Identification of the hepatocyte Na+-dependent bile acid transport protein using monoclonal antibodies

Monoclonal antibodies have been utilized to characterize the hepatocyte Na+-dependent bile acid transport system. Sinusoidal plasma membrane proteins in the 49-54-kDa range, which are thought to be components of this transport system, based on photo-affinity labeling and reconstitution studies, have been partially purified by affinity chromatography and utilized as an immunogen for the production of a panel of monoclonal antibodies (mAb). One of these mAbs, 25A-3, recognized both a 49- and a 54-kDa protein as assessed by immunoprecipitation. In addition, it was shown to protect the bile acid transport system from inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in a dose-dependent manner. DIDS covalently labeled membrane proteins of 49 and 54 kDa, and this process could be significantly inhibited when performed in the presence of mAb 25A-3. Furthermore, the DIDS-labeled membrane proteins were immunoprecipitated by 25A-3. These results establish that one of these membrane components is the bile acid carrier protein. Another mAb (25D-1) which immunoprecipitated only a 49-kDa protein was shown to block the protective effect of 25A-3 on DIDS inhibition of bile acid transport. In addition both antibodies effected each other's binding capacity to hepatocytes and reacted with the same 49-kDa protein as established by sequential immunoprecipitation. Binding studies indicated that there are approximately 3.3 X 10(6) 49-kDa transport molecules/hepatocyte. These results firmly establish that the 49-kDa protein is the Na+-dependent hepatocyte bile acid transporter.     

Purification and reconstitution of the bile acid transport system from hepatocyte sinusoidal plasma membranes

The taurocholic acid transport system from hepatocyte sinusoidal plasma membranes has been studied using proteoliposome reconstitution procedures. Membrane proteins were initially solubilized in Triton X-100. Following detergent removal, the resultant proteins were incorporated into lipid vesicles prepared from soybean phospholipids (asolectin) using sonication and freeze-thaw procedures. The resultant proteoliposomes demonstrated Na+-dependent transport of taurocholic acid which could be inhibited by bile acids. Greatly reduced amounts of taurocholic acid were associated with the phospholipid or membrane proteins alone prior to proteoliposome formation. Membrane proteins were fractionated on an anionic glycocholate-Sepharose 4B affinity column which was prepared by coupling (3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholan-24-oyl)-N alpha-lysine to activated CH-Sepharose 4B via the epsilon-amino group of lysine resulting in the retention of a free carboxyl group. The adsorbed proteins enriched in components in the 54 kDa zone, which were originally identified by photoaffinity labeling to be components of the bile acid transport system, were also incorporated into liposomes. This vesicle system showed almost a 4-fold increase in Na+-dependent taurocholic acid uptake when compared to proteoliposomes formed from total membrane protein, as well as sensitivity to inhibition by bile acids. These results demonstrate that the bile acid carrier system can be reconstituted in proteoliposomes and that utilizing proteins in the 54 kDa zone leads to a significant enhancement in the transport capacity of the reconstituted system, consistent with the role of 54 kDa protein(s) as component(s) of the bile acid carrier system.     

Ventilatory response to helium-oxygen breathing during exercise: effect of airway anesthesia

Krishnan, Bharath S., Ron E. Clemens, Trevor A. Zintel,Martin J. Stockwell, and Charles G. Gallagher. Ventilatory response to helium-oxygen breathing during exercise: effect of airwayanesthesia. J. Appl. Physiol. 83(1):82-88, 1997.The substitution of a normoxic helium mixture(HeO₂) for room air (Air) during exercise results in a sustained hyperventilation, which is present evenin the first breath. We hypothesized that this response is dependent onintact airway afferents; if so, airway anesthesia (Anesthesia) shouldaffect this response. Anesthesia was administered to the upper airwaysby topical application and to lower central airways by aerosolinhalation and was confirmed to be effective for over 15 min. Subjectsperformed constant work-rate exercise (CWE) at 69 ± 2 (SE) % maximal work rate on a cycle ergometer on three separate days: twiceafter saline inhalation (days 1 and3) and once after Anesthesia(day 2). CWE commenced after a briefwarm-up, with subjects breathing Air for the first 5 min (Air-1),HeO₂ for the next 3 min, and Airagain until the end of CWE (Air-2). The resistance of the breathingcircuit was matched for Air andHeO₂. BreathingHeO₂ resulted in a small butsignificant increase in minute ventilation(I) anddecrease in alveolar PCO₂ in both theSaline (average of 2 saline tests; not significant) and Anesthesiatests. Although Anesthesia had no effect on the sustainedhyperventilatory response to HeO₂breathing, theI transientswithin the first six breaths ofHeO₂ were significantly attenuatedwith Anesthesia. We conclude that theI response to HeO₂ is not simply due to areduction in external tubing resistance and that, in humans, airwayafferents mediate the transient but not the sustained hyperventilatoryresponse to HeO₂ breathing duringexercise.

     

Antifreeze polypeptides from the Newfoundland ocean pout,Macrozoarces americanus: presence of multiple and compositionally diverse components

Summary Eight major antifreeze polypeptides (AFP) were purified from the sera of Newfoundland ocean pout. Except for their approximately identical size (6,000 Dalton), these components were shown to be separate entities by their behaviour on polyacrylamide gel electrophoresis, ion exchange chromatography, gel permeation and reverse phase high performance liquid chromatography. They could also be divided into two cross-reactive, yet distinct, immunological groups. Amino acid analysis demonstrated that ocean pout AFP are different from all of the other antifreezes studied to date. The ocean pout AFP do not contain the abundance of alanine (60 mol%) found in winter flounder and shorthorn sculpin AFP nor the high half-cystine residues (8 mol%) observed in sea raven AFP. It is suggested that ocean pout AFP represent a new type of macromolecular antifreeze.Abbreviations AFGP antifreeze glycoprotein(s) - AFP antifreeze polypeptide(s) - HPLC high performance liquid chromatography - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis     

Interaction of prolyl 4-hydroxylase with synthetic peptide substrates. A conformational model for collagen proline hydroxylation.

With the aim of understanding the structural basis for the substrate specificity of collagen prolyl 4-hydroxylase, we have studied the conformational features of synthetic oligopeptide substrates and their interaction with the enzyme purified from chicken embryo. Circular dichroism and infrared spectral data, taken in conjunction with relevant crystal structure data, indicated an equilibrium mixture of the polyproline-II (PP-II) helix, the beta-turn, and the random coil conformations in aqueous and trifluoroethanol solutions of the "collagen-related" peptides: t-Boc-Pro-Pro-Gly-Pro-OH, t-Boc-Pro-Pro-Gly-Pro-NHCH3, t-Pro-Pro-Gly-Pro-Pro-OH, t-Boc-Pro-Pro-Ala-Pro-OH, and t-Boc-Pro-Pro-Gln-Pro-OCH3, where t-Boc is tert-butoxycarbonyl. In another set of peptides related to elastin, t-Boc-Val-Pro-Gly-Val-OH and t-Boc-Gly-Val-Pro-Gly-Val-OH, the data indicated the beta-structure, rather than the PP-II helix, was in equilibrium with the beta-turn. Kinetic parameters for the enzymatic hydroxylation of the peptides showed that as a group, the first (proline-rich) set of peptides has higher Km values and lower Vmax and Kcat/Km values than the valine-rich peptides. Data on the inhibition of hydroxylation of the standard assay substrate (Pro-Pro-Gly)10 by the oligopeptides pointed to common binding sites for the peptides. Hydroxyproline-containing peptides had no effect on the hydroxylation of the standard substrate, showing the absence of product inhibition. Based on these and earlier data, we propose that in collagen and related peptides, a supersecondary structure consisting of the PP-II helix followed by the beta-turn is the minimal structural requirement for proline hydroxylation. The PP-II structure would aid effective interaction at the substrate binding subsites, while the beta-turn would be essential at the catalytic site of the enzyme. In elastin and related peptides, the beta-strand structure may be interchangeable with the PP-II structure. This conformational model for proline hydroxylation resolves the discrepancies in earlier proposals on the substrate specificity of prolyl 4-hydroxylase. It is also consistent with the available information on the active site geometry of the enzyme.     

Conformational requirement for lysine hydroxylation in collagen. Structural studies on synthetic peptide substrates of lysyl hydroxylase.

An attempt has been made to understand the conformational determinants that govern the hydroxylation of selected lysyl residues in the nascent collagen molecule by lysyl hydroxylase (EC 1.14.11.4). A series of peptide substrates of the enzyme, ranging in length from 3 to 12 residues, were synthesized. These included: tert-butyloxylcarbonyl (t-Boc)-Ile-Lys-Gly; Boc-Ala-Lys-Gly; N-acetyl-Ala-Lys-Gly-Ser; Hyp-Gly-Pro-Lys-Gly-Glu; Leu-Hyp-Gly-Ala-Lys-Gly-Glu; Gly-Phe-Hyp-Gly-Leu-Hyp-Gly-Ala-Lys-Gly-Glu; (Hyp-Gly-Pro-Lys-Gly-Glu)2; and Ala-Arg-Gly-Ile-Lys-Gly-Ile-Arg-Gly-Phe-Ser-Gly. The conformational features of these peptides were studied by spectroscopic methods so as to relate this information with the kinetic parameters for the interaction of these peptides with purified lysyl hydroxylase. Spectroscopic data, supported by conformational energy calculations, indicated that the tripeptides t-Boc-Ile-Lys-Gly and t-Boc-Ala-Lys-Gly adopt a gamma-turn structure in water and trifluoroethanol with Lys in the second position of the turn. In the tetra- and larger peptides two structures, the beta-turn and a polyproline-II (PP-II) type extended conformation, were identified. The proportions of these two structures in a given peptide depended on the polarity of the solvent. All of the peptides were hydroxylated by lysyl hydroxylase isolated from chicken embryos. In contrast, a control peptide, t-Boc-Ala-Gly-Lys which adopted a beta-turn with Lys at the end of the turn, was not hydroxylated. Competitive inhibition of the hydroxylation of protocollagen by some of the peptides showed a common binding site for these substrates in the enzyme's active site. Kinetic data on the peptides indicated improved hydroxylation rate (higher Vmax) in peptides having relatively higher beta-turn content and improved binding (lower Km) in peptides with higher content of the PP-II structure. The efficacy of the substrate was also governed by its chain length. These data suggest that the conformational criterion for lysine hydroxylation in collagen-related peptides is the presence of a "bent" structure, such as the gamma- or beta-turn at the catalytic site of lysyl hydroxylase and an "extended" PP-II type structure at the binding site(s) of the enzyme's active site. This suggestion also provides a conformational rationale for earlier observations on the substrate specificity of lysyl hydroxylase.     

Polypeptide models of collagen. Solution properties of (Gly-Pro-Sar)n and (Gly-Sar-Pro)n.

Our studies on the solution conformation of (Gly-Pro-Sar)_n and (Gly-Sar-Pro)_n synthesized as polypeptide models for collagen are reported. It is found that, while (Gly-Pro-Sar)_n exists in ordered triple-helical conformation, (Gly-Sar-Pro)_n remains as a disordered random coil in water. Addition of certain helix-promoting solvents seems to generate order in (Gly-Sar-Pro)_n.     

Structure-function relationships in a winter flounder antifreeze polypeptide. I. Stabilization of an alpha-helical antifreeze polypeptide by charged-group and hydrophobic interactions

The major antifreeze polypeptide (AFP) from winter flounder (37 amino acid residues) is a single alpha-helix. Aspartic acid and arginine are found, respectively, at the amino and carboxyl-termini. These charged amino acids are ideally located for stabilizing the alpha-helical conformation of this AFP by means of charge-dipole interaction (Shoemaker, K. R., Kim, P.S., York, E.J., Stewart, J. M., and Baldwin, R. L. (1987) Nature 326, 563-567). In order to understand these and other molecular interactions that maintain the AFP structure, we have carried out the chemical synthesis of AFP analogs and evaluated their conformations by circular dichroism spectroscopy. We synthesized the entire AFP molecule (37-mer) and six COOH-terminal peptide fragments (36-, 33-, 27-, 26-, 16-, and 15-mers). Peptides containing acidic NH2-terminal residues displayed greater helix formation and thermal stability compared to those peptides of similar size, but with neutral NH2-terminal residues. Helix formation was maximum above pH 9.2. The peptide conformations also displayed a pH-dependent sensitivity to changes in ionic strength. Helix formation was reduced in the presence of acetonitrile. We conclude that the AFP helix is most likely stabilized by: charge-dipole interactions between charged terminal amino acids and the helix dipole, a charge interaction between Lys18 and Glu22 (either a salt bridge or a hydrogen bond), and hydrophobic interactions.