Lipoprotein substrate binding site of LCAT karen murray

i.ecithin:chotesterol acyltransferase (LCAT) catalyses the esterifkation of plasma lipoprotein cholesterol in plasma and plays a central role in the re\<Tbp cholesterol transport pathway. Studies of the natural mutations of LCAT revealed a putative substrate binding region of the enzyme (residues #121l.'idi that is totally conserved in six mammalian species. This putative binding domain of I,("AT was probed by enzyme linked immunoassays utilizing a polyclonal antibody against plasma IX,"AT and a site specific antibody prepared against a peptide representing the 121-136 region. Both antibodies reacted with a recombinant form of purified LCAT, secreted by cultured baby hamster Kidney ( BHK) cellsby both Western Blot and by an ntibody sandwich ELISA for M'AT However, only the polyclonal antibody recognized the enzyme when it. \vas first adsorbed to HDL or to a hvdrophohic surface. These studies provide experimental evidence that the 121-136 region of LCAT represents a lipoprolein snbstraie binding domain. Consequently, we believe that future studies which probe this lipoprotein substrate binding region by mutagenesis and conformational analysis will vield valuable information regarding the mechanism of action of ibis key enzyme in cholesterol transport.