Inhibition of rat liver UDP-glucuronosyltransferase by silymarin and the metabolite silibinin-glucuronide

The inhibitory effects of silymarin, its main constituent silibinin and the metabolite silibinin-glucuronide on UDP-glucuronosiltransferase (UGT) were evaluated in rat hepatic microsomes. Three substrates were chosen to cover both UGT1A and UGT2B family isozymes: bilirubin (substrate of UGT1A1), p-nitrophenol (UGT1A6) and ethinylestradiol (UGT2B1 and 2B3 for position C17 and UGT1A1 for position C3). The study of p-nitrophenol and bilirubin glucuronidation indicated that silymarin (SM) and silibinin glucuronide (SB-G) were enzyme inhibitors. The kinetic analysis showed that the type of inhibition was competitive in all cases and the Ki obtained were: for p-nitrophenol glucuronidation, KiSB-Gapp: 14+/-1 microg/ml and KiSMapp: 51+/-10 microg/ml and for bilirubin glucuronidation, KiSB-Gapp: 16+/-3 microg/ml. In turn, ethinylestradiol glucuronidation was not affected by any of the compounds studied suggesting that the inhibitory effect was restricted to UGT1A isozymes. Similar studies performed using human hepatic microsomes showed that SM and SB-G were also inhibitors of human UGT1A isozymes. In conclusion, administration of silymarin or its main constituent silibinin could lead to the decrease in the glucuronidation of substrates whose conjugation depends on UGT1A isozymes in a process mediated by silibinin-glucuronide, though their effect in humans needs further investigation.     

Thioredoxin-dependent enzymatic activation of mercaptopyruvate sulfurtransferase. An intersubunit disulfide bond serves as a redox switch for activation

Rat 3-mercaptopyruvate sulfurtransferase (MST) contains three exposed cysteines as follows: a catalytic site cysteine, Cys(247), in the active site and Cys(154) and Cys(263) on the surface of MST. The corresponding cysteine to Cys(263) is conserved in mammalian MSTs, and Cys(154) is a unique cysteine. MST has monomer-dimer equilibrium with the assistance of oxidants and reductants. The monomer to dimer ratio is maintained at approximately 92:8 in 0.2 m potassium phosphate buffer containing no reductants under air-saturated conditions; the dimer might be symmetrical via an intersubunit disulfide bond between Cys(154) and Cys(154) and between Cys(263) and Cys(263), or asymmetrical via an intersubunit disulfide bond between Cys(154) and Cys(263). Escherichia coli reduced thioredoxin (Trx) cleaved the intersubunit disulfide bond to activate MST to 2.3- and 4.9-fold the levels of activation of dithiothreitol (DTT)-treated and DTT-untreated MST, respectively. Rat Trx also activated MST. On the other hand, reduced glutathione did not affect MST activity. E. coli C35S Trx, in which Cys(35) was replaced with Ser, formed some adducts with MST and activated MST after treatment with DTT. Thus, Cys(32) of E. coli Trx reacted with the redox-active cysteines, Cys(154) and Cys(263), by forming an intersubunit disulfide bond and a sulfenyl Cys(247). A consecutively formed disulfide bond between Trx and MST must be cleaved for the activation. E. coli C32S Trx, however, did not activate MST. Reduced Trx turns on a redox switch for the enzymatic activation of MST, which contributes to the maintenance of cellular redox homeostasis.     

A new subfamily of agmatinases present in methanogenic Archaea is Fe(II) dependent

Here we report that the Methanocaldococcus jannaschii enzyme derived from the MJ0309 gene is an Fe(II) dependent agmatinase (SpeB). This is the first report of an iron-dependent agmatinase. We demonstrate that aerobically isolated recombinant enzyme contains two disulfide bonds and only a trace amount of any metal and requires the presence of both dithiothreitol (DTT) and 4 equiv of Fe(II) for maximum activity. The DTT activation could be indicative of the presence of a redox system, which would regulate the activity of this as well as other enzymes in the methanogens. Site-directed mutagenesis of the four conserved cysteines C71, C136, C151, and C229 to alanine or serine showed that only the C71 and C151 mutants showed a significant drop in activity indicating that the disulfide bond responsible for regulating activity was likely between C136 and C229. We propose that the C71 and C151 cysteine thiols, produced by the DTT-dependent reduction of their disulfide, are two additional metal binding ligands that alter the metal specificity of the M. jannaschii agmatinase from Mn(II) to Fe(II).     

Molecular and functional characterization of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family

Transport of the co-substrate UDPGA (UDP-glucuronic acid) into the lumen of the endoplasmic reticulum is an essential step in glucuronidation reactions due to the intraluminal location of the catalytic site of the enzyme UGT (UDP-glucuronosyltransferase). In the present study, we have characterized the function of several NSTs (nucleotide sugar transporters) and UGTs as potential carriers of UDPGA for glucuronidation reactions. UDPGlcNAc (UDP-N-acetylglucosamine)-dependent UDPGA uptake was found both in rat liver microsomes and in microsomes prepared from the rat hepatoma cell line H4IIE. The latency of UGT activity in microsomes derived from rat liver and V79 cells expressing UGT1A6 correlated well with mannose-6-phosphatase latency, confirming the UGT in the recombinant cells retained a physiology similar to rat liver microsomes. In the present study, four cDNAs coding for NSTs were obtained; two were previously reported (UGTrel1 and UGTrel7) and two newly identified (huYEA4 and huYEA4S). Localization of NSTs within the human genome sequence revealed that huYEA4S is an alternatively spliced form of huYEA4. All the cloned NSTs were stably expressed in V79 (Chinese hamster fibroblast) cells, and were able to transport UDPGA after preloading of isolated microsomal vesicles with UDPGlcNAc. The highest uptake was seen with UGTrel7, which displayed a V(max) approx. 1% of rat liver microsomes. Treatment of H4IIE cells with beta-naphthoflavone induced UGT protein expression but did not affect the rate of UDPGA uptake. Furthermore, microsomes from UGT1-deficient Gunn rat liver showed UDPGA uptake similar to those from control rats. These data show that NSTs can act as UDPGA transporters for glucuronidation reactions, and indicate that UGTs of the 1A family do not function as UDPGA carriers in microsomes. The cell line H4IIE is a useful model for the study of UDPGA transporters for glucuronidation reactions.     

Reversible inactivation of AT(2) angiotensin II receptor from cysteine-disulfide bond exchange

Dithiothreitol (DTT) treatment of angiotensin II (Ang II) type 2 (AT(2)) receptor potentiates ligand binding, but the underlying mechanism is not known. Two disulfide bonds proposed in the extracellular domain were examined in this report. Based on the analysis of ligand affinity of cysteine (Cys, C) to alanine (Ala, A) substitution mutants, we provide evidence that Cys(35)-Cys(290) and Cys(117)-Cys(195) disulfide bonds are formed in the wild-type AT(2) receptor. Disruption of the highly conserved Cys(117)-Cys(195) disulfide bond linking the second and third extracellular segments leads to inactivation of the receptor. The Cys(35)-Cys(290) bond is highly sensitive to DTT. Its breakage results in an increased binding affinity for both Ang II and the AT(2) receptor-specific antagonist PD123319. Surprisingly, in the single Cys mutants, C35A and C290A, a labile population of receptors is produced which can be re-folded to high-affinity state by DTT treatment. These results suggest that the free -SH group of Cys(35) or Cys(290) competes with the disulfide bond formation between Cys(117) and Cys(195). This Cys-disulfide bond exchange results in production of the inactive population of the mutant receptors through formation of a non-native disulfide bond.     

Non-steroidal anti-inflammatory drugs interact with testosterone glucuronidation

Testosterone and epitestosterone are secreted mainly as glucuronide metabolites and the urinary ratio of testosterone glucuronide to epitestosterone glucuronide, often called T/E, serves as a marker for possible anabolic steroids abuse by athletes. UDP-glucuronosyltransferase (UGT) 2B17 is the most important catalyst of testosterone glucuronidation. The T/E might be affected by drugs that interact with UGT2B17, or other enzymes that contribute to testosterone glucuronidation. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used by sportsmen and we have examined the effect of two NSAIDs, diclofenac and ibuprofen, on testosterone and epitestosterone glucuronidation in human liver microsomes. In parallel, we have studied the inhibitory effect of these NSAIDs on recombinant UGT2B17 and UGT2B15, as well as other human hepatic UGTs that revealed low but detectable testosterone glucuronidation activity, namely UGT1A3, UGT1A4, UGT1A9 and UGT2B7. Both diclofenac and ibuprofen inhibited testosterone glucuronidation in microsomes, as well as UGT2B15 and UGT2B17. Interestingly, UGT2B15 was more sensitive than UGT2B17 to the two drugs, particularly to ibuprofen. Human liver microsomes lacking functional UGT2B17 exhibited significantly higher sensitivity to ibuprofen, suggesting that UGT2B15 plays a major role in the residual testosterone glucuronidation activity in UGT2B17-deficient individuals. Nonetheless, a minor contribution of other UGTs, particularly UGT1A9, to testosterone glucuronidation in such individuals cannot be ruled out at this stage. The epitestosterone glucuronidation activity of human liver microsomes was largely insensitive to ibuprofen and diclofenac. Taken together, the results highlight potential interactions between NSAIDs and androgen glucuronidation with possible implications for the validity of doping tests.     

Cysteine 116 participates in intermolecular bonding of the human VEGF(121) homodimer

VEGF(121), the 121-amino acid form of vascular endothelial growth factor is a homodimer with nine cysteine residues per monomer. While three intramolecular and two intermolecular disulfide bonds have been mapped, the state of the ninth cysteine, Cys116, is not known. In this study, we determined that human VEGF(121) contains a third interchain disulfide bond between Cys116 of each monomer. We also isolated a VEGF(121) variant with two extra cysteines bound to each Cys116. No evidence was found for the exsistence of Cys116 in the reduced state. In fact, selective reduction of the Cys116 interchain disulfide bond yielded an unstable VEGF(121) molecule, which reoxidized quickly. Biological activities of VEGF(121) Cys116 variants were assessed. The oxidative state of Cys116 has no effect on binding or proliferation activities but may be important for overall stability of the molecule.     

Glucuronidation of apomorphine

Apomorphine, a dopaminergic receptor agonist, is largely used in the therapy of Parkinson's disease. In this study, we characterized the glucuronidation of apomorphine and other catechols in rat liver and brain microsomes, using UDP-[U-14C]glucuronic acid and separation of the glucuronides formed by a thin layer chromatographic method. rat liver microsomes glucuronidate apomorphine at a significant rate, that was increased in the presence of dithiothreitol. Two apomorphine glucuronides were separated by high pressure liquid chromatography. We showed by electrospray mass spectrometry that both products were monoglucuronides. Other catechols were also glucuronidated in liver microsomes at various rates, and among them, 4-nitrocatechol was the most efficiently conjugated. in rat brain microsomes, only 4-nitrocatechol was significantly glucuronidated, suggesting that in the liver, several uridine-diphosphate glucuronosyltransferase (UGT) isoforms participate to the conjugation of catechols. To determine which isoforms catalyze apomorphine glucuronidation, two recombinant enzymes expressed in V79 cells were used. The isoform UGT1A6 was unable to glucuronidate apomorphine, but we observed a significant activity catalyzed by the isoform UGT2B1. These results provide, to our knowledge, the first demonstration of apomorphine conjugation by recombinant UGT2B1, and the first evidence of the lack of apomorphine glucuronidation in the rat brain.     

Essential cysteine residues for human RNase κ catalytic activity

Human RNase κ is an endoribonuclease expressed in almost all tissues and organs and belongs to a highly conserved protein family bearing representatives in all metazoans. To gain insight into the role of cysteine residues in the enzyme activity or structure, a recombinant active form of human RNase κ expressed in Pichia pastoris was treated with alkylating agents and dithiothreitol (DTT). Our results showed that the human enzyme is inactivated by DDT, while it remains fully active in the presence of alkylating agents. The unreduced recombinant protein migrates on SDS/PAGE faster than the reduced form. This observation in combination with the above findings indicated that human RNase κ does not form homodimers through disulfide bridges, and cysteine residues are not implicated in RNA catalysis but participate in the formation of intramolecular disulfide bond(s) essential for its ribonucleolytic activity. The role of the cysteine residues was further investigated by expression and study of Cys variants. Ribonucleolytic activity experiments and SDS/PAGE analysis of the wild-type and mutant proteins under reducing and non-reducing conditions demonstrated that Cys7, Cys14 and Cys85 are not essential for RNase activity. On the other hand, replacement of Cys6 or Cys69 with serine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of human RNase κ by forming a disulfide bond. Due to the absolute conservation of these cysteine residues, the Cys6-Cys69 disulfide bond is likely to exist in all RNase κ family members.     

Redox properties of the tissue factor Cys186-Cys209 disulfide bond

TF (tissue factor) is a transmembrane cofactor that initiates blood coagulation in mammals by binding Factor VIIa to activate Factors X and IX. The cofactor can reside in a cryptic configuration on primary cells and de-encryption may involve a redox change in the C-terminal domain Cys(186)-Cys(209) disulfide bond. The redox potential of the bond, the spacing of the reduced cysteine thiols and their oxidation by TF activators was investigated to test the involvement of the dithiol/disulfide in TF activation. A standard redox potential of -278 mV was determined for the Cys(186)-Cys(209) disulfide of recombinant soluble TF. Notably, ablating the N-terminal domain Cys(49)-Cys(57) disulfide markedly increased the redox potential of the Cys(186)-Cys(209) bond, suggesting that the N-terminal bond may be involved in the regulation of redox activity at the C-terminal bond. Using As(III) and dibromobimane as molecular rulers for closely spaced sulfur atoms, the reduced Cys(186) and Cys(209) sulfurs were found to be within 3-6 ? (1 ?=0.1 nm) of each other, which is close enough to reform the disulfide bond. HgCl2 is a very efficient activator of cellular TF and activating concentrations of HgCl2-mediated oxidation of the reduced Cys(186) and Cys(209) thiols of soluble TF. Moreover, PAO (phenylarsonous acid), which cross-links two cysteine thiols that are in close proximity, and MMTS (methyl methanethiolsulfonate), at concentrations where it oxidizes closely spaced cysteine residues to a cystine residue, were efficient activators of cellular TF. These findings further support a role for Cys(186) and Cys(209) in TF activation.     

An intramolecular disulfide bridge as a catalytic switch for serotonin N-acetyltransferase

Serotonin N-acetyltransferase (EC. 2.3.1.87) (AA-NAT) is a melatonin rhythm-generating enzyme in pineal glands. To establish a melatonin rhythm, AA-NAT activity is precisely regulated through several signaling pathways. Here we show novel regulation of AA-NAT activity, in which an intramolecular disulfide bond may function as a switch for the catalysis. Recombinant AA-NAT activity was irreversibly inhibited by N-ethylmaleimide (NEM) in an acetyl-CoA-protected manner. Oxidized glutathione or dissolved oxygen reversibly inhibited AA-NAT in an acetyl-CoA-protected manner. To identify the cysteine residues responsible for the inhibition, AA-NAT was first oxidized with dissolved oxygen, treated with NEM, reduced with dithiothreitol, and then labeled with [(14)C]NEM. Cys(61) and Cys(177) were specifically labeled in an acetyl-CoA-protected manner. The AA-NAT with the Cys(61) to Ala and Cys(177) to Ala double substitutions (C61A/C177A-AA-NAT) was fully active but did not exhibit sensitivity to either oxidation or NEM, whereas the AA-NATs with only the single substitutions retained about 40% of these sensitivities. An intramolecular disulfide bond between Cys(61) and Cys(177) formed upon oxidation and cleaved upon reduction was identified. Furthermore, C61A/C177A-AA-NAT expressed in COS7 cells was relatively insensitive to H(2)O(2)-evoked oxidative stress, whereas wild-type AA-NAT was strongly inhibited under the same conditions. These results indicate that the formation and cleavage of the disulfide bond between Cys(61) and Cys(177) produce the active and inactive states of AA-NAT. It is possible that intracellular redox conditions regulate AA-NAT activity through switching via an intramolecular disulfide bridge.     

Effects of removing a conserved disulfide bond on the biological characteristics of rat lipocalin-type prostaglandin D synthase

Three cysteine residues, Cys(65), Cys(89), and Cys(186) in lipocalin-type prostaglandin D synthase (L-PGDS), are conserved among all species and the disulfide bond between Cys(89) and Cys(186) is highly conserved among most, but not all, lipocalins. In this study, four rat L-PGDS variants were constructed by site-directed mutagenesis, and the conserved disulfide bond in several variants was removed by substituting cysteine with alanine. The effects of removing this disulfide bond on their biological characteristics were investigated. The NMR experiments indicated that the removal of disulfide did not change their conformations significantly. However, both thermal-induced and urea-induced unfolding experiments showed that the stabilities of enzymes without the disulfide bond decreased significantly. Moreover, the ligand-binding affinities of these variants were assessed by fluorescence experiments. Dissociation constants (K(d)) of 0.668, 0.689, 0.543 and 0.571 microM were obtained for ANS binding to wild-type rat L-PGDS, C(65)A, C(186)A, and C(89,186)A variants, respectively, and 71.2 and 62.3 nM for retinoic acid binding to wild-type rat L-PGDS and the C(186)A variant, respectively. These results suggested that the removal of the disulfide bond slightly increased the affinities for ligand binding by changing the hydrophobic regions. This study may offer valuable information for further studies on other rat lipocalins.     

The Human UGT1A3 Enzyme Conjugates Norursodeoxycholic Acid into a C23-ester Glucuronide in the Liver

Norursodeoxycholic acid (norUDCA) exhibits efficient anti-cholestatic properties in an animal model of sclerosing cholangitis. norUDCA is eliminated as a C₂₃-ester glucuronide (norUDCA-23G) in humans. The present study aimed at identifying the human UDP-glucuronosyltransferase (UGT) enzyme(s) involved in hepatic norUDCA glucuronidation and at evaluating the consequences of single nucleotide polymorphisms in the coding region of UGT genes on norUDCA-23G formation. The effects of norUDCA on the formation of the cholestatic lithocholic acid-glucuronide derivative and of rifampicin on hepatic norUDCA glucuronidation were also explored. In vitro glucuronidation assays were performed with microsomes from human tissues (liver and intestine) and HEK293 cells expressing human UGT enzymes and variant allozymes. UGT1A3 was identified as the major hepatic UGT enzyme catalyzing the formation of norUDCA-23G. Correlation studies using samples from a human liver bank (n = 16) indicated that the level of UGT1A3 protein is a strong determinant of in vitro norUDCA glucuronidation. Analyses of the norUDCA-conjugating activity by 11 UGT1A3 variant allozymes identified three phenotypes with high, low, and intermediate capacity. norUDCA is also identified as a competitive inhibitor for the hepatic formation of the pro-cholestatic lithocholic acid-glucuronide derivative, whereas norUDCA glucuronidation is weakly stimulated by rifampicin. This study identifies human UGT1A3 as the major enzyme for the hepatic norUDCA glucuronidation and supports that some coding polymorphisms affecting the conjugating activity of UGT1A3 in vitro may alter the pharmacokinetic properties of norUDCA in cholestasis treatment.     

Intermolecular cross-links mediate aggregation of phospholipid vesicles by pulmonary surfactant protein SP-A

As the most abundant glycoprotein component of pulmonary surfactant, SP-A (Mr = 30,000-36,000) plays a central role in the organization of phospholipid bilayers in the alveolar air space. SP-A, isolated from lung lavage, exists in oligomeric forms (N = 6, 12, 18, ...), mediated by collagen-like triple helices and intermolecular disulfide bonds. These protein-protein interactions, involving the amino-terminal domain of SP-A, are hypothesized to facilitate the alignment of surfactant lipid bilayers into unique tubular myelin structures. SP-A reorganization of surfactant lipid was assessed in vitro by quantitating the calcium-dependent light scattering properties of lipid vesicle suspensions induced by SP-A. Accelerated aggregation of unilamellar vesicles required SP-A and at least 3 mM free calcium. The initial rate of aggregation was proportional to the concentration of canine SP-A over lipid:protein molar ratios ranging from 200:1 to 5000:1. Digestion with bacterial collagenase or incubation with dithiothreitol (DTT) completely blocked lipid aggregation activity. Both treatments decreased the binding of SP-A to phospholipids. The conditions used in the DTT experiments (10 mM DTT, nondenaturing Tris buffer, 37 degrees C) resulted in the selective reduction and 14C-alkylation of the intermolecular disulfide bond involving residue 9Cys, whereas the four cysteines found in the noncollagenous domain of SP-A were inefficiently alkylated with [14C]-iodoacetate. HPLC analysis of tryptic SP-A peptides revealed that these four cysteine residues participate in intramolecular disulfide bond formation (138Cys-229Cys and 207Cys-221Cys). Our data demonstrate the importance of the quaternary structure (triple helix and intermolecular disulfide bond) of SP-A for the aggregation of unilamellar phospholipid vesicles.     

The formation of an intrachain disulfide bond in the leptin protein is necessary for efficient leptin secretion

Leptin is a cytokine secreted by the adipose tissue that is involved in the control of body weight. We previously showed that a point mutation (R105W) in leptin results in leptin deficiency, marked obesity and hypogonadism in humans adults. Expression in COS1 cells showed impaired secretion and intracellular accumulation of the mutated protein. However, impaired secretion of the mutant leptin had not been demonstrated in adipose cells. In this work, we demonstrate that secretion of R105W mutant is impaired in rat and human adipocytes. We also show that R105W mutant expressed in COS1 cells and in PAZ6 adipocytes forms large molecular aggregates that cannot cross a filtration membrane with a cut-off of 100 kDa. Moreover, we have engineered, by site directed mutagenesis, the cDNAs coding for leptin in which either Cys 117, Cys 167, or both, were replaced by a serine. When expressed in COS1 cells or PAZ6 adipocytes, cysteine mutants also show impaired secretion and formation of large molecular aggregates. Therefore, our work indicates that the formation of an intramolecular disulfide bridge is necessary for normal processing and secretion of leptin. Moreover, the similarity of the behavior of R105W mutant and cystein mutants suggests that the lack of secretion observed with the naturally occurring mutant could result from impaired disulfide bond formation.     

Reactive nitrogen species derived activation of rat liver microsomal glutathione S-transferase

The effect of reactive nitrogen species on rat liver microsomal glutathione S-transferase (MGST1) was investigated using microsomes and purified MGST1. When microsomes or the purified enzyme were incubated with peroxynitrite (ONOO(-)), the GST activity was increased to 2.5-6.5 fold in concentration-dependent manner and a small amount of the MGST1 dimer was detected. MGST1 activity was increased by ONOO(-) in the presence of high amounts of reducing agents including glutathione (GSH) and the activities increased by ONOO(-) or ONOO(-) plus GSH treatment were decreased by 30-40% by further incubation with dithiothreitol (DTT, reducing disulfide) or by sodium arsenite (reducing sulfenic acid). Furthermore, GSH was detected by HPLC from the MGST1 which was incubated with ONOO(-) plus GSH or S-nitrosoglutathione followed by DTT treatment. In addition, the MGST1 activity increased by nitric oxide (NO) donors such as S-nitrosoglutathione, S-nitrosocysteine or the non-thiol NO donor 1-hydroxy-2-oxo-3 (3-aminopropyl)-3-isopropyl was restored by the DTT treatment. Since DTT can reduce S-nitrosothiol and disulfide bond to thiol, S-nitrosylation and a mixed disulfide bond formation of MGST1 were suggested. Thus, it was demonstrated that MGST1 is activated by reactive nitrogen species through a forming dimeric protein, mixed disulfide bond, nitrosylation and sulfenic acid.     

Mutating the four extracellular cysteines in the chemokine receptor CCR6 reveals their differing roles in receptor trafficking,ligand binding,and signaling

CCR6 is the receptor for the chemokine MIP-3 alpha/CCL20. Almost all chemokine receptors contain cysteine residues in the N-terminal domain and in the first, second, and third extracellular loops. In this report, we have studied the importance of all cysteine residues in the CCR6 sequence using site-directed mutagenesis and biochemical techniques. Like all G protein-coupled receptors, mutating disulfide bond-forming cysteines in the first (Cys118) and second (Cys197) extracellular loops in CCR6 led to complete elimination of receptor activity, which for CCR6 was also associated with the accumulation of the receptor intracellularly. Although two additional cysteines in the N-terminal region and the third extracellular loop, which are present in almost all chemokine receptors, are presumed to form a disulfide bond, this has not been demonstrated experimentally for any of these receptors. We found that mutating the cysteines in the N-terminal domain (Cys36) and the third extracellular loop (Cys288) neither significantly affected receptor surface expression nor completely abolished receptor function. Importantly, contrary to several previous reports, we demonstrated directly that instead of forming a disulfide bond, the N-terminal cysteine (Cys36) and the third extracellular loop cysteine (Cys288) contain free SH groups. The cysteine residues (Cys36 and Cys288), rather than forming a disulfide bond, may be important per se. We propose that CCR6 forms only a disulfide bond between the first (Cys118) and second (Cys197) extracellular loops, which confines a helical bundle together with the N-terminus adjacent to the third extracellular loop, creating the structural organization critical for ligand binding and therefore for receptor signaling.     

Chemical modification of rat hepatic microsomes with N-ethylmaleimide results in inactivation of both UDP-N-acetylglucosamine-dependent stimulation of glucuronidation and UDP-glucuronic acid uptake.

Chemical modification of rat hepatic microsomes with N-ethylmaleimide (NEM) resulted in inactivation of UDP-N-acetylglucosamine (UDP-GlcNAc)-dependent stimulation of glucuronidation of p-nitrophenol. Inactivation kinetics and pH dependence were in agreement with the modification of a single sulfhydryl group. NEM also inactivated the uptake of UDP-glucuronic acid (UDP-GlcUA) but not UDP-glucose. With various sulfhydryl-modifying reagents, the inactivation of UDP-GlcUA uptake was linked to that of glucuronidation. UDP-GlcUA protected against NEM-sensitive inactivation of both UDP-GlcNAc-dependent stimulation of glucuronidation and UDP-GlcUA uptake, suggesting that the sulfhydryl group is located within or near the UDP-GlcUA binding site of the microsomal protein involved in the stimulation. Using microsomes labeled with biotin-conjugated maleimide and immunopurification with anti-peptide antibody against UDP-glucuronosyltransferase family 1 (UGT1) isozymes, immunopurified UGT1s were found to be labeled with the maleimide and UDP-GlcUA protected against the labeling as it did with the NEM-sensitive inactivation. These data suggest the involvement of a sulfhydryl residue of microsomal protein in the UDP-GlcNAc-dependent stimulation mechanism via the stimulation of UDP-GlcUA uptake into microsomal vesicles.     

E. coli propionyl-CoA synthetase is regulated in vitro by an intramolecular disulfide bond

The E. coli propionyl-CoA synthetase (PCS) was cloned, expressed, purified, and analyzed. Kinetic analyses suggested that the enzyme preferred propionate as substrate but would also use acetate. The purified, stored protein had relatively low activity but was activated up to about 10-fold by incubation with dithiothreitol (DTT). The enzyme activation by DTT was reversed by diamide. This suggests that the protein contains a regulatory disulfide bond and that the reduction to two sulfhydryl groups activates PCS while the oxidation to a disulfide leads to its inactivation. This idea was tested by sequential mutagenesis of the 9 Cys in the protein to Ala. It was revealed that the C128A and C315A mutants had wildtype enzyme activity but were no longer activated by DTT or inhibited by diamide. The data obtained indicate that two Cys residues could be involved in redox-regulated system through formation of an intramolecular disulfide bridge in PCS.