Limited Tryptic Digestion of Human Serum Low‐Density Lipoprotein: Islation and Characterisation of the‐Deficient Particle and of Its Apoprotien

Abstract

Limited tryptic digestion of human serum low-density (LD) lipoprotein (e 1.024-1.045 g/ml)under defined conditions permitted isolation by gel filtration chromatography of a stable, proteindeficient lipoprotein; the liberated protein was separated as a mixture of peptides of low molecular weight ( < 5000). Comparison of the chemical, physical and immunological characteristics of the trypsin-treated LD-lipoprotein with those of the native preparation revealed several differences, including (a) a diminished protein content (loss of some 20-25% of the total protein of LD-lipoprotein) and increased proportions of the various lipid components, except for triglyceride (probably resulting from a loss of bound free fatty acids with the liberated peptides); (b) a greater heterogeneity in particle size and slightly larger mean diameter; (c) a lower hydrated density and greater peak sf rate than the native LD-lipoprotein (d) an increased net negative charge; and (e) a partial immunological identity between LD-lipoprotein and the corresponding trypsin-treated fraction.While the amino acid compositions of the protein moieties of LD-lipoprotein and of trypsintreated LD-lipoprotein were essentially identical, trypsin-treated apo-LD-lipoprotein was distinct in its complete solubility in urea-containing buffers at high concentrations, and also in its partial solubility in buffers lacking denaturing agents.Comparison of the apoproteins of the native and trypsin-treated LD-lipoproteins by electrophoretic techniques based on molecular weight revealed a transformation of the high-molecular weight material ( > 250 000) characteristic of apo-LD lipoprotein into several polypeptide species (10 major forms) ranging in size from 161 500 to about 10000. The largest of these (band bl : 161 500) could be completely dissociated into smaller components (b2: 93500 and b3: 77000) upon extensive heat treatment at 90 "C. Electrophoresis of the soluble fraction of apo-LD-lipoprotein and of that from its trypsin-treated counterpart in polyacrylamide gels containing urea at basic pH showed the disappearance of the small amounts (< 5 %) of C apoproteins of apo-LDlipoprotein upon tryptic treatment. These results, which were highly reproducible in LD-lipoprotein preparations from different individuals, suggest that trypsin-treated LD-lipoprotein may provide a model for investigation of the organisation and structural role of the principal apoprotein (apolipoprotein-B) in the LD-lipoprotein molecule.The detailed arrangement of the lipid and protein components in the serum low-density lipoprotein Abbreviations. VLD-lipoproteins, very low-density lipoproteins sity as defined; HD-lipoproteins, high-density lipoproteins, e,, 1.063-3.21 g/ml; apo-LD-lipoprotein, the total apoprotein of native LD-lipoprotein; Lp(a), a variant of human serum LD-lipoprotein; TosPheCHzCl, ~-l-(tosylamido-2-phenyl)ethyl chloromethyl ketone. Nomrncluture. Apolipoprotein nomenclature is according to Alaupovic [13].remains the subject of debate [1,2]. Physical, chemical and enzymatic...

Lipoproteins containing apoprotein B are a major regulator of neutrophil responses to monosodium urate crystals.

“…1 B, open-headed arrows). These peptides probably represented apo B fragments (47,48). In addition, a group of low molecular weight anionic polypeptides (Fig.…”

“…These were probably apo B-related as essentially all the protein of LDL exists as apo B (60). A possible explanation for the failure to detect these as apo B species would be the known differential immunochemical reactivity of various apo B species (27,47); alternatively, proteolytic cleavage of apo B during preparation (47,48) and incubation with urate crystals, or the denaturation carried out before electrophoresis may have rendered certain apo B peptides devoid of a suitable epitope for the apo B-specific antibodies.…”

Determination of the molecular mass of apolipoprotein B-100 A chemical approach

“…Structural analysis of apo B-100 has been greatly hampered by its insolubility in aqueous buffers after delipidation, although its amino acid composition [2,4] is not particularly dominated by hydrophobic amino acids. In our laboratory, the application of h.p.l.c.…”