Studies on the Metabolism of Triglyceride (III)

Partial Purification and Characterization of Triglyceride Lipase from Rat Liver

Abstract

Hepatic triglyceride lipase (TGL) was extracted from acetone-ether dried powder of rat liver with 0.05M NH₄Cl-NH₄OH buffer (pH 8.5) containing 0.5U/ml of heparin for 60min at 0°C. The crude enzyme extracts thus obtained were partially purified on a heparin-Sepharose affinity chromatography. This gave a 78-fold purification over crude enzyme extracts. A purified enzyme did not require serum for substrate activation and was not inhibited by 1M sodium chloride, being differed from lipoprotein lipase in rat postheparin plasma.
A purified enzyme had a single pH optimum at 9.0, whereas crude enzyme extracts had lipolytic activities over a wide range of pH values with two maxima at pH9.0 and 4.0. The lipolytic activities in crude enzyme extracts were separated on heparin-Sepharose column into two fractions, a breakthrough fraction and a purified hepatic TGL. In an attempt to define the location of these lipolytic activities, NaCl-noninhibitable hepatic TGL in postheparin plasma and crude enzyme extracts of postheparin liver were assayed for their activities at different pH values. The lipolytic activities of crude extracts of postheparin liver had a pH optimum at 4.0 with a marked decrease at 9.0, being the same with that of the break-through fraction of the column. Heparin-released hepatic TGL in plasma, on the other hand, had a similar pH dependence to that of a purified hepatic TGL.
These results indicate that TGL activity in the liver is heterogeneous. This enzyme activity can be separated into two forms. One, having a high affinity for heparin bound to Sepharose, is in the hepatic plasma membrane and released by heparin into the circulation. The other, having no affinity for heparin, might be present intracellularly. The functional importance of the former form of enzyme was discussed.