Protein-disulfide isomerase is a component of an NBD-cholesterol monomerizing protein complex from hamster small intestine.
A rapid in vitro assay was developed for monitoring protein-mediated cholesterol monomerization from bile acid aggregates. This assay uses a fluorescent cholesterol analog, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3 beta-ol (NBD-cholesterol), which was shown to be absorbed by hamster in a fashion similar to cholesterol. The fluorescence of aggregates of NBD-cholesterol was strongly quenched in 2.5 mM of taurocholic acid. Addition of proteins from enterocytes of hamster small intestine led to a time- and dose-dependent dequenching of NBD-cholesterol fluorescence. Comparable dequenching can be detected with SDS and appears to involve monomerization of the NBD-cholesterol. Purification of enterocyte extract by sequential chromatography revealed an approximately 140-kDa protein complex (p140) able to mediate the monomerization of NBD-cholesterol. Each p140 complex mediated monomerization of 2.7 NBD-cholesterol molecules. The p140 complex appeared to be formed by dimerization of two approximately 58-kDa molecules since SDS-PAGE revealed a single dominant band at 58 kDa (p58). Protein sequence analysis suggested that p58 is protein-disulfide isomerase (PDI), and this conclusion was confirmed by cloning of hamster PDI, and detection of PDI enzyme activity in the purified fraction. Additional studies with either pure PDI or lysates of cells transfected with hamster PDI showed that PDI by itself was not sufficient for monomerizing cholesterol. Further, despite a similar mobility on SDS-PAGE (approximately 58 kDa), the p140 complex appeared approximately 45-kDa larger than pure PDI (approximately 95 kDa) when analyzed by a gel-filtration chromatography. The p140 complex may thus contain an unidentified molecule(s) in addition to PDI that may contribute importantly to cholesterol monomerization.
Cai
Tian-Quan
TQ
Department of Atherosclerosis and Endocrinology, Merck Research Laboratories, 126 East Lincoln Avenue, RY80W-250, Rahway, NJ 07065, USA. tianquan_cai@merck.com
Guo
Qiu
Q
Wong
Birming
B
Milot
Denise
D
Zhang
Liwen
L
Wright
Samuel D
SD
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism*; D006224:Cricetinae; D006801:Humans; D007421:Intestine, Small / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008647:Mesocricetus; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011108:Polymers / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
C077527:7-nitrobenz-2-oxa-1,3-diazol-4-ylcholesterol; D001647:Bile Acids and Salts; D046911:Macromolecular Substances; D011108:Polymers; D002784:Cholesterol; D019704:Protein Disulfide-Isomerases; D009327:4-Chloro-7-nitrobenzofurazan