Anion-binding Protein Research Articles

Supramolecular Incarceration and Extraction of Tetrafluoroberyllate from Water by Nanojars.

The previously unexplored noncovalent binding of the highly toxic tetrafluoroberyllate anion (BeF42-) and its extraction from water into organic solvents are presented. Nanojars resemble anion-binding proteins in that they also possess an inner anion binding pocket lined by a multitude of H-bond donors (OH groups), which wrap around the incarcerated anion and completely isolate it from the surrounding medium. The BeF4-binding propensity of [BeF4⊂{CuII(OH)(pz)}n]2- (pz = pyrazolate; n = 27-32) nanojars of different sizes is investigated using an array of techniques including mass spectrometry, paramagnetic 1H, 9Be, and 19F NMR spectroscopy, and X-ray crystallography, along with thermal stability studies in solution and chemical stability studies toward acidity and Ba2+ ions. The latter is found to be unable to precipitate the insoluble BaBeF4 from nanojar solutions, indicating a very strong binding of the BeF42- anion by nanojars. 9Be and 19F NMR spectroscopy allows for the unprecedented direct probing of the incarcerated anion in a nanojar and, along with 1H NMR studies, reveals the fluxional structure of nanojars and their inner anion-binding pockets. Single-crystal X-ray diffraction provides the crystal and molecular structures of (Bu4N)2[BeF4⊂{Cu(OH)(pz)}32], which contains a novel Cux-ring combination (x = 9 + 14 + 9), (Bu4N)2[BeF4⊂{Cu(OH)(pz)}8+14+9], and (Bu4N)2[BeF4⊂{Cu(OH)(pz)}6+12+10] and offers detailed structural parameters related to the supramolecular binding of BeF42- in these nanojars. The extraction of BeF42- from water into organic solvents, including the highly hydrophobic solvent n-heptane, demonstrates that nanojars are efficient binding and extracting agents not only for oxoanions but also for fluoroanions.

Pseudomonas aeruginosa T6SS-mediated molybdate transport contributes to bacterial competition during anaerobiosis.

Type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria and functions as a versatile protein export machinery that translocates effectors into eukaryotic or prokaryotic target cells. Growing evidence indicates that T6SS can deliver several effectors to promote bacterial survival in harmful environments through metal ion acquisition. Here, we report that the Pseudomonas aeruginosa H2-T6SS mediates molybdate (MoO42-) acquisition by secretion of a molybdate-binding protein, ModA. The expression of H2-T6SS genes is activated by the master regulator Anr and anaerobiosis. We also identified a ModA-binding protein, IcmP, an insulin-cleaving metalloproteinase outer membrane protein. The T6SS-ModA-IcmP system provides P.aeruginosa with a growth advantage in bacterial competition under anaerobic conditions and plays an important role in bacterial virulence. Overall, this study clarifies the role of T6SS in secretion of an anion-binding protein, emphasizing the fundamental importance of this bacterium using T6SS-mediated molybdate uptake to adapt to complex environmental conditions.

Principles Governing Anion Selectivity in Proteins

The highly selective anion binding by proteins is essential for a number of biological processes including normal renal function and maintenance of blood acidity. Various transport systems are at the core of anion homeostasis in living cells, by binding selectively a range of substrates such as chloride (Cl-), bromide (Br-), fluoride (F -), carbonate (CO32-), and bicarbonate (HCO3-), among others. The efficient binding requires a high specificity recognition of anions by the protein host. However, the detailed molecular-level understanding of general anion recognition across protein sites is lacking as most of the studies are focusing only on a specific protein. In the present work, we use high-resolution crystal structures of proteins with known specificity sequence such as haloalkane dehalogenase (DhlA), the human anion exchanger 1 (hAE1), and the Cl-/F- antiporter from the CLC family to identify the thermodynamical underpinnings of anion selectivity as a first step to the rational design of anion-selective proteins. To that end, we employ a combination of in-vacuum and implicit solvent QM calculations to evaluate the binding free energies of a set of binding site models interacting with different inorganic anions including Cl-, F-, HCO3-, and CO32-. We place our findings in a broader context of anion-selective binding sites by comparison to the statistical distribution of amino acid topology and organization in various anion binding proteins available in the Protein Data Bank. The analysis involved 30 proteins structures binding F-, 67 CO32--binding proteins and 49 structures that bind HCO3-. Our results can inform the rational design of anion-moving and anion-binding systems including applications of membrane proteins to desalination and biosensing.

Polymers for Anion Recognition and Sensing

In biological systems, the selective and high-affinity recognition of anionic species is accomplished by macromolecular hosts (anion-binding proteins) that have been "optimized" through evolution. Surprisingly, it is only recently that chemists have systematically attempted to develop anion-responsive synthetic macromolecules for potential applications in medicine, national security, or environmental monitoring. Recent results indicating that unique features of polymeric systems such as signal amplification, multivalency, and cooperative behavior may be exploited productively in the context of anion recognition and sensing are documented. The wide variety of interactions-including Lewis acid/base, ion-pairing, and hydrogen bonding-that have been employed for this purpose is reflected in the structural diversity of anion-responsive macromolecules identified to date.

Amino acid containing anion receptors

Nature has devised highly efficient and selective ways of recognizing anionic substrates by using surprisingly few building blocks the most important of which are the amino acids serine, tryptophane, and arginine in addition to NH groups along the protein backbone. Deliberate use of amino acids for the construction of abiotic anion receptors could lead to systems that mimic the anion coordinating properties of anion binding proteins. With this aim in mind several groups have focused their attention on the development of anion receptors containing amino acid building blocks and came up with remarkably efficient systems. This critical review summarizes the different approaches (174 references).

ATP modulates sulfobromophthalein uptake in rat liver plasma membrane vesicles.

The hepatic uptake of the bilirubin-bilirubin-sulfobromophthalein (BSP) group of organic anions is a carrier-mediated process and is accounted for by at least four distinct plasma membrane proteins (bilitranslocase, BSP/bilirubin-binding protein, organic anion-binding protein and the organic anion transport protein). In order to investigate the regulation of basolateral organic anion uptake, BSP transport was measured in rat basolateral liver plasma membrane vesicles in the presence of ATP. ATP significantly stimulated the electroneutral uptake of BSP with an increment in Vmax compared with control (1.57 +/- 0.14 vs 0.73 +/- 0.06 nmol BSP/mg protein per 15 s, respectively; P < 0.001) while the apparent K(m) was not changed significantly (12 +/- 1 vs 12 +/- 2 mumol/L). The stimulatory effect was dose-dependent for ATP (K(m) 1.01 +/- 0.37 mmol/L). ATP had no detectable effect on the electrogenic component of BSP transport. Other nucleotides (ADP, AMP, GTP) and non-hydrolysable ATP did not enhance BSP uptake, suggesting that ATP hydrolysis was necessary for the effect. This was supported by the lack of effect on BSP uptake when ATP was added in the presence of vanadate. The addition of phorbol-12-myristate 13-acetate, an activator of protein kinase C (PKC), increased BSP uptake in a dose-dependent manner in the presence, but not in the absence, of ATP. Incubation of vesicles with staurosporine, an inhibitor of PKC activity, resulted in a dose-dependent inhibition of ATP-sensitive BSP transport. These data indicate that electroneutral BSP hepatic uptake is modulated by ATP. The effect is related to ATP hydrolysis and involves the activity of PKC.

Microsequencing of Organic Anion-Binding Proteins from Basolateral Plasma Membranes in Hepatocytes after Photoaffinity Labelling with Bumetanide and 7, 7-Azotaurocholate

Binding proteins for weak organic acids, in particular bile acids and loop diuretics, were separated by two-dimensional gel electrophoresis after photoaffinity labelling of rat liver basolateral plasma membranes. After staining with Coomassie Blue the protein spots were isolated, electroeluted and concentrated in an SDS gel. The proteins were digested in the gel by endoproteinase Lys-C. Appropriate fragments were separated by HPLC and the sequence was determined. A 57-kD protein with an apparent pi of 5.3, which was marked by photoaffinity labelling with 7,7-azotaurocholate and stained with an antibody against recombinant protein disulfide isomerase (PDI), showed complete identity with PDI in three fragments. [3H]bumetanide, a loop diuretic, marked two 54-kD proteins. One 54-kD protein with an apparent pi of 6.35 showed homology to the tyrosine kinase inhibitor of the insulin receptor. The amino acid sequence of three fragments belonging to the other 54-kD protein with an apparent pi of 6.2 was identical with cytokeratin 8. Two further proteins that were present in large amounts were also sequenced. A 35-kD protein was identified to be arginase. The internal sequences of a 58-kD protein showed 100% homology to endoproteinase ER 60. The results show that the proposed method of isolation of proteins from 2D gels (a) provides sequence data, even if there are only minute amounts of protein obtainable, and (b) allowed identification of yet unknown binding proteins of organic anions in the liver.

Bilitranslocase and sulfobromophthalein/bilirubin-binding protein are both involved in the hepatic uptake of organic anions.

The hepatic uptake of cholephilic organic anions is a carrier-mediated process. Three distinct proteins [bilitranslocase (BTL), sulfobromophthalein (BSP)/bilirubin-binding protein (BBBP), and organic anion-binding protein] have been isolated from the basolateral plasma-membrane domain of the hepatocyte. To investigate the relative role of the first two of them in accounting for the hepatic uptake of organic anions, we measured the initial rates of uptake of 35S-labeled BSP into rat liver plasma-membrane vesicles. Because transport by BTL is electrogenic but transport by BBBP is electroneutral, studies were done either with or without a positive-inside membrane potential produced by adding valinomycin in the presence of an inwardly directed K+ gradient (outside K+ > inside K+). Both electrogenic and electroneutral transport systems followed saturation kinetics. Electroneutral uptake showed an apparent Km of 20 +/- 3 microM (mean +/- SD) and a Vmax of 1.0 +/- 0.13 nmol.(mg of prot)-1.15 sec-1, whereas the electrogenic portion of BSP uptake exhibited a Km of 5.2 +/- 0.8 microM and a Vmax of 1.1 +/- 0.1 nmol.(mg of prot)-1.15 sec-1. In this case, an overshoot was observed 15 sec after valinomycin addition. Electroneutral BSP uptake was inhibited by incubation with anti-BBBP antibody, whereas anti-BTL antibody did not show any inhibitory effect. Conversely, the electrogenic uptake was inhibited by anti-BTL antibody at a BSP concentration of 5 microM; no inhibition was seen either at 20 microM BSP or upon addition of anti-BBBP antibody. From these data we conclude that the hepatic uptake of organic ions occurs via two immunologically distinct carrier proteins (BTL and BBBP) operating in parallel. BTL is a higher affinity electrogenic transporting system of organic ions, whereas BBBP is a lower affinity electroneutral transporter.

Organic anion transport in HepG2 cells: Absence of the high-affinity, chloride-dependent transporter

In previous studies, we identified a 55 kD organic anion–binding protein in liver cell sinusoidal plasma membrane subfractions. Other investigators identified another 55 kD bromosulfophthalein/bilirubin binding protein on the surface of rat hepatocytes and HepG2 cells and suggested that this protein served as a transporter for these ligands. In this study, transport of 35S-sulfobromophthalein by the human hepatoma cell line, HepG2, was quantified in the presence and absence of bovine serum albumin to further clarify the possible function of these plasma membrane binding proteins. In contrast to results in normal rat hepatocytes, virtually no uptake of 35S-sulfobromophthalein by HepG2 cells in the presence of bovine serum albumin was found. In the absence of albumin, HepG2 cells expressed temperature-dependent uptake of 35S-sulfobromophthalein. However, the high-affinity CI−-dependent sulfobromophthalein transport that characterizes normal rat hepatocytes was absent, as indicated by an approximately 95-fold lower affinity and 170-fold higher capacity of HepG2 cells for sulfobromophthalein compared with previous results with rat hepatocytes. These results suggest that 55 kD sulfobromophthalein/bilirubin–binding protein on the liver cell surface differs from organic anion-binding protein and is not responsible for sulfobromophthalein extraction in the presence of albumin, although it may play some role in lower affinity transport by cells. Immunoblot analysis and metabolic labeling of HepG2 cells demonstrated synthesis of organic anion-binding protein. However, light microscopic immunocytochemistry and immunoprecipitation of surface iodinated rat hepatocytes and HepG2 cells with antibody to a recombinant organic anion-binding protein fusion protein indicated absence of organic anion-binding protein on the surface of HepG2 cells. Because cell surface organic anion-binding protein and the mitochondrial F1-ATPase β-subunit are immunologically highly cross-reactive, the presence of intracellular but not cell surface organic anion-binding protein immunoreactivity in HepG2 cells suggests that the mitochondrial and cell surface forms of this protein are independently regulated. These studies also indicate that demonstration of sulfobromophthalein transport alone cannot be used as evidence for the presence of the high-affinity transporter. (HEPATOLOGY 1991;14:1217–1223.)

Organic anion transport in HepG2 cells: Absence of the high-affinity, chloride-dependent transporter

In previous studies, we identified a 55 kD organic anion-binding protein in liver cell sinusoidal plasma membrane subfractions. Other investigators identified another 55 kD bromosulfophthalein/bilirubin binding protein on the surface of rat hepatocytes and HepG2 cells and suggested that this protein served as a transporter for these ligands. In this study, transport of 35S-sulfobromophthalein by the human hepatoma cell line, HepG2, was quantified in the presence and absence of bovine serum albumin to further clarify the possible function of these plasma membrane binding proteins. In contrast to results in normal rat hepatocytes, virtually no uptake of 35S-sulfobromophthalein by HepG2 cells in the presence of bovine serum albumin was found. In the absence of albumin, HepG2 cells expressed temperature-dependent uptake of 35S-sulfobromophthalein. However, the high-affinity Cl(-)-dependent sulfobromophthalein transport that characterizes normal rat hepatocytes was absent, as indicated by an approximately 95-fold lower affinity and 170-fold higher capacity of HepG2 cells for sulfobromophthalein compared with previous results with rat hepatocytes. These results suggest that 55 kD sulfobromophthalein/bilirubin-binding protein on the liver cell surface differs from organic anion-binding protein and is not responsible for sulfobromophthalein extraction in the presence of albumin, although it may play some role in lower affinity transport by cells. Immunoblot analysis and metabolic labeling of HepG2 cells demonstrated synthesis of organic anion-binding protein. However, light microscopic immunocytochemistry and immunoprecipitation of surface iodinated rat hepatocytes and HepG2 cells with antibody to a recombinant organic anion-binding protein fusion protein indicated absence of organic anion-binding protein on the surface of HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The rat hepatocyte plasma membrane organic anion binding protein is immunologically related to the mitochondrial F1 adenosine triphosphatase beta-subunit.

A 55-kD organic anion binding protein (OABP) was identified previously in liver cell plasma membrane sinusoidal subfractions. Although this protein was localized to the surface of hepatocytes by immunofluorescence, immunoblot analysis revealed reactivity toward both plasma membrane and mitochondrial fractions. To clarify these findings, an immunoreactive clone from a rat liver cDNA expression library was isolated, the 1,500-base pair cDNA insert was sequenced, and the corresponding beta-galactosidase fusion protein was expressed and purified. The resulting sequence corresponded to that of the rat mitochondrial F1-adenosine triphosphatase (F1-ATPase) beta-subunit. This protein and OABP are of similar size and are mutually immunologically cross-reactive. That the antigen was present on the cell surface as well as in mitochondria was suggested from studies of immunoprecipitation after cell-surface iodination, and light- and electron-microscopic immunocytochemistry. Photoaffinity labeling of bovine F1-ATPase with high-specific-activity [35S]sulfobromophthalein revealed binding only to the beta-subunit. Hepatocyte uptake of bilirubin and sulfobromophthalein requires cellular ATP and mitochondria also transport these organic anions, which at high doses inhibit respiration. The presence of an organic anion binding site on the F1-ATPase beta-subunit suggests that it may play a role in these processes.

Inverse Relationship Between Total Glutathione S‐Transferase Content and Bile Acid Release in Isolated Hepatocytes from Untreated, Phenobarbital Pretreated and Hypothyroid Rats*

The role of cytosolic anion binding proteins (glutathione S-transferases) in the hepatic transport of bile acids remains controversial. To investigate whether increased levels of the hepatocyte total glutathione S-transferase content were associated with changes in the release of bile acids from the hepatocyte, we measured the rate of release of radioactive bile acids in isolated hepatocytes from thyroidectomized, phenobarbital pretreated and untreated rats. The isolated hepatocytes were preincubated with either 14C-cholic acid or 14C-taurocholic acid, and the release rate of radiolabeled bile acids was determined. Hepatocyte total glutathione S-transferase content was measured by rocket immunoelectrophoresis. The release rate of the radiolabeled bile acids was significantly (P less than 0.005) decreased in both hypothyroid and phenobarbital pretreated hepatocytes. The levels of total glutathione S-transferase content were significantly (P less than 0.001) increased in the hepatocytes from both hypothyroid and phenobarbital pretreated animals. Our findings reveal a striking inverse relationship between the total glutathione S-transferase content of the hepatocyte and the release rate of radiolabeled bile acids in isolated hepatocytes from two independent animal models. These observations support the hypothesis that cytosolic anion binding proteins (glutathione S-transferases) may influence the net flux across the hepatocyte plasma membrane largely by limiting efflux.

Characterization of Rose Bengal binding to sinusoidal and bile canalicular plasma membrane from rat liver

The binding of Rose bengal, a model organic anion, to sinusoidal and bile canalicular membrane fractions isolated from rat liver was compared. The fluorescence change of Rose bengal after being bound to liver plasma membranes was utilized for measuring the binding. The dissociation constants ( K d = 0.1−0.12 μM) and the binding capacities ( n = 11−15 nmol/mg protein) for Rose bengal are comparable between the two membrane fractions, although the n value for sinusoidal membrane is somewhat larger than that for bile canalicular membrane. The Rose bengal binding to both membrane fractions was inhibited by various organic anions at relatively low concentrations, i.e., the half-inhibition concentrations (IC 50) for Indocyanine green, sulfobromophthalein, Bromophenol blue and 1-anilino-8-naphthalene sulfonate were 0.1, 100, 1.5–2.5 and 100 μM, respectively, while taurocholate did not inhibit the Rose bengal binding to either membrane fraction at these low concentration ranges. The type of inhibition of sulfobromophthalein and Indocyanine green for Rose bengal binding is different between the two membrane domains. That is, in sinusoidal and bile canalicular membrane fractions, these organic anions exhibit mixed-type and competitive-type inhibition, respectively. It was suggested that the fluorescence method using Rose bengal may provide a simple method for detecting the specific organic anion binding protein(s) in the liver plasma membrane.

Selective regulation of intrinsic membrane proteins in HepG2.

Expression of three hepatocellular membrane proteins--the asialoglycoprotein receptor (hepatic binding protein) the insulin receptor and organic anion binding protein--have been studied in the HepG2 cell line. HepG2 grown in minimal essential medium supplemented with 10% fetal bovine serum maximally expressed hepatic binding protein and insulin receptor only at confluence while organic anion binding protein appeared independent of the state of cellular proliferation. When cells were grown in medium supplemented with dialyzed fetal bovine serum, adult bovine serum or in chemically defined medium, expression of hepatic binding protein and insulin receptor but not organic anion binding protein was reduced by 60 to 80%. Immunoblotting techniques revealed that cells grown in dialyzed fetal bovine serum contained virtually no mature, glycosylated 45 kD hepatic binding protein, but a small amount of 36 kD protein. Metabolic labeling of cells grown in dialyzed fetal bovine serum with [35S] methionine indicated reduced synthesis of the 45 kD hepatic binding protein and the absence of the 36 kD protein. Restoration of normal expression of hepatic binding protein and insulin receptor was achieved by addition to dialyzed fetal bovine serum of: fetal bovine serum; 2,000 dalton ultrafiltrate of fetal bovine serum, or 300 to 350 dalton fraction of the ultrafiltrate. The normal concentration of organic anion binding protein demonstrable in cells grown in dialyzed fetal bovine serum indicates that the low molecular weight factor(s) is not a generalized modulator of plasma membrane biogenesis. However, its effect on the steady-state level of the hepatic binding protein and insulin receptor, characteristic of mature hepatocytes, suggests a role for this fetal serum factor in hepatocellular differentiation.

Intracellular pH topography of Penicillium cyclopium protoplasts. Maintenance of Δ pH by both passive and active mechanisms

The intracellular pH distribution in protoplasts of Penicillium cyclopium has been studied using the recently developed fluorescent probe microscopic technique. The technique gives detailed pH maps of the interior of the protoplasts with the exception of vacuoles (no fluorescence signal from vacuoles was observed). In the cytoplasm two separate layers were distinguished: a thinner outer layer with acidic pH (around 5) and the larger core region with near neutral pH. The pH of the core region is decreased by the addition of uncouplers, inhibitors of respiration and during the uptake of l-phenylalanine. These compounds do not change the pH of the surface layer, which is, however, acidified by addition of vanadate, an inhibitor of the proton pump of the plasmalemma. We suggest that the pH of the surface layer is maintained by the combined effects of a Donnan distribution of protons (bound to postulated anion binding proteins) and the proton extrusion via the plasmalemma proton pump. This mechanism explains the protection of the cytoplasmic core of acidophilic eukaryotes from the influence of the usually acidic environment.

Immunological studies of an organic anion-binding protein isolated from rat liver cell plasma membrane.

The mechanism of organic anion uptake by hepatocytes has kinetics that suggest facilitated diffusion, and carrier-mediated membrane transport has been postulated. In previous studies, we purified a 55,000-mol wt organic anion-binding protein (OABP) by affinity chromatography on sulfobromophthalein (BSP)-Sepharose of deoxycholate solubilized liver cell plasma membrane preparations. Using specific goat and rabbit antibodies to OABP, we have now investigated the distribution of this protein in liver fractions and other tissues by an enzyme-linked immunosorbent assay and by the immunoblot (Western blot) procedure. These studies indicated that OABP is present in significant amounts in all tissues examined except for blood. Although OABP has not as yet been isolated from each of these tissues and characterized, OABP in heart retained the ability to bind organic anions, and was purified by affinity chromatography on BSP-sepharose. In liver, OABP was membrane bound and remained so after extraction with 0.9 M NaCl, which suggests that it is an intrinsic membrane protein. OABP did not have a ubiquitous subcellular distribution within the hepatocyte. Preparation of subfractions of liver cell plasma membrane revealed that OABP is present in the sinusoidal and absent from the canalicular membrane. Immunofluorescence studies performed in short-term cultured hepatocytes suggest that OABP is associated with the surface of these cells and does not have a significant intracellular distribution.

Comparison of binding of thyroid hormone analogues to hepatic organic anion binding proteins

D v protein and ligandin are two hepatic cytosolic proteins which bind organic anions, including endogenous thyroid hormones. Binding studies were performed using the ANS displacement technique to compare the binding of a variety of thyroid hormone analogues to purified organic anion binder and ligandin. Inhibition of ANS binding by these compounds was competitive. Both proteins bound L- and D-thyroxine with comparable affinity ( K d 30–45 μM), whereas ligandin boudn 3′,3′,5-triiodo- L-thyronine, 3′,3′,5-triiodo- L-thyronine and most analogues with greater affinity. Nevertheless, the order of ligand affinities for both binders was highly correlated, suggesting that the nature of the binding site on both proteins is similar. The binding affinities of these organic anion binders are 2–3 orders of magnitude lower than an hepatic cytosolic thyroid binder reported by others, suggesting that ligandin and organic anion binder may not be important in intracellular thyroid hormone transfer.

Does Z-protein have a role in transport of bilirubin and bromosulfophthalein by isolated perfused rat liver?

Bilirubin and other organic anions are transported in serum avidly bound to albumin from which they are extracted and transferred into the hepatocyte where they bind to cytosolic proteins. Two abundant organic anion binding proteins, ligandin and Z-protein, were previously purified from liver cytosol and characterized. Other studies in isolated perfused rat liver revealed that selectively increased cytosolic ligandin concentration, following phenobarbital treatment or thyroidectomy, directly correlated with net bilirubin uptake which resulted from reduced bilirubin efflux. To clarify the role of Z-protein in hepatic organic anion transport, we have now determined the kinetics of bilirubin and bromosulfophthalein (BSP) uptake in isolated perfused liver of normal rats and compared results to rats in which Z-protein, but not ligandin, was selectively increased following treatment with clofibrate (ethylchlorophenoxy-isobutyrate). These studies revealed that despite a 147% induction of Z-protein in treated animals, there was no effect on influx or efflux of tracer doses of bilirubin or BSP. Addition of albumin to the protein-free 10% fluorocarbon perfusate reduced influx of 3H-bilirubin (p less than 0.03) and tended to reduce influx of BSP. In this situation, there was still no influence of Z-protein concentration on efflux. These studies indicate that Z-protein does not appear to play a role in the hepatic uptake of bilirubin and BSP.

Preparation and metabolism of 125-sulfobromophthalein

Metabolism of 125I-sulfobromophthalein (BSP) prepared by the chloramine-T method was studied in rats. 125I-BSP is removed rapidly from the circulation. However, as compared to BSP, its plasma clearance and biliary excretion are delayed, and its accumulation in the liver is prolonged. Although BSP and 125I-BSP show similar binding to albumin in serum, their binding properties to liver cytosolic proteins and to the liver cell plasma membrane organic anion binding protein (OABP) differ. In contrast to the X-, Y- and Z-protein binding of BSP, 125I-BSP binds predominantly to a high molecular weight protein and only a small proportion of 125I-BSP binds to OABP.

Newly identified organic anion-binding proteins in rat liver cytosol

Previously, all organic anion binding activity in the Y protein (ligandin-containing) fraction of rat liver cytosol has been attributed to the glutathione S-transferases. Gel filtration on Sephadex G-75 superfine has resolved the Y protein fraction into organic anion-binding fractions of two distinct molecular weights: (a) M r 45 000 containing the glutathione S-transferases, and (b) M r 35 000 referred to as Y′. Two proteins (D v and D 1) were purified from the Y′ fraction. D v is a basic protein consisting of four subunits of M r 8000–9000 and selectively binds 1-anilino-8-naphthalene sulfonate and sulfobromophthalein. D 1 is a monomer ( M r 40 000) which exhibits high affinity binding for Rose Bengal. D v protein bound a broad spectrum of organic anions. Antibody raised in rabbits to rat D v showed no cross-reactivity with purified glutathione S-transferases A, B, or C. Thus, we have identified organic anion-binding proteins which are unrelated to the glutathione S-transferases, but which were previously associated with the crude Y-fraction. The relative abundance of these proteins and their binding characteristics suggest their role in hepatic organic anion transport.