Quantification of apolipoprotein B-48 and B-100 in rat liver endoplasmic reticulum and Golgi fractions.
We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by quantitative immunoblotting using 125I-Protein A. Standard curves were prepared in each assay using apo B prepared from total rat lipoproteins by extraction with tetramethylurea. Subcellular fractions (rough and smooth endoplasmic reticulum and Golgi fractions) were prepared from rat liver and separated into membrane and cisternal-content fractions. For quantification, membrane fractions were solubilized in Triton X-100, and the apo B was immunoprecipitated before separation by SDS/PAGE and immunoblotting. Content fractions were concentrated by ultrafiltration and separated by SDS/PAGE without immunoprecipitation. Quantification of apo B in subcellular fractions and detection of apo B by immunoblotting yielded consistent results. In all fractions apo B-48 was the major form, accounting for approximately three-quarters of the total apo B. By using marker enzymes as internal standards, it was calculated that all of the apo B was recovered in the endoplasmic reticulum and Golgi fractions, with approximately 80% of each form of apo B in the endoplasmic reticulum. More than 90% of the apo B of the rough- and smooth-endoplasmic-reticulum fractions was membrane-bound, whereas approx. 33 and 15% of the apo B of the cis-enriched Golgi fractions and trans-enriched Golgi fractions respectively were membrane-bound.
Cartwright
I J
IJ
Department of Molecular Biology and Biotechnology, University of Sheffield, U.K.
Higgins
J A
JA
D000818:Animals; D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B / Q000032:analysis* / Q000097:blood; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D006056:Golgi Apparatus / Q000737:chemistry*; D008099:Liver / Q000737:chemistry*; D008297:Male; D008862:Microsomes, Liver / Q000737:chemistry; D011233:Precipitin Tests; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions
D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B