Inactivation of Hormone-sensitive Lipase in Extracts of Adipose Tissue

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Abstract When supernatant fluid fractions of rat adipose tissue homogenates prepared in water, 0.25 m sucrose, or 0.15 m KCl were incubated at 30° with ATP, MgCl2, and Tris buffer, pH 7.4, lipase activity decreased. On passage over Sephadex G-25, all of the lipase activity of the fluid fraction emerged in the exclusion volume, but was inactivated by ATP and MgCl2 only when material from the later column fractions was added back. Inactivation of the ammonium sulfate-precipitated hormone-sensitive lipase required ATP (g0.5 mm), MgCl2 at a molar concentration in excess of that of ATP, and a factor(s) present in the same Sephadex G-25 fractions. Since inactivation was completely prevented by 50 µm EDTA in the presence of excess Mg++ ion, another cation may be required. The conditions chosen were essentially optimal for demonstration of inactivation of ammonium sulfate-precipitated lipase dependent on the addition of ATP, MgCl2, and the Sephadex G-25 fraction. At lower pH or temperature, the rate of this process was decreased; at 40° or at higher pH, inactivation not dependent on these additions increased. Inactivation was almost completely prevented in 50 mm phosphate buffer, pH 7.4. GTP or creatine phosphate, but not AMP or sodium pyrophosphate, could replace ATP, but no substitute for MgCl2 was found. Inactivation could be decreased or abolished by the addition of 30 µm cyclic adenosine 3',5'-monophosphate (cyclic AMP). This effect of cyclic AMP was prevented by the addition of protein kinase inhibitor from skeletal muscle (Gilman, A. (1970) Proc. Nat. Acad. Sci. U. S. A. 67, 305–312). Inactivation was proportional to the amount of the lipase fraction added in the presence of Sephadex G-25 fraction from an equal or greater amount of tissue, and was proportional to the latter in the presence of lipase derived from more than twice as much tissue.