Purification and Properties of Rat Liver Lysosomal Lipase

Abstract

Abstract A triglyceride lipase (EC 3.1.1.3) was purified 1200-fold from homogenates of rat liver, by cosedimentation with lysosomes, detergent solubilization, selective elution from carboxymethylcellulose by lipid activator, and gradient elution from carboxymethylcellulose. Removal of phospholipids and detergent was achieved by rechromatography on carboxymethylcellulose. The purified enzyme contains a major band of protein (58,000 mol wt) and three minor bands as indicated by sodium dodecyl sulfate gel electrophoresis. The enzyme exhibited a pH optimum of approximately 4.0, except in the presence of cardiolipin and acetate buffer (pH optimum 4.5). Activator saturation functions are hyperbolic in acetate buffer and sigmoidal in citrate buffer. Cardiolipin is a more efficient activator than phosphatidylserine. The enzyme preparation catalyzes the hydrolysis of all saturated fatty acylesters of p-nitrophenol tested (C2 – C18). In the presence of cardiolipin, p-nitrophenylcaprate (C10) is the best. It also catalyzes the hydrolysis of cholesteryl-3-palmitate, but only in the presence of phospholipid and only at 7% the rate of tripalmitin. No phospholipase A was detected. The purified lipase is inhibited by fluoride, ethanol, and heparin. Divalent metal ions, chloride, sulfate, acetate, and citrate activate.