Biosynthesis of Lipoprotein by Rat Intestinal Mucosa

Abstract

Abstract Lymph chylomicrons are at the lipid-rich end of the spectrum of the lipoproteins. The nature of their protein moiety and its biosynthesis have not yet been fully elucidated. Observations are reported on the incorporation of radioactive leucine into a density by mucosal cells of the small intestine isolated from the rat during absorption of fat. The amino acid incorporation exhibited the characteristic energy requirement and sensitivity to inhibitors of other systems for biosynthesis of proteins. The protein moiety was not homogeneous, but appeared to consist of a small number of species that could be separated by thin layer electrophoresis in starch granules. Similar, but nonidentical, electrophoretic patterns were obtained with the residual soluble intracellular protein of the intestinal mucosa and with chylomicrons of intestinal lymph. The lowest density lipoprotein was isolated from a homogenate of the mucosal cells. After removal of lipids by extraction with organic solvents, the protein moiety exhibited several unusual properties in addition to its affinity for lipids: (a) a predominance of apolar amino acids, (b) resistance to fractionation procedures involving ionic properties, (c) considerable solubility in organic solvents, and (d) peculiarities of the peptide map that were related to the foregoing features. Peptide maps were obtained after proteolytic digestion of the protein moieties of the lowest density lipoprotein fraction, the residual soluble protein, and lymph chylomicrons by means of two-dimensional electrophoresis and chromatography. Unusual maps were obtained in which most of the peptide fragments failed to move upon electrophoresis over a broad pH range, but were readily resolved by chromatography in organic solvents. Strong, although imperfect, resemblance was noted among the peptide maps of all three protein fractions. Preliminary studies of low and high density lipoproteins of rat serum revealed different, more complex peptide maps. No definitive conclusion could be drawn about the specificity of the protein which becomes associated with lipid during absorption through the intestinal mucosa. A speculative interpretation of the aforementioned similarity in peptide maps led us to suggest that lipids in transit become associated with some of the more abundant soluble proteins of the mucosal cells and that the resulting lipoprotein complexes appear in the chylomicrons of intestinal lymph. Thin layer methods of chromatography and electrophoresis were extensively employed. Their convenience and great sensitivity permitted detailed studies to be carried out on submilligram quantities of material.