ApoLDL: evidence for an aggregating system of heterogeneous subunits

The protein moieties of low density lipoprotein (LDL) (d 1.019-1.063 g/ml) and subfractions: I (d 1.018-1.023 g/ml), II (d 1.023-1.028 g/ml), III (d 1.028-1.034 g/ml), and IV (d 1.034-1.052 g/ml) were obtained by a mild delipidation procedure involving attachment of LDL to an ionic-exchange column and a gradient of the non-ionic detergent Brij-36T (J. Lipid Res. 1979. 20: 631.). The apoLDL, eluted in the presence of 6 M urea, 0.1% SDS, or 4 M guanidine-HCl and analyzed by pore-gradient polyacrylamide gel electrophoresis and high performance gel exclusion chromatography, appeared to be made up of several polypeptide components with molecular weights between 250,000 and 14,000. The complex pattern was observed in the apoprotein of LDL subfractions I to IV; however the distribution of the most prominent components was different for each density range. These results, those from immunoelectrophoresis, N-terminal analysis, and measurements of tetramethylurea-soluble apoproteins discounted the possibility that the presence of bands with molecular weights below 250,000 could be caused by contamination with VLDL or HDL or nonspecific proteolysis. Delipidation of subfractions I to IV with organic solvents produced, on the other hand, simple patterns of highly aggregated apoLDL subfractions, where most of the protein had molecular weights above 250,000. The more disaggregated apoLDL preparations were those obtained from LDL rapidly isolated by single-spin centrifugation in KBr gradients, from fresh plasma immediately mixed with EDTA, phenylmethyl sulfonylfluoride, and chloramphenicol. ApoLDL fractionated by single-pore polyacrylamide gel electrophoresis appeared to be a system of heterogeneous antigens unevenly distributed on the different polypeptide components. These results, and those obtained by peptide maps of tryptic hydrolyzates from the major constituents, suggest that apoLDL is made of several polypeptides of different lengths with similar repetitive sequences but with some dissimilar segments specific for the major protein constituent of LDL. These polypeptides show a high tendency to form multiple aggregates with size distribution dependent on the preparation history.-Socorro, L., F. López, A. López, and G. Camejo. ApoLDL: evidence for an aggregating system of heterogeneous subunits.