APF CBP, THE SMALL, AMPHIPATHIC, ANIONIC PROTEIN(S) IN BILE AND GALLSTONES, CONSISTS OF LIPID-BINDING AND CALCIUM-BINDING FORMS/

Two very similar small anionic, amphipathic proteins, a phospholipid-b inding apoprotein (anionic polypeptide fraction [APF]) and a calcium-b inding polypeptide (CBP), are found abundantly in bile and all types o f gallstones, The often disparate properties among various preparation s of APF/CBP could reflect different sources and separation procedures , leading to partly degraded and/or denatured protein and varied assoc iation of bile salts, lipids, bile pigments, and detergents, The prese nt study presents new methods for isolation and purification of APF/CB P, and characterizes the preparations thus obtained, It was found that isolation by selective precipitation of proteins from fresh T-tube bi le by added calcium chloride, followed by demineralization with ethyle nediaminetetraacetic acid (EDTA), removal of salts, lipids, and some p igment by Sephadex LH-20, and serial ultrafiltration yields the purest preparations. Though free of lipids, bile salts, detergents, and most pigments, these new preparations all show the same 7-kd and 12-kd ban ds on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-P AGE), the same major peaks on hydrophobic high-performance liquid chro matography (HPLC), and retain the self-associative, lipid- and calcium -binding functions, typical of older preparations obtained by potentia lly denaturative procedures, The varied properties among APF/CBP prepa rations are thus apparently related mainly to their content of differe nt proportions of two major components, lipid-binding APF and calcium binding CBP, Immunologic cross-reactions indicate common epitopes, and amino acid analyses are also similar, suggesting that APF and CBP may have the same polypeptide backbone, but differ because of posttransla tional modification(s), Sufficiently pure APF and CBP have now been ob tained to permit possible structural identification by sequencing and molecular biological techniques, though such attempts have thus far be en unsuccessful.