Tryptic peptide analysis of the human apolipoprotein E isomorphs.
The nature of the polypeptide backbone of human apolipoprotein (apo) E present in the very low density lipoproteins (VLDL) of normal and homozygous type III hyperlipoproteinemic patients was investigated by tryptic cleavage fingerprinting and specific chemical modification studies. Apo E from normal subjects was resolved on polyacrylamide isoelectric focussing gels into five bands (apo E-I', E-I, E-II, E-III, and E-IV), whereas apo E from type III patients was resolved into three bands (apo E-I3', E-I3, and E-II3). The apo E isoforms, contained within unstained polyacrylamide gel slices, were washed to remove ampholytes, desialylated, and digested with L-1-tosylamido-2-phenylethylchloromethyl ketone treated trypsin. Autoradiography of 125I-labelled tryptic apo E peptides showed complete identity between all isoforms from normal subjects. High performance liquid chromatographic (HPLC) analysis showed that complete peptide identity exists between apo E-I', E-I, and E-II and between apo E-I3', E-I3, and E-II3. Distinct HPLC peptide profiles were found for apo E-II, E-III, E-IV, and E-II3. These resolved peak differences were reproducible between runs, between digests, and between apo E isolations, suggesting that the distinct profiles were neither a result of artifacts nor of contamination. Specific chemical modification studies revealed that human apo E isomorphism is due, in part, to differences in arginine and cysteine residues but not to lysine residues. These findings indicate that human apo E isomorphism results from differences in the primary amino acid sequence of the individual isoforms in addition to charged carbohydrate heterogeneity. Furthermore, the apo E isomorphic profile observed in homozygous type III hyperlipoproteinemic patients reflects both a deficiency of apo E-III and E-IV and the presence of the altered apo E-II isoprotein (apo E-II3).
D000181:Acrylonitrile; D001053:Apolipoproteins / Q000032:analysis*; D001057:Apolipoproteins E; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D003512:Cyclohexanones; D006801:Humans; D007461:Iodoacetates; D007525:Isoelectric Focusing; D008079:Lipoproteins, VLDL / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis*; D014357:Trypsin / Q000378:metabolism*
D001053:Apolipoproteins; D001057:Apolipoproteins E; D003512:Cyclohexanones; D007461:Iodoacetates; D008079:Lipoproteins, VLDL; D010446:Peptide Fragments; C018031:1,2-cyclohexanedione; D014357:Trypsin; D000181:Acrylonitrile