Abstract
Lecithin: cholesterol acyltransferase (LCAT) circulates in plasma as an LCAT: high density lipoprotein complex. Plasma high density lipoprotein was fractionated by ultracentrifugation and was coupled to Sepharose 2B according to Porath's technique. This insoluble, but fully hydrated gel was relatively stable to 3M KBr, 0.1 M CaCl2 or 0.5 mM sodium deoxycholate, however appreciable amount of lipid and protein were dissociated from the gel by exposure to 5 mM sodium deoxycholate.
Partially purified LCAT (equivalent to 40 ml fresh plasma) was applied to a column of HDL-Sepharose (0.9×60 cm), and the column was washed with Tris HCl-EDTA-NaCl buffer, pH 7.4 at 37°C, then the bound LCAT was dissociated with 0.5 mM sodium deoxycholate.The recovery of enzyme was 65 % and the purification was raised by 28 folds by this step. The net purification was approximately 1,500 folds of the original plasma.
HDL-Sepharose which was labelled with 4-C-14 cholesterol was used for the study of enzymesubstrate complex formation and catalytic reaction of LCAT. The inhibition of LCAT reaction by sodium deoxycholate did not run parallel with LCAT: HDL complex formation, and these findings suggest the presence of additional mechanisms for the inhibition of LCAT reaction by sodium deoxycholate.
