Purification and characterization of a novel human 15 kd cholesterol crystallization inhibitor protein in bile.
Crystallization-inhibiting proteins can explain longer nucleation times associated with bile from gallstone-free subjects as compared with bile from patients with cholesterol gallstones. We partially characterized and examined the crystallization inhibitory potency of a newly purified 15 kd human biliary protein. Gallbladder bile was passed through an anti-apolipoprotein A-I (apo A-I) immunoaffinity column to extract lipid-associated proteins. The bound fraction was separated by 30 kd ultrafiltration. Sodium dodecyl sulfate-polyacrylamide gel electrophesis (SDS-PAGE) was performed under nonreducing and reducing conditions. Cholesterol crystallization activity was tested in a photometric cholesterol crystal growth assay. Isoelectric focusing was performed by using a standard gel. The purified 15 kd protein was subjected to N-terminal amino acid sequencing. Although the whole apo A-I-bound fraction contained a variety of proteins and lipids, its 30 kd filtrate yielded a nearly pure 15 kd protein with only minor contamination from apo A-1. Amino acid sequencing showed that the protein was unique. Enzymatic deglycosylation revealed no evidence for glycosylation. At a protein concentration of 10 micrograms/ml, crystallization time was delayed as compared with control and apo A-I, and final crystal mass was reduced to 75% of control. Its isoelectric point was 6.1 without isoforms. Under nonreducing conditions, the protein formed a 30 kd dimer and a 60 kd tetramer. We conclude that this protein is a novel potent biliary crystallization inhibitor protein.
Secknus
R
R
Department of Gastroenterology, Cleveland Clinic Foundation, OH 44195-5218, USA.
Yamashita
G
G
Ginanni Corradini
S
S
Chernosky
A
A
Williams
C
C
Hays
L
L
Secknus
M A
MA
Holzbach
R T
RT
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I; D001646:Bile / Q000737:chemistry*; D002769:Cholelithiasis / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism*; D002846:Chromatography, Affinity; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D012016:Reference Values
D016632:Apolipoprotein A-I; D006023:Glycoproteins; D010446:Peptide Fragments; D002784:Cholesterol