Abstract
The apoprotein (apoB) of low density lipoprotein (LDL) is reported to be a large polypeptide, and it is proposed that there are two similar-sized subunit proteins in LDL (Smith, Dawson, and Tanford. 1972. J. Biol. Chem. 247: 3376-3381.). When apoB is isolated under conditions that minimize artifactual proteolysis, only a single, large molecular weight protein appears on polyacrylamide gel electrophoresis in SDS. To investigate the organization of apoB as it exists within native LDL, limited proteolysis with trypsin has been used as a structural probe. Tryptic digestion for 1 hr at pH 7.6 with enzyme-to-protein ratios of 1:100 and 1:5 results in the liberation of approximately 10% and 30% of apoB as smaller, water-soluble peptides. These peptides may be separated from the partially digested but still intact tryptic core (T-core) of the lipoprotein by chromatography on Sephadex G-75. Repeatedly, the 1:5 T-core of native LDL is found to contain a family of polypeptides of 14,000-100,000 molecular weight. Although they have lost significant quantities of apoprotein, these T-cores sustain an appearance of homogeneity, as studied by analytical ultracentrifugation. Their measured molecular weights do not differ appreciably from those of the native LDL, and the carbohydrate content of the 1:5 tryptic T-core of LDL is similar to that of the native LDL. In normolipemic individuals, LDL generally exists in a monodisperse state, but, in different individuals, monodisperse LDL may range in molecular weight from 2.4 to 3.9 x 10(6). Limited tryptic digestions were used to probe the organization of apoB in these different molecular weight LDL. As assayed by SDS-acrylamide gel electrophoresis of the larger polypeptides and fingerprinting of the smaller released peptides, those regions of LDL exposed to trypsin digestion are identical in monodisperse LDL of 2.5 and 3.4 x 10(6) molecular weight. Thus, the different quantities of lipid bound in these various LDL must interact with apoB so that the same regions of the apoprotein are exposed to the action of trypsin in these different molecular weight lipoproteins.
Highlights
The apoprotein(apoB) oflow density lipoprotein (LDL) is reported to be a large polypeptide, and it is proposed that there are two similar-sized subunit proteins in low density lipoproteina (LDL)
The variable occurrence of large molecular weight polypeptides in LDL samples isolated on repeated occasions is shown in Fig. 1, which presentsa series of 3% acrylamide gels performed on LDL isolated from the same subject under identical conditions
In order to assess the physical changes in LDL resulting from controlled partial tryptic digestion, three separate monodisperse LDL preparations were studied by analytical ultracentrifugation to determine the molecular weight ofthe native lipoprotein and of the isolated tryptic core (T-core)
Summary
Isolation and purificationof LDL phate buffer, pH 7.8, containing 20% glycerol and LDL was isolated from blood bank plasma and from the blood of individual nonlipemic and hyperlipemic fasting donors by ultracentrifugation following the method of Fisher, Hammond, and Warmke[10]. Modification was Whenattempting tosuppress proteolysis com- performed according to Cole [20] using a0.1 M phospletely, the drawn blood was added to tubes contain- phate buffer, pH 8.1, in the presence of either 6 M ing 20 mg of soybean trypsin inhibitor per 100 ml guanidine o r 3% SDS. T h e effectiveness of the inhibitor in stopping further proteolysis of LDL was demonstrated by comparing the proteolytic products by SDS-acrylamide gel electophoresis immediately after digestion and at varying time intervals thereafter. Descending paper chromatography was performed in butanol-acetic acid-water for 16 hr, followed by high voltage electrophoresis in pyridine-aceticacid-water, pH 3.7, for 60 min at 2,500 V, as described by Katz, Dreyer, and Anfinsen [26]. The chromatograms were developed with a cadmium-ninhydrin reagent [27]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
