Amplification of human APO(a) kringel 4–37 from blood lymphocyte DNA

We have been able to amplify the lysine binding pocket region of human apo(a) kringle type 5 starting from the DNA isolated from peripheral blood lymphocytes. This development now permits the identification of Lp(a) mutants that by lacking their ability of bind to lysine/fibrin would have a lesser thrombogenic potential.     

Advantages and limitations of density gradient ultracentrifugation in the fractionation of human serum lipoproteins: role of salts and sucrose

Two density gradient ultracentrifugation methods, Redgrave et al. (1975. Anal. Biochem. 65: 42-49) and Nilsson et al. (1981. Anal. Biochem. 110: 342-348), currently used for the separation and analysis of plasma lipoproteins were compared with respect to their resolving power and capacity to obtain pure products as a function of time of ultracentrifugation using the same rotor (Beckman SW-40), speed (150,000 g), and temperature (14 degrees C). The effects of sucrose and salts were also investigated. The Redgrave gradient insured the separation of the major classes of plasma lipoproteins after 24 hr of centrifugation; however, equilibrium conditions were only reached after 48 hr, at which time the lipoproteins were contaminated by albumin. When the effluents from each rotor tube were continuously monitored at 280 nm, each lipoprotein band gave values that were higher than those from mass analyses. This was due to a light scattering effect, the extent of which was dependent on the concentration of lipoproteins and salts. Sucrose prevented the scattering effect and was found to bind irreversibly to the apolipoproteins. In contrast, after 66 hr centrifugation, the lipoproteins obtained from the Nilsson gradient exhibited a close correspondence between protein mass and absorbance values at 280 nm, had no scattering effect, and were uncontaminated by albumin. The difference in spectroscopic behavior between the Redgrave and the Nilsson procedures was attributed to three factors: 1) the presence of sucrose in the latter gradient and incorporation of this sugar into lipoproteins as assessed by mass and radioactivity measurements; 2), the salt density to which the serum samples were exposed to at the beginning of the ultracentrifugation; and 3) the final lipoprotein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between apo[a] isoforms and Lp[a] density in subjects with different apo[a] phenotype: a study before and after a fatty meal

Plasma Lp[a] levels and apo[a] isoform distribution among lipoproteins isolated by density gradient ultracentrifugation were studied in subjects with one-band or two-band apo[a] phenotypes as assessed by gradient gel electrophoresis before and after an oral fat load. There were no significant differences in the ultracentrifugal profile between fasting plasma and postprandial plasma that was freed of triglyceride-rich particles (TRP). One-band phenotypes exhibited a single symmetrical peak in the density gradient, whereas two-band phenotypes exhibited a multi-modal distribution. Low molecular weight apo[a] isoforms were preferentially associated with low density Lp[a] whereas high molecular weight apo[a] isoforms were found with high density Lp[a] particles. Feeding a high fat meal caused no significant increase in the total plasma level of Lp[a]. However, the isolated TRP contained the apoB-100-apo[a] complex in a quantity that represented only about 1% of its total amount in the fasting plasma. In all cases the apo[a] isoforms present in TRP were also present in the fasting plasma; however, in the two-band apo[a] phenotypes the ratio of the slow over the fast migrating band was in all cases about eightfold higher in TRP than in the fasting plasma. These observations indicate that postprandially a small percentage of apoB-100-apo[a] associates with TRP and suggest that this complex may derive from de novo synthesis rather than from a pre-existing Lp[a] plasma pool. The liver would be the source of the complex due to the presence in the latter of apoB-100.     

Heterogeneity of human plasma lipoprotein (a). Isolation and characterization of the lipoprotein subspecies and their apoproteins

Lipoprotein (a) (Lp(a] from the plasma of normolipidemic human donors was isolated by rate zonal and isopycnic density gradient ultracentrifugation. The final preparations usually contained varying amounts of isopycnic low-density lipoproteins (LDL), which were totally removed either by heparin-Sepharose column chromatography or by chromatofocusing. The Lp(a) preparations exhibited both inter- and intraindividual density heterogeneity which was accounted for by the differences in their protein and lipid composition. In addition, there was heterogeneity in the size of apoprotein (a) (apo(a] which was found to be linked to apoprotein B (apo-B) through disulfide bonds. Three different apo(a) species were obtained; they had a size either smaller, equal to, or larger than apo-B-100, the protein moiety of LDL. The apo(a) that was smaller than apo-B resided in a low-density Lp(a) particle whose peak was in the 1.019-1.063 g/ml density range. The larger apo(a) was a component of the dense Lp(a) particle and was responsible for the increased density in this Lp(a) species. The third apo(a) which was equivalent in size to apo-B resided in a density range intermediate between the other two Lp(a)s. It is concluded that Lp(a) may differ not only from one individual to another, but also within the same individual who may have more than one Lp(a) species. Part of this heterogeneity may be accounted for by differences in the (a) polypeptide.     

Biosynthesis of human preproapolipoprotein A-II

The primary translation product of human apolipoprotein A-II was purified from wheat germ and ascites cell-free lysates programmed with RNA isolated from either a hepatocellular carcinoma cell line (HepG2) or intestinal epithelium. A-II mRNA represents 0.2% of the translatable RNA in these hepatocytes and in jejunal epithelium. Plasma high density lipoprotein-associated A-II is a 77-amino acid polypeptide. The primary translation product is 100 amino acids long and contains a 23-amino acid NH2-terminal extension. Cotranslational cleavage of the cell-free product indicated that this NH2-terminal sequence consists of an 18-amino acid long signal peptide, Met-Lys-Leu-Leu-Ala-Ala-X-Val-Leu-Leu-Leu-X-X-Cys-X-Leu-X-X-, and a 5-amino acid long propeptide, Ala-Leu-Val-Arg-Arg. This functional division was confirmed by sequencing the stable intracellular form of apolipoprotein A-II isolated from HepG2 cells. Approximately 45% of the proapo-A-II is cleaved to the mature form during export from HepG2 cells. The COOH-terminal dipeptide conforms to the rule that prosegments are cleaved after paired basic residues. We have previously shown (Gordon, J. I., Sims, H. F., Lentz, S. R., Edelstein, C., Scanu, A. M., and Strauss, A. W. (1983) J. Biol. Chem. 258, 4037-4044) that proapolipoprotein A-I is not cleaved during export from these cells and contains a prosegment with a COOH-terminal Gln-Gln dipeptide. Therefore, proteolytic processing of the two principal high density lipoprotein-associated apolipoproteins proceeds along different pathways.     

Sedimentation behavior of native and reduced apolipoprotein A-II from human high density lipoproteins.

The solution properties of human serum apolipoprotein A-II, both in the native and in the reduced forms, were investigated by the technique of sedimentation equilibrium in the analytical ultracentrifuge. For both proteins, the apparent weight average molecular weights determined in neutral buffer systems were found to be dependent on protein concentration and invariant with the rotor speeds used (16,000 to 44,000 rpm) indicating a reversible self-association. These results were also found to be independent of temperature between 5 and 30 degrees C. The pattern of self-association of native apolipoprotein A-II could best be described by a monomer-dimer-trimer equilibrium, in agreement with previously reported data (Vitello, L B., and Scanu, A. M. (1975), Biochemistry 15, 1161). The self-association pattern of apolipoprotein A-II reduced in the presence of 50 mM dithiothreitol conformed with a monomer-dimer-tetramer equilibrium similar to that reported for the native single chain apolipoprotein A-II of the rhesus monkey (Barbeau, D. L., et al. (1977), J. Biol. Chem. 252, 6745), but differing significantly from that reported for the reduced and carboxymethylated human product (Osborne, J. C. , et al. (1975), Biochemistry 14, 3741).     

Studies of the cyanogen bromide fragments of the apoprotein of human serum low density lipoproteins.

The apoprotein of human serum low density lipoproteins was reduced and carboxymethylated and then cleaved by cyanogen bromide (CNBr). The peptides which were produced from this cleavage (90% yield, based upon loss of methionine) were resolved by SDS polyacrylamide gel electrophoresis into 10 major bands, each having an amino acid composition very similar to that of intact reduced and carboxymethylated LDL apoprotein. The fractionation of the CNBr fragments by preparative gel filtration was dependent upon the nature of the eluting solvent. NH4OH and SDS solvents eluted all of the material in the void volume. In 6 M guanidinium chloride solvents several peaks were, however, resolved, each having an amino acid composition similar to that of the unfractionated products. Whereas no NH2-terminal was detected in reduced and carboxylmethylated LDL apoprotein, automated Edman degradation of the protein following treatment with CNBr revealed the presence of several NH2-termini. The results suggest that LDL apoprotein may be made of segments of, at least, very similar amino acid composition and that both the protein itself and derivative fragments have a great tendency to aggregate even in denaturing solvents.