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The catabolism of intravenously injected 125I-labelled low density lipoproteins (LDL) was followed in normal miniature swine for 2 weeks. When compared with the two-exponential model, the decay curve of the plasma radioactivity associated with the LDL fraction was best described by a three-exponential model. In this system, the half-lives were 4.5 +/- 3.7, 19.7 +/- 6.6, and 127 +/- 70 h (mean of four studies). Assuming a kinetic model with metabolism of LDL in the rapidly equilibrating compartment and two slower equilibrating compartments (a model requiring three exponentials), the mean fractional catabolic rate for apo-LDL was calculated to be 0.015 h-1. Therefore, if at steady state, the synthetic rate for apo-LDL in the same pigs would be 5.6 +/- 4.1 mg/h. Different kinetic models using two or three exponentials would provide different values for the synthetic rate of apo-LDL. However, in view of the known existence of at least three major equilibrating pools for LDL in plasma, liver, and lymph, and in view of the present results, the kinetic model for LDL metabolism should be better represented by a three-exponential system.

Apolipoprotein (apo) B-100 cDNAs were identified in a human liver cDNA library cloned in the expression vector lambda gt11. The beta-galactosidase-apoB-100 fusion protein was detected by two independently produced low density lipoprotein polyclonal antisera and by three apoB-100 monoclonal antibodies that crossreact with apoB-74. It was not recognized by two apoB-100 monoclonal antibodies that crossreact with apoB-26. The longest clone, lambda B8, was completely sequenced. It contains a 2.8-kilobase DNA fragment containing the codons for the carboxyl-terminal 836 amino acid residues of apo-B-100, as well as the 3' untranslated region of apoB-100 mRNA. We have thus mapped apoB-74 to the carboxyl-terminal portion of apoB-100. The deduced amino acid sequence of the cloned DNA matches the sequences of 14 apoB-100 peptides determined in our laboratory. Minor differences in amino acid sequence were noted in three of the peptides, suggesting polymorphism of apoB-100 at the protein and DNA levels. Secondary structure predictions reveal an unusual pattern for apolipoproteins, consisting of beta-structure (24%), alpha-helical content (33%), and random structure (30%). Ten amphipathic helical regions of 10-24 residues were identified. This carboxyl-terminal fragment of apoB-100 is considerably more hydrophobic than other apolipoproteins with known structure. Its lipid binding regions might include stretches of highly hydrophobic beta-sheets as well as amphipathic helices. Our findings on apoB structure might be important for understanding the role of apoB-100-containing lipoproteins in atherosclerosis.

Based on our previous observation that monoclonal antibody anti-apoD-4E11 reacted with several HDL proteins we studied them further with three questions in mind: i) is there common protein polymorphism in healthy individuals? ii) how many proteins are present and what are their characteristics? iii) are they all apolipoproteins and do they have the same lipoprotein distribution as apoD? Isolated, delipidated apoD was used as a standard for radioimmunometric assay of plasma with antibody 4E11. The antigen varied from 3 to 11 mumol-equivalents of apoD per liter of plasma (equivalent to 5-20 mg apoD/dl plasma) with means of 6.1 and 6.8 mumol/l in men and women, respectively. Two-dimensional electrophoresis of plasma found up to eight 4E11-antigenic-proteins of different Mr, each heterogeneous in pI. All plasmas tested contained apoD and an Mr 38,000 antigen, the latter being the most immunoreactive. Six proteins of Mr 70,000-94,000 were found, but the number varied between subjects. Eighty nine percent of the plasma antigen was associated with lipoproteins: 83% with HDL and VHDL, 5% with LDL and VLDL. Lipoproteins of all sizes, separated by polyacrylamide gradient gel electrophoresis, contained the antigen. ApoD was almost the only 4E11-antigen in LDL, and was in two states: the one free, the other an apoD-apoB mixed disulfide complex. The apparent proportions of higher Mr antigens increased with increasing lipoprotein density, and the proportion of apoD decreased reciprocally. None of these 4E11-antigenic-proteins cross-reacted with antiserum to retinol-binding protein.

Monoclonal antibodies (MAbs) have been prepared against apolipoprotein (apo) B which had been delipidated and resolubilized (apo Bsol). The MAbs were classified into nine groups according to their behavior in competitive binding assays and, following SDS electrophoresis and immunoblots, their corresponding epitopes were assigned to apo B48, apo B74, apo B26 and to fragments of LDL apo B (apo BLDL) generated by limited tryptic proteolysis. In addition to their reactivity with soluble apo B all antibodies also reacted with LDL which had been adsorbed to polystyrene. Competitive binding of MAbs to insolubilized antigen also indicated conformational similarities between apo Bsol and apoBLDL. These similarities were, however, less apparent when the respective antigens were in solution. In competitive radioimmunoassays the majority of anti-apo Bsol MAbs reacted better with apo Bsol than with LDL although the opposite was true for one group of MAbs. In immunoprecipitation studies the MAbs which preferentially recognize apo BLDL could precipitate 95% of 125I-LDL whereas the immunoprecipitation with those MAbs which preferentially recognize soluble apo B varied between 0 and 95%. Between 65 and 82% of 125I-apo Bsol was immunoprecipitated under the same conditions. Thus, epitopes defined by MAbs prepared against apo Bsol may be expressed on apo BLDL when it is adsorbed to plastic but not necessarily when it is in solution. For those epitopes preferentially expressed on apo BLDL, reincorporation of soluble apo B into phospholipid-cholesteryl ester microemulsions or phospholipid-cholesterol liposomes increased their immunoreactivity, whereas, the reincorporation of apo B into lipid vesicles resulted in a decreased reactivity with those MAbs which recognized better apo Bsol than apo BLDL. Thus, while the respective conformations of apo Bsol and apo BLDL are only partially similar, they can be reversibly interchanged by delipidation and relipidation.