Albumin binding is a crucial determinant of bilirubin clearance in health and bilirubin toxicity in certain disease states. However, prior attempts to measure the affinity of albumin for bilirubin have yielded highly variable results, reflecting both differing conditions and the confounding influence of impurities. We therefore have devised a method based on serial ultrafiltration that successively removes impurities in [ 14 C]bilirubin until a stable binding affinity is achieved, and then we used it to assess the effect of albumin concentration and buffer composition on binding. The apparent binding affinity of human serum albumin for [ 14 C]bilirubin was strongly dependent on assay conditions, falling from (5.09 ؎ 0.24) ؋ 10 7 liters/mol at lower albumin concentrations (15 M) to (0.54 ؎ 0.05) ؋ 10 7 liters/mol at higher albumin concentrations (300 M). To determine whether radioactive impurities were responsible for this change, we estimated impurities in the stock bilirubin using a novel modeling approach and found them to be 0.11-0.13%. Formation of new impurities during the study and their affinity for albumin were also estimated. After correction for impurities, the binding affinity remained heavily dependent on the albumin concentration (range (5.37 ؎ 0.26) ؋ 10 7 liters/mol to (0.65 ؎ 0.03) ؋ 10 7 liters/ mol). Affinities decreased by about half in the presence of chloride (50 mM). Thus, the affinity of human albumin for bilirubin is not constant, but varies with both albumin concentration and buffer composition. Binding may be considerably less avid at physiological albumin concentrations than previously believed.
Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB-HSA affinity constants (K'f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 microM, K'f (litre/mol) was inversely related to [HSA], irrespective of the [Bf]/[HSA] ratio. K'f was 2.066 x 10(6) + (3.258 x 10(8)/[HSA]). When 50 mM KC1 was isoosmotically substituted for sucrose, the K'f value was significantly lower {2.077 x 10(6) + (1.099 x 10(8)/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] < or = 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with K(m) values of 28 +/- 7 and 57 +/- 8 nM respectively (mean +/- S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112 +/- 12 versus 45 +/- 4 pmol of UCB. mg-1 of protein. 15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K'f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with K(m) values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K'f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.
Anionic polypeptide fraction (APF) and calcium-binding preparations of APF/CBP could reflect different sources protein (CBP) are two very similar, small, amphipathic, and separation procedures, leading to partly degraded highly anionic proteins that are present in high concentraand/or denatured protein and varied association of bile tions in both normal and pathological bile and all types of salts, lipids, bile pigments, and detergents. The present gallstones.1-3 APF, the phospholipid-binding apoprotein of the study presents new methods for isolation and purifica-bile pigment-lipoprotein complex of bile, 4,5 is involved in the tion of APF/CBP, and characterizes the preparations secretion of cholesterol and phospholipids into bile.6 CBP thus obtained. It was found that isolation by selective binds calcium tightly and promotes the precipitation of calprecipitation of proteins from fresh T-tube bile by added cium salts when bound to mucin, 7 but inhibits calcium salt calcium chloride, followed by demineralization with eth-precipitation when not bound to mucin.7-9 APF, however, is ylenediaminetetraacetic acid (EDTA), removal of salts, also found in gallstones, 10,11 where it is uniquely localized to lipids, and some pigment by Sephadex LH-20, and serial the pigmented zones, interposed between the mucin matrix ultrafiltration yields the purest preparations. Though and the calcium-pigment deposits.11 These properties suggest free of lipids, bile salts, detergents, and most pigments, that APF and/or CBP may play key roles in the regulation these new preparations all show the same 7-kd and 12-of the precipitation of calcium salts from supersaturated bile, kd bands on sodium dodecyl sulfate-polyacrylamide gel and the deposition of these salts on the structural mucin electrophoresis (SDS-PAGE), the same major peaks on matrix of the gallstones. 1,2,11hydrophobic high-performance liquid chromatographyNeither APF nor CBP has been fully purified or sequenced, (HPLC), and retain the self-associative, lipid-and cal-and the various preparations from different laboratories, 4,5,7-cium-binding functions, typical of older preparations ob-13 isolated from different sources using different procedures, tained by potentially denaturative procedures. The var-often exhibit disparate properties, even though all display ied properties among APF/CBP preparations are thus 12-kd as well as 7-kd bands on sodium dodecyl sulfate-polyapparently related mainly to their content of different acrylamide gel electrophoresis (SDS-PAGE), and all exhibit proportions of two major components, lipid-binding APF immunologic cross-reactivities and very similar amino acid and calcium-binding CBP. Immunologic cross-reactions compositions. 13 The present collaborative communication indicate common epitopes, and amino acid analyses are from these laboratories presents new isolation methods and also similar, suggesting that APF and CBP may have compares the properties of these preparations with those of the same polypeptide backbone, but differ because of older pr...
The binding of 3H-dexamethasone (3H-DM) was studied in the cytoplasmic fraction of bone cells isolated from fetal rat calvaria by enzymatic digestion. An high-affinity glucocorticoid binding protein resembling those described in other target tissues was demonstrated. Scatchard analysis revealed a single class of binding sites with an apparent dissociation constant for 3H-DM (0C) of 7 x 10-9M and a concentration of binding sites of 0.11 pmoles/mg cytosol protein. The number of cytoplasmic binding sites per cell was calculated at 6,000 which is probably an underestimate due to occupancy of some sites by endogenous steroids. The binding sites appeared protein in nature since incubation with pronase destroyed 100% of the binding. Nuclear transfer was demonstrated in a reconstituted system utilizing bone cytosol as donor and liver nuclei as the acceptor. Competitive binding analysis revealed corticosterone to be equivalent to DM in binding affinity; progesterone was 75% as potent as DM. Aldosterone and SC-26304 (a spirolactone analogue) had, respectively, 1
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