HUMAN HEPATIC LIPASE SUBUNIT STRUCTURE DETERMINATION

Chinese hamster ovary cells were stably transfected with a human hepat ic lipase (HL) cDNA. The recombinant enzyme was purified from culture medium in milligram quantities and shown to have a molecular weight, s pecific activity, and heparin affinity equivalent to HL present in hum an post-heparin plasma. The techniques of intensity light scattering, sedimentation equilibrium, and radiation inactivation were employed to assess the subunit structure of HL. For intensity light scattering, p urified enzyme was subjected to size exclusion chromatography coupled to three detectors in series: an ultraviolet absorbance monitor, a dif ferential refractometer, and a light scattering photometer. The polype ptide molecular weight (without carbohydrate contributions) was calcul ated using the measurements from the three detectors combined with the extinction coefficient of human HL. A single protein peak containing HL activity was identified and calculated to have a molecular mass of 107,000 in excellent agreement with the expected value for a dimer of HL (106.8 kDa). In addition, sedimentation equilibrium studies reveale d that HL had a molecular mass (with carbohydrate contributions) of 12 1 kDa, Finally, to determine the smallest structural unit required for lipolytic activity, HL was subjected to radiation inactivation, Purif ied HL was exposed to various doses of high energy electrons at -135 d egrees C; lipase activity decreased as a single exponential function o f the radiation dose to less than 0.01% remaining activity, The target size of functional HL was calculated to be 109 kDa, whereas the size of the structural unit was determined to be 63 kDa. These data indicat e that two HL monomer subunits are required for lipolytic activity, co nsistent with an HL homodimer. A model for active dimeric hepatic lipa se is presented with implications for physiological function.