Hormone-sensitive lipase of rat adipose tissue. Purification and some properties.

Hormone-sensitive lipase from rat adipose tissue was solubilized with nonionic detergent and purified 2,000fold to 50% protein purity by repeated gradient sievorptive chromatography on diethyL(2-hydroxypropy1)aminoethyl-Sephadex and by affinity chromatography on triacylglycerol-containing polyacrylamideagarose gel. The identity of the enzyme protein, a polypeptide with M, = 84,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, was established by its copurification with lipase activity, selective labeling with [3H]diisopropyl fluorophosphate, and phosphorylation by the catalytic subunit of CAMP-dependent protein kinase from the same tissue and ATP-Mg. There was no indication for a role of a specific lipase kinase analogous to phosphorylase kinase. The phosphorylation enhanced the trioleoylglycerol lipase activity to 150% over that in control when approximately 0.5 mol of phosphate had been incorporated into each mol of its M, = 84,000 enzyme protein. The purified lipase catalyzed the hydrolysis of emulsified tri-, di-, and monooleoylglycerol and cholesterol oleate at the relative rates of 1:10:4:1.5, with a molar specific activity of 600 mol/s/mol of M, = 84,000 enzyme protein toward dioleoylglycerol. Its specific activity was more than 1,000-fold higher than that of a lipase recently purified from hen adipose tissue (Berglund, L., Khoo, J. C., Jensen, D., and Steinberg, D. (1980) J. BioL Chem 255, 5420-5428). It had a pH optimum of 7.0, was inhibited by micromolar diisopropyl fluorophosphate, Hg“’, and millimolar NaF, and was stable at neutral pH in detergent and high glycerol concentration. The results of this and previous work (Belfrage, P., Fredrikson, G., Nilsson, N. O., and Stridfors, P. (1980) FEBS Lett. 111, 120-124; Nilsson, N. O., Strilfors, P., Fredrikson, G., and Belfrage, P. (1980) FEBS Lett. 111, 125-130) demonstrate modulation of hormone-sensitive lipase activity by phosphorylation in uitm as well as in the intact fat cell and indicate that this is a significant mechanism for the hormonal short term regulation of adipose tissue lipolysis.