On the probable involvement of arginine residues in the bile-salt-binding site of human pancreatic carboxylic ester hydrolase

Modification of arginine residues with 2,3-butanedione inhibits the carboxylic-ester hydrolase activity on soluble and emulsified substrates when assayed with bile salts. The alpha-dicarbonyl reagent modifies seven of the nineteen arginine residues present per enzyme molecule. Nevertheless the inactivation with butanedione is greatly diminished when the protein is in the presence of negatively charged micellar bile salt. In these conditions we observe the protection of one arginine residue by sodium taurodeoxycholate and of two arginine residues by sodium cholate. This suggests that the carboxylic-ester hydrolase from human pancreatic juice contains at least two arginine residues essential for the activation by bile salts. All our data confirm the presence of two bile-salt-binding sites on the enzyme in which one arginine per site is involved and plays the general role of an anionic binding site. This study provides evidence that arginine residues may play an essential role in the interaction between bile salts and protein.     

Site-directed mutagenesis of the distal basic cluster of pancreatic bile salt-dependent lipase

Previous studies have postulated the presence of two bile salt-binding sites regulating the activity of the pancreatic bile salt-dependent lipase. One of these sites, located in an N-terminal basic cluster, has been identified as the specific bile salt-binding site. Interaction of primary bile salts with this proximal site induces the formation of a micellar binding site from a pre-existing nonspecific or pre-micellar bile salt-binding site. Here we have investigated the functional significance of another basic cluster comprised of amino acid residues Arg(423), Lys(429), Arg(454), Arg(458), and Lys(462), distal from the catalytic site. For this purpose these residues were mutagenized in Ile or Ala residues. The mutagenized enzyme lost activity on both soluble and emulsified substrates in the presence of bile salts. However, in the absence of bile salts, the mutagenized enzyme displayed the same activity on soluble substrate as the wild-type recombinant enzyme. Consequently, the distal basic cluster may represent the nonspecific (or pre-micellar) bile salt-binding site susceptible to accommodate primary and secondary bile salts. According to the literature, tyrosine residue(s) should participate in this site. Therefore, two tyrosine residues, Tyr(427) and Tyr(453), associated with the distal basic cluster were also mutagenized. Each tyrosine substitution to serine did not inhibit the enzyme activity on soluble substrate, independently of the presence of primary or secondary bile salts. However, the enzyme activity on cholesteryl oleate solubilized in primary bile salt micelles was decreased by mutations substantiating that these residues are part of the nonspecific bile salt-binding site.     

Activation of nonspecific lipase (EC 3.1.1.-) by bile salts

The enzyme nonspecific lipase (EC 3.1.1.-) from rat pancreas has been isolated and its amino acid composition determined. The amino acid composition confirms more indirect evidence that nonspecific lipase is not the same enzyme as cholesteryl ester hydrolase. Activation of the enzymatic activity by bile salts has been studied by equilibrium dialysis, gel filtration, light scattering, circular dichroism and fluorescence polarization. The binding of bile salt by the enzyme is saturable and is associated with a conformational change. Upon binding cholate, the protein experiences a decrease in beta-structure with no significant change in alpha-helix content, an increase in apparent Stokes radius, a decrease in light scattering properties, and a slight decrease in polarization of the intrinsic tryptophan fluorescence. Attachment of bile salt is associated with decreased reactivity of essential sulfhydryl groups, but no detectable change in reactivity of amino groups. A change to a more nearly spherical shape upon binding bile salt would be consistent with the experimental observations, but the exact sites of binding remain uncertain.     

Nonspecific high affinity binding of bile salts to carboxylester lipases

The interactions with bile salts of carboxylester lipases (EC 3.1.1.13) from human pancreatic juice and pig pancreas were characterized by physical methods. Bile salts cause a decrease in the fluorescence intensity of the proteins at the emission maximum of 333-335 nm. The concentration dependence of this decrease shows saturation behavior, is relatively nonspecific with respect to bile salt conjugation or the presence of the 7 alpha-hydroxyl group, and is consistent with a 1:1 interaction between enzyme and bile salt. Direct measurement of the binding of [3H]cholate by equilibrium dialysis supports the stoichiometry. Other detergents also bind, causing fluorescence changes, but with much lower affinities. Binding of taurocholate to the monomeric pig enzyme is enhanced by increasing ionic strength, indicating the predominance of hydrophobic interactions. In the range of pH 5.5-6.8, binding is pH-independent with dissociation constants of 3-20 microM. At higher pH, affinity is greatly reduced and the fluorescence spectrum changes, indicating the importance of a protonated group for efficient interaction. Occupancy of the bile salt binding site partially stabilizes the enzyme against inactivation by heat but not trypsin. However, circular dichroism spectra do not indicate that bile salt binding is accompanied by any change in secondary structure. The monomeric pig enzyme binds to the argon/water interface in the presence of bile salts and binding of taurocholate to diisopropylphosphoryl-enzyme is similar to that measured with native enzyme. These results suggest that surface binding and catalysis occur at sites distinct from the bile salt binding site of the enzyme. Stabilization of the monomeric pig enzyme against denaturation at high energy surfaces occurs concomitantly with occupancy of the bile salt binding site. Overall, the data suggest that an important role of bile salts in vivo is to stabilize these enzymes at lipid-water interfaces.     

Molecular characterization of bile salt hydrolase from Bifidobacterium animalis subsp. lactis Bi30

The present work describes the identification, purification, and characterization of bile salt hydrolase (BSH) from Bifidobacterium animalis subsp. lactis. The enzyme was purified to electrophoretic homogeneity by hydrophobic chromatography, ion-exchange chromatography and ultrafiltration. SDS-PAGE analysis of putative BSH and gel filtration revealed that the analyzed protein is presumably a tetramer composed of four monomers each of about 35 kDa. The purified enzyme was analyzed by liquid chromatography coupled to LTQ FT ICR mass spectrometry and unambiguously identified as a bile salt hydrolase from B. animalis. The isoelectric point of the studied protein was estimated to be around pH 4.9. The pH optimum of the purified BSH is between 4.7 to 6.5, and the temperature optimum is around 50 degrees C. The BSH of B. animalis could deconjugate all tested bile salts, with clear preference for glycine-conjugated bile salts over taurine-conjugated forms. Genetic analysis of the bsh showed high similarity to the previously sequenced bsh gene from B. animalis and confirmed the usefulness of bile salt hydrolase as a genetic marker for B. animalis identification.     

唾液乳杆菌BSH1及其突变体的底物特异性

为了解析胆盐水解酶催化中心中关键氨基酸位点与其底物特异性的关系,以大肠杆菌pET-20b(+)表达系统为分子改造平台,采用理性设计,结合氨基酸定点突变的方法,成功构建了唾液乳杆菌Lactobacillus salivarius胆盐水解酶BSH1的7种突变体。通过对比L.salivarius BSH1及其突变体对6种结合胆盐的底物特异性表明,7种突变体对不同的结合胆盐的水解活性有所改变。结果说明,Cys2和Thr264分别是BSH1催化TCA和GCA的关键残基,且对酶的催化活性的保持具有关键作用。其中,高保守性的氨基酸位点Cys2不是BSH1唯一的活性位点,而其他突变的氨基酸位点可能作为BSH1的结合位点参与了底物的结合,也可能影响了底物进入BSH1活性中心的通道或底物结合口袋的体积与形状,进而影响了BSH1对不同结合胆盐的水解活性。     

Cholate-induced dimerization of detergent- or phospholipid-solubilized bovine cytochrome C oxidase

Bovine heart cytochrome c oxidase (CcO), solubilized by either nonionic detergents or phospholipids, completely dimerizes upon the addition of bile salts, e.g., sodium cholate, sodium deoxycholate, or CHAPS. Bile salt induced dimerization occurs whether dodecyl maltoside, decyl maltoside, or Triton X-100 is the primary solubilizing detergent or the enzyme is dispersed in phosphatidylcholine, phosphatidylethanolamine, or mixtures thereof. In each case, complete CcO dimerization can be verified by sedimentation velocity and sedimentation equilibrium after correction for bound detergent and/or phospholipid. The relative concentration of the bile salt is critical for production of homogeneous, dimeric CcO. For example, enzyme solubilized by 2 mM detergent requires an equal molar concentration of sodium cholate. Similarly, enzyme dispersed in 20 mM phospholipid requires 50 mM sodium cholate, concentrations that are commonly used to reconstitute CcO into small unilamellar vesicles. Bile salts do more than just stabilize dimeric CcO and prevent detergent-induced dissociation into monomers. They are able to completely reverse detergent-induced monomerization and cause completely monomeric CcO to reassociate. Dimeric CcO so generated is no more stable than the original complex and easily dissociates into monomers if the bile salt is removed. The dimerization process is dependent upon a full complement of subunits; e.g., if subunits VIa and VIb are removed, the resulting monomeric CcO will not reassociate upon the addition of sodium cholate. These results support four important consequences: (1) dissociation of dimeric CcO into monomers is reversible; (2) stable dimers can be produced under solution conditions; (3) dimers can be stabilized even at relatively high pH and low enzyme concentration; and (4) subunits VIa and VIb are required for dimerization.     

Nitration of the tyrosine residues of porcine pancreatic colipase with tetranitromethane, and properties of the nitrated derivatives

The nitration of the long form (N-terminal valine) of porcine pancreatic colipase with tetranitromethane was investigated under a variety of conditions. Fractionation of the nitrated monomers on DE-cellulose led to well-defined derivatives containing one, two and three nitrotyrosines per mol. Automated Edman degradation of the nitrated peptides, especially that of the staphylococcal proteinase peptide (49-64) showed that Tyr-54 was nitrated very fast under all conditions. This residue was the only one to be nitrated in water. Partial nitration of Tyr-59 was induced by bile salt micelles, while both Tyr-59 and Tyr-58 reacted extensively in the presence of lysophosphatidylcholine micelles (in which tetranitromethane is concentrated 150-fold compared to water) or of a liquid tetranitromethane-water interface. The strong negative Cotton effect at 410 nm which has already been observed using unfractionated preparations of nitrated colipase (Behnke W.D. (1982) Biochim. Biophys. Acta 708, 118-123) is linked with the nitration of Tyr-59 and it is markedly reduced by taurodeoxycholate micelles, suggesting a conformational change induced by the micelles in the tyrosine region. Moreover, the pKa of the nitrotyrosine residues in nitrated colipase is the same as that of free nitrotyrosine (pKa = 6.8) and it is shifted to 7.6 in the presence of taurodeoxycholate micelles. Micelles protected colipase against polymerization during nitration. These data suggest that Tyr-58 and Tyr-59 are part of the interface recognition site of colipase. The participation of Tyr-55 in binding is not excluded. The upwards nitrotyrosine pKa shift in the colipase micelle complex may explain why nitrated colipase can reactivate lipase in a triacylglycerol-taurodeoxycholate system at pH 7.5.     

Importance of microbial defence systems to bile salts and mechanisms of serum cholesterol reduction

An important feature of the intestinal microbiota, particularly in the case of administered probiotic microorganisms, is their resistance to conditions in the gastrointestinal tract, particularly tolerance to and growth in the presence of bile salts. Bacteria can use several defence mechanisms against bile, including special transport mechanisms, the synthesis of various types of surface proteins and fatty acids or the production of exopolysaccharides. The ability to enzymatically hydrolyse bile salts occurs in a variety of bacteria. Choloylglycine hydrolase (EC 3.5.1.24), a bile salt hydrolase, is a constitutive intracellular enzyme responsible for the hydrolysis of an amide bond between glycine or taurine and the steroid nucleus of bile acids. Its presence was demonstrated in specific microorganisms from several bacterial genera (Lactobacillus spp., Bifidobacterium spp., Clostridium spp., Bacteroides spp.). Occurrence and gene arrangement encoding this enzyme are highly variable in probiotic microorganisms. Bile salt hydrolase activity may provide the possibility to use the released amino acids by bacteria as sources of carbon and nitrogen, to facilitate detoxification of bile or to support the incorporation of cholesterol into the cell wall. Deconjugation of bile salts may be directly related to a lowering of serum cholesterol levels, from which conjugated bile salts are synthesized de novo. Furthermore, the ability of microorganisms to assimilate or to bind ingested cholesterol to the cell wall or to eliminate it by co-precipitation with released cholic acid was also documented. Some intestinal microflora produce cholesterol reductase that catalyses the conversion of cholesterol to insoluble coprostanol, which is subsequently excreted in faeces, thereby also reducing the amount of exogenous cholesterol.     

Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis.

A high-molecular-weight (250 000) bile salt hydrolase (cholylglycine hydrolase, EC 3.5.-.-) was isolated and purified 128-fold from the "spheroplast lysate" fraction prepared from Bacteroids fragilis subsp. fragilis ATCC 25285. The intact enzyme had a molecular weight of approx. 250 000 as determined by gel infiltration chromatography. One major protein band, corresponding to a molecular weight of 32 500, was observed on 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis of pooled fractions from DEAE-cellulose column chromatography (128-fold purified). The pH optimum for the 64-fold purified enzyme isolated from Bio-Gel A 1.5 M chromatography was 4.2 and bile salt hydrolase activity measured in intact cell suspensions had a pH optimum of 4.5. Substrate specificity studies indicated that taurine and glycine conjugates of cholic acid, chenodeoxycholic acid and deoxycholic acid were readily hydrolyzed; however, lithocholic acid conjugates were not hydrolyzed. Substrate saturation kinetics were biphasic with an intermediate plateau (0.2--0.3 mM) and a complete loss of enzymatic activity was observed at high concentration for certain substrates. The presence or absence of 7-alpha-hydroxysteroid dehydrogenase was absolutely correlated with that of bile salt hydrolase activity in six to ten strains and subspecies of B. fragilis.     

Effect of bile salts on the hydrolysis of gangliosides, glycoproteins and neuraminyl-lactose by the neuraminidase of Clostridium perfringens.

Studies were done on the effect of bile salts on the rates of hydrolysis of the N-acetylneuraminyl linkages of several sialic acid-containing compounds by the neuraminidase of Clostridium perfringens. When GM3-ganglioside, two glycolipids (glycophorin and orosomucoid) and neuraminyl-lactose were used as substrates, hydrolysis was obtained even in the absence of bile salts, but addition of this detergent, below its critical micellar concentration, increased the reaction rates; above the critical micellar concentration of the detergent rates decreased again. When a second ganglioside, GM1, was used as substrate, the requirement for bile salts was absolute; hydrolysis was not observed at all without this detergent. With increasing concentrations of bile salt and in the presence of high concentrations of enzyme, rates of hydrolysis increased, reaching maximal values at fixed ratios of bile salt to GM1-ganglioside. Physical measurements showed that mixtures of bile salt and GM1-ganglioside form mixed micelles that have a higher critical micellar concentration, a lower molecular weight and greater axial ratio than the corresponding micelles of pure GM1-ganglioside.     

Effect of bile salts on the interfacial inactivation of pancreatic carboxylester lipase

Pig pancreatic carboxylester lipase (cholesterol esterase, E.C. 3.1.1.13) was inactivated at a tributyrin/water interface. The apparent rate constant for inactivation increased with increase in the particle surface area of the tributyrin emulsion. The large energy of activation and entropy change for inactivation (33.7 Kcal.mol-1 and 35.8 cal.mol-1.deg-1, using 5 mM sonicated tributyrin at 37 degrees C, respectively) suggest that the observed inactivation reflects denaturation of the enzyme at the tributyrin/water interface. Bile salts protected the enzyme from irreversible inactivation at the tributyrin/water interface. The protection by bile salts was related both to their concentration and to the tributyrin concentration (substrate surface area). The protection by bile salts was not related to their concentration below or above their critical micellar concentration; the binding of bile salts to enzyme was probably the dominant protection factor. Similar stabilization was observed with other detergents such as Brij-35, Triton X-100, and sodium dodecyl sulfate. These results suggest that inactivation of carboxylester lipase occurs at a high-energy lipid-water interface and that an important role of bile salts in vivo is to stabilize carboxylester lipase at interfaces.     

On the interactions between pancreatic lipase and colipase and the substrate, and the importance of bile salts.

The interactions between pancreatic lipase and colipase and the substrate and the effect of bile salts on these interactions have been investigated by the use of kinetic experiments and studies on the semiquantitative phase distribution of lipase and colipase activities. The results suggest that lipase binds to hydrophobic interfaces with partial irreversible inactivation. Bile salts in the range of micellar concentrations and above a pH of about 6.5 displace lipase from this binding, resulting in a reversible in activation. At pH values below about 6.5, lipase binds strongly to the substrate even in the presence of bile salt, and a low activity peak is seen around pH 5.5. This is the result of the binding of lipase to the "supersubstrate" and the activity of the catalytic site. In the presence of bile salt, colipase promotes the binding of lipase to the "supersubstrate" but not to other hydrophobic interfaces, and catalytic activity is reestablished. Kinetic data indicate that the binding between colipase and lipase in the presence of substrate is strong and occurs in an approximately stoichiometric relationship.     

Calcium binding by monosulfate esters of taurochenodeoxycholate

The effect of sulfate esterification of the 3 alpha- or 7 alpha-hydroxyl groups of taurochenodeoxycholate on calcium binding was studied at 0.154 M NaCl in the presence and absence of phosphatidylcholine using a calcium electrode. For comparison, similar studies were made with taurochenodeoxycholate, taurodeoxycholate, and taurocholate. No high affinity calcium binding was demonstrable for any of these bile salts in pre-micellar solutions. Taurine-conjugated bile salts have greater affinity for calcium when in a micellar form. At elevated bile salt concentrations, the calcium binding of unsulfated dihydroxy taurine conjugates was similar to that of the monosulfate esters of taurochenodeoxycholate. The presence of phosphatidylcholine decreased calcium binding of the unsulfated dihydroxy bile salts and slightly increased calcium binding by taurocholate. However, the addition of phosphatidylcholine to monosulfate esters of taurochenodeoxycholate results in large increments in calcium binding. The results indicate that increased calcium binding due to the presence of phosphatidylcholine in bile salt solutions depends, in part, on the hydrophilicity of the bile salt and that the interaction of monosulfate esters of taurochenodeoxycholate with phosphatidylcholine leads to the formation of a high affinity calcium binding site.     

Studies on the substrate specificity of a carboxyl ester hydrolase from human pancreatic juice. I. Action on carboxyl esters,glycerides and phospholipids

Purified carboxyl ester hydrolase (carboxylic-ester hydrolase, EC 3.1.1.1) from human pancreatic juice was found to hydrolyze triacetin, methyl butyrate and glycerides solubilized by bile salts. It has no activity on substrate presented as emulsion or monomolecular films.The human enzyme was found to deacylate phospholipids and lysophospholipids at different rates. The hydrolysis of short-chain phospholipids and lysophospholipids at different rates. The hydrolysis of short-chain phosphatidylcholines was dependent of substrate solubility and dioctanoyl phosphatidylcholine was deacylated with the highest rate. Long-chain phosphatidylcholines and lysophosphatidylcholines present in microsomal membranes were deacylated with very low rates, only lysophosphatidylcholine deacylation was faster. Evidence is presented that human carboxyl ester hydrolase is the lyophosphatidylcholine-hydrolyzing enzyme corresponding to bovine lysophospholipase.Bile salts play an important part on the activity of human carboxyl ester hydrolase, in addition to the role of detergent that they have on insoluble substrates.     

Carboxylic ester hydrolases of rat pancreatic juice

An attempt was made to establish the number and characteristics of the enzymes in pancreatic juice that hydrolyze nitrogen- and phosphorus-free esters of fatty acids. For this purpose model compounds were hydrolyzed by lyophilized rat pancreatic juice under conditions that accelerated or inhibited the reactions. Although it is not established with certainty, it is suggested that three enzymes are responsible for the hydrolysis of fatty acid esters. The first enzyme is glycerol-ester hydrolase (EC 3.1.1.3) or lipase. This enzyme hydrolyzes water-insoluble esters of primary alcohols. The reaction occurs at an oil/water interface and is inhibited by bile salts at pH 8. The enzyme is relatively stable at pH 9, but unstable at pH 4. It has a broad pH optimum between 7.5 and 9.5. The second enzyme hydrolyzes esters of secondary alcohols and of other alcohols as well. It has an absolute requirement for bile salts and has a pH optimum at about 8. The enzyme is unstable in pancreatic juice when maintained at pH 9, probably due to the action of trypsin. It may be identical with sterol-ester hydrolase (EC 3.1.1.13). The third enzyme hydrolyzes water-soluble esters. It too has an absolute requirement for bile salts, although a smaller amount is necessary for maximum activity. This enzyme also is unstable at pH 9, but can be differentiated from the preceding enzyme by its stability at pH 4 and its pH optimum of 9.0. Carboxylic-ester hydrolase (EC 3.1.1.1) is not found in pancreatic juice, although it is present in pancreatic tissue.     

Lipolytic enzymes of the human pancreas. II. Purification and properties of cholesterol ester hydrolase

Cholesterol ester hydrolase (sterol-ester acylhydrolase, EC 3.1.1.13) was purified from human pancreatic tissue by column chromatography and acetone precipitation, leading to a 400-fold enrichment. Isoelectric focusing of this product reveals a double-band at pH 4.5 and 4.6. The molecular weight was estimated at 320 kDa by means of Sephadex filtration on calibrated columns. Obviously these large molecules represent a tetrameric form of the monomeric subunit (molecular mass 76-80 kDa), which is also enzymatically active. It was found together with the dimeric form in pancreatic juice, where the tetrameric enzyme is responsible for the major part of the hydrolytic activity, splitting cholesterol ester as well as synthetic substrates, such as fluorescein or p-nitrophenyl esters. Attempts to split the tetrameric cholesterol ester hydrolase, isolated from pancreatic tissue, into active subunits found additionally in pancreatic juice by the influence of bile acids and proteolytic enzymes failed. The spectral shift method using Rhodamine fluorescence was employed in order to prove that fluorescein dilaurate forms micellar solutions and mixed micelles when bile salts are present.     

鸡源和猪源乳杆菌胆盐水解酶的表达及酶学性质比较

【目的】随着抗生素生长促进剂(AGPs)在动物饲料中逐步禁止使用,AGPs替代物的研究成为热点。由于胆盐水解酶(BSH)在脂类代谢中的关键作用,成为AGPs替代物研究的一个重要方向。在原核表达和纯化的基础上鉴定鸡源和猪源乳杆菌BSH在酶学性质方面的差异性。【方法】分别对鸡源胆盐水解酶(BSHc)和猪源胆盐水解酶(BSHp)基因进行原核表达和蛋白纯化,通过测定对6种甘氨结合胆盐和牛磺结合胆盐的水解效率获得两种酶的酶学动力学性质,进而测定了温度、pH和金属离子对酶活力的影响。【结果】BSHc和BSHp对甘氨结合胆盐的水解效率高于牛磺结合胆盐,BSHc对甘氨结合胆盐的水解效率较BSHp稍高;BSHc和BSHp的最适酶解温度分别为45°C和42°C;BSHc和BSHp的最适反应pH分别为6.0和5.4;含有Cu~(2+)、Fe~(3+)、Mn~(2+)和Zn~(2+)的金属盐对BSHc和BSHp的酶活力均具有不同程度的抑制作用,特别是Cu~(2+)和Fe~(3+)抑制作用比较强;含有Na~+、K~+、Mg~(2+)和Ca2+的金属盐对BSHc和BSHp酶活力的抑制作用相对较弱或无抑制作用,但KIO3对BSHc和BSHp酶活力具有强抑制作用,KI和CaCl_2对BSHp酶活力也具有较强的抑制作用。【结论】原核表达和纯化的BSHc和BSHp对甘氨结合胆盐的水解效率高于牛磺结合胆盐,BSHc的最适酶解温度和pH稍高于BSHp,Cu~(2+)、Fe~(3+)、Mn~(2+)和Zn~(2+)等金属离子对BSHc和BSHp酶活力具有明显抑制作用,试验结果为鉴定BSH抑制物进而研制AGPs替代物奠定了基础。     

Site-directed mutagenesis of the basic N-terminal cluster of pancreatic bile salt-dependent lipase. Functional significance

Previous studies have postulated the presence of a heparin-binding site on the bile salt-dependent lipase (BSDL), whereas two bile salt-binding sites regulate the enzyme activity. One of these sites may overlap with the tentative heparin-binding site at the level of an N-terminal basic cluster consisting of positive residues Lys(32), Lys(56), Lys(61), Lys(62), and Arg(63). The present study uses specific site-directed mutagenesis to determine the functional significance of this basic cluster. Mutations in this sequence resulted in recombinant enzymes that were able to bind to immobilized and to cell-associated heparin before moving throughout intestinal cells. Recombinant BSDL was fully active on soluble substrate, but mutants were less active on micellar cholesteryl oleate in comparison with the wild-type enzyme. Activation studies by primary (sodium taurocholate) and by secondary (sodium taurodeoxycholate) bile salts revealed that the activation of BSDL by sodium taurocholate at concentrations below the critical micellar concentration, and not that evoked by micellar bile salts, was affected by substitutions, suggesting that this N-terminal basic cluster likely represents the specific bile salt-binding site of BSDL. Substitutions also affected the activation of the enzyme promoted by anionic phospholipids, extending the function of this site to that of a cationic regulatory site susceptible to accommodate anionic ligands.     

Self-association of unconjugated bilirubin-IX alpha in aqueous solution at pH 10.0 and physical-chemical interactions with bile salt monomers and micelles.

Spectrophotometric measurements of bilirubin-IX alpha in water and in aqueous/organic solvent mixtures at pH 10.0 as a function of bilirubin-IX alpha concentration (approx. 0.6--400 microM) are consistent with the formation of dimers (KD - 1.5 microM) in dilute (less than 10 microM) aqueous solution and further self-aggregation to multimers at higher concentrations. Added urea (to 10M) and increases in temperature (to 62 degrees C) obliterate the dimer-multimer transition at 10 microM, but added NaCl (to 0.30 M) promotes strong aggregation of dimers over a narrow concentration range, suggesting a 'micellization' phenomenon. Concentrations of dioxan or ethanol greater than 60% (v/v) in water were required to obtain the absorption spectrum of bilirubin-IX alpha monomers, suggesting that both hydrophobic and electrostatic (pi-orbital) interactions are involved in stabilizing the dimeric state in water. Micellar concentrations of sodium dodecyl sulphate induced spectrophotometric shifts in the dimer absorption spectrum of bilirubin-IX alpha consistent with progressive partitioning of bilirubin-IX alpha monomers into a relatively non-polar region of the micelles and allowed a deduction of the apparent critical micellar concentration that closely approximated the literature values. The pattern of bilirubin IX alpha association with bile salts is complex, since the absorption spectrum shifts hypsochromically below and bathochromically above the critical micellar concentration of the bile salts. Consistent with these observations, bilirubin IX alpha appears to bind to the polar face of bile salt monomers and to the polar perimeter of small bile salt micelles. At higher bile salt concentrations some-bilirubin-IX alpha monomers partition into the hydrophobic interior of the bile salt micelles. Our results suggest that under physiological conditions the natural conjugates of bilirubin-IX alpha may exhibit similar physical chemical properties in bile, in that dimers, highly aggregated multimers and bile salt-associated monomers may co-exist.