The effect of phenobarbital and 3′-methyl-N,N-dimethyl-4-aminoazobenzene administration on catalytic,binding and immunological properties of ligandin subunits in rat liver

Following administration of phenobarbital to rats, liver ligandin content, bilirubin binding, glutathione-S-transferase and steriod isomerase activities by 150% and the 22 000-dalton subunit was selectively increased. Following adminstration of 3′-methyl-N,N-dimethyl-4-aminoazobenzene, rat liver ligandin content and steroid isomerase decrased by 65%, glutathione-S-transferase incrased by 100%, bilirubin binding was abolished, and the relative proportion of the 22 000- and 25 000-dalton subunits remained unchanged.     

Interactions of bilirubin and other ligands with ligandin.

Circular dichroism methods were used to study the structure of rat ligandin and the binding of organic anions to the protein. Ligandin has a highly ordered secondary structure with about 40%alpha helix, 15% beta structure, and 45% random coil. Bilirubin binding occurred primarily at a single high affinity site on the protein. The binding constant for bilirubin (5 X 10-7 Mminus 1) was the highest among the ligands studied. The bilirubin-ligandin complex exhibited a well-defined circular dichroic spectrum with two major overlapping ellipticity bands of opposite sign in the bilirubin absorption region. This spectrum was virtually a mirror image of that of human or rat serum albumin-bilirubin complexes. Studies on the direct transfer of bilirubin from ligandin to rat serum albumin showed that sasociation constants of bilirubin-ligandin complexes were approximately tenfold less than those of the bilirubin-albumin system. Ligandin exhibited a broad specificity with respect to the typeof ligand bond. A series of organic anions inclucing dyes used clinically for liver function tests, fatty acids, hormones, heme derivatives, bile acids, and other ligands that were considered likely to interact with ligandin, were examined. Most induced ellipticity changes consistent with competitive displacement of bilirubin from ligandin and relative affinities of these compounds for ligandin were determined based on their effectiveness in desplacing the bilirubin. Some substances such as glutathione, conjugated sulfobromophthaleins and lithocholic acid bound to ligandin but induced anomalous spectral shifts, when added to ligandin-bilirubin complexes. Other compounds, including some that act as substrates for the glutathione transferase activity exhibited by ligandin, revealed no apparent competitive effects with respect to the bilitubin binding site.     

The preparation of ligandin with glutathione-S-transferase activity from porcine liver cytosol affinity chromatography on bromosulphophthalein-Sepharose.

A simple and rapid method for the purification of porcine ligandin with glutathione-S-transferase activity is presented. After ion-exchange chromatography on DEAE-Sephadex, ligandin is isolated from porcine liver cytosol by affinity chromatography on bromosulphophthalein-Sepharose and gel filtration of Sephadex G-100. Evidence is presented that the purified ligandin is homogeneous with respect to polyacrylamide-gel electrophoresis (7.5%) and sodium dodecylsulphate-gel electrophoresis. Physico-chemical investigations show that the purified ligandin has properties similar to those of ligandin isolated from other species with respect to molecular weight, amino-acid composition, secondary structure and catalytic activity. As is the case for human and rat ligandin, porcine ligandin binds bilirubin. Evidence is also presented that porcine liver cytosol contains several bromosulphophthalein-binding proteins with basic isoelectric points lacking catalytic activity.     

Isolation and amino acid sequence analysis of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase.

3 beta-Hydroxysteroid dehydrogenase/steroid isomerase has been purified to homogeneity from bovine adrenal glands. A single protein of molecular weight 42,090 +/- 40 containing both enzyme activities has been isolated. Approximately 86% of the amino acid sequence of the bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase has been obtained by sequencing peptides isolated from digests with trypsin and lysyl endopeptidase and by chemical cleavage with CNBr. The sequence obtained is identical with that of the deduced amino acid sequence of the bovine ovarian 3 beta-hydroxysteroid dehydrogenase/steroid isomerase [Zhao et al. (1989) FEBS Lett. 259, 153-157], with the exception that the N-terminal methionine residue found in the bovine ovarian sequence is not present in the mature bovine adrenal enzyme. On the basis of the primary structure and comparisons with other NAD+ binding proteins, we propose a structural model of the bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase localizing the NAD+ binding site as well as the membrane-anchoring segment.     

Binding of pyridoxal 5'-phosphate to bovine serum albumin and albumin fragments obtained after proteolytic hydrolysis. Localization and nature of the primary PLP binding site.

The binding of pyridoxal 5'-phosphate (PLP) to bovine serum albumin (BSA), and to large BSA fragments obtained after proteolytic hydrolysis, was investigated in order to study the structure of these fragments in relation to the albumin structure itself, and to get information about the PLP binding sites on albumin. From absorbance and circular dichroism spectra, combined with peptide mapping of the tryptic digests of the reduced PLP-protein complexes, it could be concluded that the primary binding site is localized with the NH2-terminal part of the albumin molecule. The COOH-terminal part contains one or more secondary sites. It appeared that in albumin and in the largest NH2-terminal fragment, the environment of the primary binding site is rather apolar in character. However, in the smallest NH2-terminal fragment this site is more exposed to the solvent. This suggests that the part of the peptide chain which is not common in both fragments has a stabilizing effect on the structure around the primary binding site.     

Structural heterogeneity of the alpha subunits of the nicotinic acetylcholine receptor in relation to agonist affinity alkylation and antagonist binding

The structural basis for the heterogeneity of the two agonist binding sites of the Torpedo californica acetylcholine receptor with respect to antagonist binding and reactivity toward affinity alkylating reagents was investigated. There is one agonist binding site on each of the two alpha subunits in a receptor monomer. One of these sites is easily affinity labeled with bromoacetylcholine, while more extreme conditions are required to label the other. Evidence is presented that the site which is easily labeled with bromoacetylcholine is the site with higher affinity for the antagonist d-tubocurarine. Digestion of purified alpha subunits with staphylococcal V8 protease gave two limit fragments with apparent molecular weights of 17K and 19K. Both of these fragments began at residue 46 of the alpha sequence, and both reacted with monoclonal antibodies specific for the sequence alpha 152-159 but not with antibodies specific for alpha 235-242. Their tryptic peptide maps and reactivity with a number of monoclonal antibodies were virtually identical. Only the 17-kilodalton (17-kDa) fragments stained heavily for sugars with Schiff's reagent. However, both fragments bound 125I-labeled concanavalin A. Complete removal of carbohydrate detectable with concanavalin A from V8 protease digests of alpha subunits resulted in two fragments of lower apparent molecular weights, indicating that these fragments differed not only in carbohydrate content but also in their C-termini or by another covalent modification. Covalent labeling of one of the two agonist sites of the intact receptor with bromo[3H]acetylcholine followed by digestion with V8 protease resulted in labeling of only the 19-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a common high affinity binding site on glutathione S-transferase B for lithocholic acid and bilirubin

Binding of lithocholic acid, bilirubin, and gossypol to glutathione S-transferase B (ligandin or transferase YaYc) was compared using four methods. Tryptophan quenching revealed a single high affinity site for bilirubin and gossypol but could not be used for lithocholic acid. Both displacement of the fluorescent probe, 1-anilino-8-naphthalenesulfonate, and spectral changes induced by bilirubin binding demonstrated a common high affinity site for which all three ligands compete. Similar results were obtained by equilibrium dialysis. The dissociation constants for the binding of both bilirubin and lithocholic acid were comparable with the various methods (range 0.2-0.7 microM). Thus, lithocholic acid and bilirubin share a high affinity binding site on gluthathione S-transferase B that appears to be separate from the binding site for substrates.     

Circular dichroism analysis of the secondary structure of Z protein and its complexes with bilirubin and other organic anions.

Circular dichroism (CD) methods were employed to study the conformation of Z protein and characterize its complexes with bilirubin and other organic anions. Z protein-bilirubin complexes exhibited a spectrum with overlapping ellipticity bands of opposite sign in the bilirubin absorption region. These results were compared with those obtained with ligandin, the other major organic anion binding protein of liver. Secondary structural differences between the two proteins were easily demonstrated since ligandin is predominantly an alpha-helical protein and Z features mainly beta-structure. Furthermore, the optical activity pattern generated by bilirubin binding to Z was virtually a mirror image of that of the ligandin bilirubin system. CD experiments were designed to study the direct transfer of bilirubin between Z protein and ligandin, and it was shown that both proteins have almost equal affinities for bilirubin. The bilirubin on Z was readily displaced by oleic acid and displaced to a lesser extent by sulfobromophthalein,     

Multiple Ya subunits of glutathione S-transferase detected by monoclonal antibodies

Monoclonal antibodies to ligandin (YaYa) and glutathione (GSH) S-transferase B (YaYc) were produced by hybridomas derived from the fusion of mouse myeloma cells and spleen cells of mice immunized with the YaYa or YaYc proteins, respectively. Enzyme-linked immunosorbent assay was used to screen for antibody-producing clones. Immunoblotting of the subunits of transferase B, ligandin, and another GSH S-transferase containing Yb subunits showed that the monoclonal antibodies produced by two anti-YaYa subclones recognized the Ya subunits of both ligandin and transferase B, but they did not bind Yc or Yb subunits. It was also revealed that antibodies produced by several anti-YaYc subclones recognized the Yc subunit, but not the Ya subunit of the antigen which was used for the immunization of the mice. However, these monoclonal antibodies did bind the Ya subunit of ligandin. These results indicate that the Ya subunits of GSH S-transferase B and of ligandin do share at least one common determinant. However, these two Ya subunits are structurally distinct as evidenced by their differences in binding by monoclonal anti-YaYc antibodies.     

Cell-surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S-layer of Caulobacter crescentus

The paracrystalline surface (S)-layer of Caulobacter crescentus is composed of a single secreted protein (RsaA) that interlocks in a hexagonal pattern to completely envelop the bacterium. Using a genetic approach, we inserted a 12 amino acid peptide from Pseudomonas aeruginosa strain K pilin at numerous semirandom positions in RsaA. We then used an immunological screen to identify those sites that presented the inserted pilin peptide on the C. crescentus cell surface as a part of the S-layer. Eleven such sites (widely separated in the primary sequence) were identified, demonstrating for the first time that S-layers can be readily exploited as carrier proteins to display ‘epitope-size’ heterologous peptides on bacterial cell surfaces. Whereas intact RsaA molecules carrying a pilin peptide could always be found on the surface of C. crescentus regardless of the particular insertion site, introduction of the pilin peptide at 9 of the 11 sites resulted in some proteolytic cleavage of RsaA. Two types of proteolytic phenomena were observed. The first was characterized by a single cleavage within the pilin peptide insert with both fragments of the S-layer protein remaining anchored to the outer membrane. The other proteolytic phenomenon was characterized by cleavage of the S-layer protein at a point distant from the site of the pilin peptide insertion. This cleavage always occurred at the same location in RsaA regardless of the particular insertion site, yielding a surface-anchored 26 kDa proteolytic fragment bearing the RsaA N-terminus; the C-terminal cleavage product carrying the pilin peptide was released into the growth medium. When the results of this work were combined with the results of a previous study, the RsaA primary sequence could be divided into three regions with respect to the location of a peptide insertion and its effect on S-layer biogenesis: (i) insertions in the extreme N-terminus of RsaA either produce no apparent effect on S-layer biogenesis or disrupt surface-anchoring of the protein; (ii) insertions in the extreme C-terminus either produce no apparent effect on S-layer biogenesis or disrupt protein secretion; and (iii) insertions more centrally located in the protein either have no apparent effect on S-layer biogenesis or result in proteolytic cleavage of RsaA. These data are discussed in relation to our previous assignment of the RsaA N- and C-terminus as regions that are important for surface anchoring and secretion respectively.     

Bilirubin and haeme binding to human serum albumin studied by spectroscopy methods

Cobinding of bilirubin and of haeme to human serum albumin was investigated by means of difference absorption spectroscopy and fluorescence spectroscopy. Two specific sites for bilirubin and two for haeme binding occur on the albumin molecule. The primary binding site for bilirubin (Ka = 2.5 microM-1) is different from the primary heame binding site (Ka = 50 microM-1; Beaven et al., Eur J. Biochem. 41, 539-546, 1974), the former, however, might be identical with the secondary center for haeme binding. Similarly, the primary haeme binding center might be identical with the secondary bilirubin binding site.     

Conformational and ligand binding properties of the isolated domains from the beta 2 subunit of Escherichia coli tryptophan synthetase investigated by the reactivity of their cysteines.

A mild proteolytic treatment of the dimeric beta 2 subunit of Escherichia coli tryptophan synthetase (L-serine hydrolase (adding indole) EC 4.2.1.20) is known to nick each polypeptide chain into two complementary fragments, F1 and F2 (H?gberg-Railbaud, A., and Goldberg, M.E. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 442-446). The reactivity of the cysteines in the isolated or associated fragments is studied and used to characterize the structural and functional properties of these fragments. It is shown that the total number of cysteines, their reactivity to dithiobisnitrobenzoate, and their protection by various ligands are the same in the nicked and intact enzyme, thus demonstrating the close structural analogy between these two proteins. In the isolated F1 fragments two cysteines are reactive and two are buried, thus confirming that this fragments has a compact, globular structure. Various ligands tested fail to produce any modification of the cysteines in the isolated fragments, thus suggesting that none of the fragments alone carries a binding site for the substrates and coenzyme.     

Affinity alkylation of human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase by 2 alpha-bromoacetoxyprogesterone: evidence for separate dehydrogenase and isomerase sites on one protein

We have copurified human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, which synthesize progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate, from microsomes as a homogeneous protein based on electrophoretic and NH2-terminal sequencing data. The affinity alkylator, 2 alpha-bromoacetoxyprogesterone, simultaneously inactivates the pregnene and androstene dehydrogenase activities as well as the C21 and C19 isomerase activities in a time-dependent, irreversible manner following first order kinetics. At four concentrations (50/1-20/1 steroid/enzyme M ratios), the alkylator inactivates the dehydrogenase activity (t1/2 = 1.5-3.7 min) 2-fold faster than the isomerase activity. Pregnenolone and dehydroepiandrosterone protect the dehydrogenase activity, while 5-pregnene-3,20-dione, progesterone, and androstenedione protect isomerase activity from inactivation. The protection studies and competitive kinetics of inhibition demonstrate that the affinity alkylator is active site-directed. Kitz and Wilson analyses show that 2 alpha-bromoacetoxyprogesterone inactivates the dehydrogenase activity by a bimolecular mechanism (k3' = 160.9 l/mol.s), while the alkylator inactivates isomerase by a unimolecular mechanism (Ki = 0.14 mM, k3 = 0.013 s-1). Pregnenolone completely protects the dehydrogenase activity but does not slow the rate of isomerase inactivation by 2 alpha-bromoacetoxyprogesterone at all. NADH completely protects both activities from inactivation by the alkylator, while NAD+ protects neither. From Dixon analysis, NADH competitively inhibits NAD+ reduction by dehydrogenase activity. Mixed cofactor studies show that isomerase binds NAD+ and NADH at a common site. Therefore, NADH must not protect either activity by simply binding at the cofactor site. We postulate that NADH binding as an allosteric activator of isomerase protects both the dehydrogenase and isomerase activities from affinity alkylation by inducing a conformational change in the enzyme protein. The human placental enzyme appears to express the pregnene and androstene dehydrogenase activities at one site and the C21 and C19 isomerase activities at a second site on the same protein.     

Location of antigenic determinants on primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping

The binding domains of 28 monoclonal antibodies (mAbs) against the alpha, beta, and delta subunits of the Torpedo acetylcholine receptor were mapped on the primary sequences of these subunits. Small peptide fragments (2000-20,000 daltons) of the purified subunits were obtained by digestion with staphylococcal V8 protease and papain, separated on a discontinuous polyacrylamide gel electrophoretic system, and electroblotted onto diaminophenyl thioether paper. The blots were probed with the various monoclonal antibodies and also with antibodies against carboxy-terminal decapeptides of the alpha, beta, and delta subunits to identify the carboxy-terminal fragments. From inspection of the binding patterns of the various antibodies to the subunits fragments and the molecular weights of these fragments, and by using the carboxy termini of the subunits as reference points, it was possible to deduce the regions on the primary sequence of each subunit in which the antibodies bound and in some cases to order the binding sites within these sequences. mAb 148, which inhibits receptor function by cross-linking receptor molecules on the cytoplasmic side, was mapped to the sequence beta 368-406. The main immunogenic region of the native receptor, which is of pathological importance in the autoimmune disease myasthenia gravis, was mapped by using mAb 210 to within 80 amino acid residues (alpha 46-127). The overall antigenic structure of alpha subunits was examined. Synthetic peptides have been used to locate determinants responsible for 83% of the antibodies in antisera to denatured alpha subunits and 46% of the antibodies to denatured alpha subunits in antisera to intact receptor. Theoretical models of the transmembrane orientation of the subunit polypeptide chains were tested by determining whether mapped monoclonal antibodies bound to the extracellular or intracellular surface of receptor-rich membranes. Our results confirm previous reports that the carboxy termini of the subunits are exposed on the intracellular surface, as is part of the region between a putative channel-forming domain (M5) and a putative membrane-spanning region (M3). However, contrary to current theoretical models, the region between M5 and the putative membrane-spanning sequence M4 also appears to be on the intracellular surface, implying that M4 and M5 are not membrane-spanning domains.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assessment of structural similarities in chick oviduct progesterone receptor subunits by partial proteolysis of photoaffinity-labeled proteins

Partial proteolytic fragmentation of the two chick oviduct progesterone receptor subunits was used to identify structural features shared by the two proteins. Both subunits can be photoaffinity labeled at their hormone-binding sites (Birnbaumer, M., Schrader, W. T., and O'Malley, B. W. (1983) J. Biol. Chem. 258, 1637-1644) using the radioactive steroid [methyl-3H] 17 alpha, 21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione. Native subunits A (Mr = 79,000) and B (Mr = 108,000) were partially purified, photoaffinity-labeled, and then subjected to various mild proteolytic digestions. Labeled fragments were analyzed by fluorography after electrophoresis of the digests under denaturing conditions. Digestion patterns were characteristic for each protease tested. However, fragments from both A and B were indistinguishable for all peptides of less than Mr = 60,000. Time course studies demonstrated the sequential production of progressively smaller discrete fragments in a manner consistent with a precursor-product relationship among them and established the existence of similar structural domains resistant to proteolysis in both proteins. Autoradiographic peptide maps were obtained by 125I-labeling of pure A and B protein isolated by two-dimensional gel electrophoresis followed by exhaustive tryptic digestion and two-dimensional separation. These studies revealed that a significant proportion of the smaller A protein differs in its primary sequence from that of the B protein which excludes the possibility of their sharing a precursor-product relationship. We conclude that B and A subunits are separate proteins with common structural features in the native state, but with considerable amino acid sequence differences. The simplest hypothesis consistent with these findings is that B and A are the products of two separate genes which have diverged to give rise to two different but related proteins that fold in such a manner as to be almost indistinguishable by proteolytic attack of their native conformation.     

Spectroscopic studies of the binding of bilirubin by ligandin and aminoazo-dye-binding protein A.

Ligandin and aminoazo-dye-binding protein A both bind bilirubin at a single site. Quantitative studies of the interactions using difference spectrophotometry show that at pH 7.0, protein A binds the tetrapyrrole with an association constant (K) greater than or equal to 2 X 10(7) litre/mol, whereas binding by ligandin is slightly weaker (K = 7 X 10(6) litre/mol) at this pH. The protein-bilirubin complexes give rise to absorption and fluorescence spectra quite different from those of unbound bilirubin and also to large Cotton effects. It appears that on binding to both proteins, the ligand is forced into a rigid twisted configuration in a hydrophobic environment. Ligandin and protein A resemble serum albumin in their interactions with bilirubin.     

Studies on subunit structure and evidence that ligandin is a heterodimer

Several lines of evidence indicate that ligandin consists of two different subunits. The protein dissociates into two components that are detected by electrophoresis in a discontinuous sodium dodecyl sulfate system, or in acid-urea gels, and by isoelectric focusing in the presence of urea. The apparent molecular weights of the two polypeptides are 25,000 and 22,000. Alkylated or succinylated ligandins also exhibit subunit heterogeneity and resolved into two bands in these electrophoretic systems. Cross-linked ligandin showed only one band in sodium dodecyl sulfate-gel electrophoresis indicating that the two subunits are part of a heterodimeric protein rather than monomers of two different proteins. No dansylated terminal amino acids were detected suggesting that the NH2-terminal residues of both chains are blocked. One mole of arginine or phenylalanine was released per mole of ligandin after digestion with carboxypeptidase B or A, respectively. Tryptic maps of succinylated ligandin were consistent with identical disposition of arginine residues in both chains, but several additional tryptic peptides were obtained with native ligandin as compared to the predicted number if both subunits were identical. These observations are consistent with the possibility that both subunits contain common sequences and that a small peptide of about 25 to 30 amino acid residues is cleaved from the COOH-terminal of the larger subunit to produce the smaller subunit.     

Modification of ovine luteinizing hormone subunits with SMPT and its effect on subunit recombination, immunological activity, receptor binding and steroidogenic activity

The increasing use of heterobifunctional cross-linking agents in the design of defined conjugates for selective targeting and inducing immune response has prompted us to study the role of epsilon-NH2 group modification of oLH subunits, their recombination and effect on immunoreactivity, receptor binding and biological activity. The epsilon-NH2 groups of alpha oLH and beta oLH subunits were separately modified by using SMPT. The alpha oLH-SMPT modified derivatives hybridize to beta oLH. Similarly, the beta oLH-SMPT derivatives recombined with alpha oLH. The recombination was judged by gel filtration chromatography and RP-HPLC analysis. The sequential modification of subunits led to progressive reduction in immunoreactivity and receptor binding activity. The modification of six or more epsilon-NH2 groups in alpha oLH although recombine fully with native beta oLH but failed to react to anti-oLH antibody. Moreover, the steroidogenic activity was also abolished. Introduction upto four SMPT groups in alpha oLH compromised immunological and biological activities but further addition of two or more SMPT groups completely abolished antibody reactivity, receptor binding and steroidogenic activity indicating the importance of later two amino groups in the receptor binding and steroidogenic activity. The present investigation clearly demonstrate that only 1:2-3 molar ratio of oLH subunits:SMPT could generate the site(s) in the subunits of the oLH that retained reasonable immunological, receptor binding and biological activity of the hormone. Therefore, this molar ratio may be used in future for the design and synthesis of bioeffective hormonotoxins.     

Hepatic ligandin subunits and mRNAs during development

Eight-week-old rats had twofold higher hepatic ligandin concentration than 10-day-old animals as determined immunologically and by steroid isomerase and glutathione S-transferase assays. Increased ligandin content was accompanied by parallel increase in subunit synthesis as determined by [³H]leucine incorporation into each subunit relative to incorporation into total cytosolic proteins. The mRNA content for each ligandin subunit was twofold higher in older animals as determined by cell-free in vitro translation followed by immunoprecipitation and dot hybridization using a ligandin cDNA probe. When poly A mRNA from the postmitochondrial fraction of liver from young or old rats was subjected to agarose gel electrophoresis under denaturing conditions and hybridized to ligandin cDNA probe, a single 11 S band was obtained. With RNA from total liver, an additional 13 S band was obtained, suggesting the existence of a precursor form of ligandin mRNA. Since precursor polypeptides were not observed with RNA from total liver in cell-free in vitro translation systems, the precursor form requires processing to the 11 S form before the mRNA becomes functional.