Intramolecular thiolester linkages in apolipoprotein B.
Intramolecular thiolester bonds in apolipoprotein B (ApoB) were studied using [14C]methylamine (MA) to cleave the thiolester and [3H]- or [14C]iodoacetate (IA) to titrate the newly generated sulfhydryls. Covalent incorporation of [14C]MA and [3H]carboxylmethyl group into the previously carboxymethylated LDL or the reduced and carboxymethylated ApoB was observed and both radioactivities coincided with ApoB-100 band on SDS-polyacrylamide gel electrophoresis. The [14C]MA-labeled ApoB was completely trypsinized and cross-linked to the activated thiol Sepharose 4B beads. The peptides were eluted with DTT and the free -SH groups blocked with IA then separated on FPLC. Two fractions contained [14C]MA. Sequence analyses showed that these labeled peptides contained Cys-51 and Cys-3734, respectively. Evidence suggests that the thiolester is formed between Cys-51 and gamma-Glu-54 for one, and Cys-3734 and beta-Asp-3737 for the other, with Lys and a hydrophobic amino acid, Val/Leu, in between. This is the first evidence for the presence of intramolecular thiolester linkages in ApoB. The presence of high energy, labile thiolester bonds may explain many of the unusual properties of ApoB and LDL.
Lipoprotein and Atherosclerosis Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
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D000595:Amino Acid Sequence; D001055:Apolipoproteins B / Q000378:metabolism*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004952:Esters; D006801:Humans; D008744:Methylamines / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D001055:Apolipoproteins B; D004952:Esters; D008744:Methylamines; D010455:Peptides; D013438:Sulfhydryl Compounds