D002787:Sterol Esterase / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D000345:Affinity Labels; D006728:Hormones; D046911:Macromolecular Substances; D007531:Isoflurophate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D002787:Sterol Esterase