重组N-乙酰鸟氨酸脱乙酰基酶的表达、纯化和复性研究

报道重组N-乙酰鸟氨酸脱乙酰基酶(NAOase)的研究进展。重组NAOase由大肠杆菌argE基因编码,在重组菌BL21(DE3)-pET22b-argE中的表达量为32.5%,大多以无活性的包涵体存在。低温诱导可增大有活性的可溶表达部分的比例。可溶性NAOase经Ni-NTA凝胶亲和纯化后得到SDS-PAGE电泳纯的酶,比酶活为1193.2u/mg蛋白。诱导条件影响整菌蛋白的成分及比例。37℃诱导生成的包涵体经尿素梯度洗涤后纯度较22℃高。低的蛋白浓度和合适的氧化还原体系是影响复性的关键因素。稀释法和透析法皆可使包涵体部分复性。在合适的条件下以稀释法复性时,约有17.78%包涵体可顺利复活。包涵体经尿素洗涤、溶解、Ni-NTA凝胶柱亲和纯化后,获得了高纯度的NAOase。     

Hemopexin-mediated heme uptake by liver. Characterization of the interaction of heme-hemopexin with isolated rabbit liver plasma membranes

Plasma membranes isolated from rabbit liver retain the ability to interact specifically with heme-hemopexin. In this system, apohemopexin does not compete effectively with heme-hemopexin for binding. The membranes bind heme-hemopexin complexes with high affinity (KD = 6.8 X 10(-7) M) and with an apparent capacity of 2.3 pmol/mg of membrane protein. These membranes also retain the ability to remove heme from heme-hemopexin. The release of heme reaches a plateau after 15-30 min at 30 degrees C and does not involve metabolic energy, proteolysis of hemopexin or pH gradients. The apohemopexin formed is rapidly released from the membranes. The accumulation of heme is saturable and is affected by pH and temperature with maximum uptake occurring between pH 5.5 and 6.5 and at 30 degrees C. Interestingly, much more heme (approximately 25 pmol/mg of membrane protein) is accumulated than hemopexin at saturation, implying that the receptor can turn over several times and that a heme-binding component exists in the rabbit liver plasma membrane.     

Purification of a catalase-peroxidase from Halobacterium halobium: characterization of some unique properties of the halophilic enzyme.

A hydroperoxidase purified from the halophilic archaeon Halobacterium halobium exhibited both catalase and peroxidase activities, which were greatly diminished in a low-salt environment. Therefore, the purification was carried out in 2 M NaCl. Purified protein exhibited catalase activity over the narrow pH range of 6.0 to 7.5 and exhibited peroxidase activity between pH 6.5 and 8.0. Peroxidase activity was maximal at NaCl concentrations above 1 M, although catalase activity required 2 M NaCl for optimal function. Catalase activity was greatest at 50 degrees C; at 90 degrees C, the enzymatic activity was 20% greater than at 25 degrees C. Peroxidase activity decreased rapidly above its maximum at 40 degrees C. An activation energy of 2.5 kcal (ca. 10 kJ)/mol was calculated for catalase, and an activation energy of 4.0 kcal (ca. 17 kJ)/mol was calculated for peroxidase. Catalase activity was not inhibited by 3-amino-1,2,4-triazole but was inhibited by KCN and NaN3 (apparent Ki [KiApp] of 50 and 67.5 microM, respectively). Peroxidative activity was inhibited equally by KCN and NaN3 (KiApp for both, approximately 30 microM). The absorption spectrum showed a Soret peak at 404 nm, and there was no apparent reduction by dithionite. A heme content of 1.43 per tetramer was determined. The protein has a pI of 3.8 and an M(r) of 240,000 and consists of four subunits of 60,300 each.     

Catalytic efficiency and some structural properties of cold-active protein-tyrosine-phosphatase

A procedure was established for expression and purification of abundant recombinant cold-active protein-tyrosine-phosphatase (RCPTPase), which showed identical enzymatic characteristics to the native enzyme (NCPTPase). The purified RCPTPase showed high catalytic activity at low temperature and maximal activity at 30 degrees C. RCPTPase has a thermodynamic characteristic in that its activation enthalpy was determined to be low, 4.3 kcal/mol, at temperatures below 19.3 degrees C, where the Arrhenius relationship exhibited an inflection point, in comparison with 20.3 kcal/mol above 19.3 degrees C. Also, the thermostability, DeltaG(water), of the catalytic site in the RCPTPase molecule was increased with a decrease in temperature. It was considered that cold-active protein-tyrosine-phosphatase could maintain its catalytic site in a stable conformation for eliciting high catalytic activity with low activation enthalpy at low temperature.     

Expression and characterization of full-length human heme oxygenase-1: the presence of intact membrane-binding region leads to increased binding affinity for NADPH cytochrome P450 reductase

Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes.     

Stabilization of apoglobin by low temperature increases yield of soluble recombinant hemoglobin in Escherichia coli.

Accumulation of soluble recombinant hemoglobin (rHb1.1) in Escherichia coli requires proper protein folding, prosthetic group (heme) addition, and subunit assembly. This served as a new model system for the study of the effects of temperature, protein synthesis rates, and protein accumulation rates on protein solubility in E. coli. Fermentation expression of rHb1.1 at 30 degrees C from cultures containing a medium or high globin gene dosage (pBR-based or pUC-based plasmids with rHb1.1 genes under the control of the tac promoter) was compared. A medium gene dosage resulted in rHb1.1 accumulating to approximately 7% of the soluble cell protein, of which 78% was soluble. A high globin gene dosage resulted in a > or = 3-fold increase in total globin to 23 to 24% of the soluble cell protein, but 70% was insoluble. Accumulation of insoluble rHb1.1 began immediately upon induction. The proportion of rHb1.1 from the high globin gene dosage that accumulated as insoluble globin was affected by reducing (i) the inducer concentration and (ii) the temperature. Reducing the inducer concentration reduced globin synthesis up to eightfold but increased the proportion of soluble rHb1.1 to 93%. In contrast, total globin protein synthesis was barely affected by reducing the temperature from 30 to 26 degrees C, while soluble globin accumulation increased > 2-fold to approximately 15% of the soluble cell protein. The contrast between the effects of reducing rates of protein synthesis and accumulation and those of reducing temperature suggests that lower temperature stabilizes one or more folding intermediates. We propose a simplified physical model which integrates protein synthesis, folding, and heme association. This model shows that temperature-dependent apoglobin stability is the most critical factor in soluble rHb1.1 accumulation.     

Characterization of recombinant human endothelial nitric-oxide synthase purified from the yeast Pichia pastoris

Human endothelial nitric-oxide synthase (eNOS) was expressed in the methylotrophic yeast Pichia pastoris, making use of the highly inducible alcohol oxidase promoter. The recombinant protein constituted approximately 3% of total protein and was largely soluble (>75%). About 1 mg of purified eNOS was obtained from 100-ml yeast cell cultures by affinity chromatography of crude cell supernatants. The purified enzyme had a V(max) of 192 +/- 18 nmol of L-citrulline x mg(-1) x min(-1), had a K(m) for L-arginine of 3.9 +/- 0.2 microM, and showed an absolute requirement for tetrahydrobiopterin (H(4)biopterin). NADPH oxidase activity was 136 +/- 9 and 342 +/- 24 nmol x mg(-1) x min(-1) in the absence and presence of 0.1 mM L-arginine, respectively, and not affected by H(4)biopterin. The protein contained 0.56 +/- 0.06 equivalents of FAD and 0.79 +/- 0.08 equivalents of FMN. On-line gel filtration/inductively coupled plasma mass spectrometry analysis confirmed that both iron (0.80 +/- 0.09 mol/subunit) and zinc (0.43 +/- 0.03 mol/subunit) were bound to the enzyme. Graphite furnace-atomic absorption spectroscopy yielded a value for bound iron of 0.84 +/- 0.04 mol/subunit. The absorbance of the enzyme at 398 nm implied a heme content of 0.85 +/- 0.09 mol/subunit, and the high pressure liquid chromatography heme assay gave an estimate of 0.71 +/- 0.02 mol heme/subunit. Gel permeation chromatography yielded one single peak with a Stokes radius of 6.62 +/- 0.7 nm, indicating that the native protein is dimeric. Upon low temperature gel electrophoresis the untreated protein appeared mainly as a monomer (88 +/- 3%), but pretreatment with H(4)biopterin and L-arginine led to a pronounced shift toward dimers (77 +/- 4%). Thus, in contrast to bovine eNOS (List, B. M., Kl?sch, B., V?lker, C., Gorren, A. C. F., Sessa, W. C., Werner, E. R., Kukovetz, W. R., Schmidt, K., and Mayer, B. (1997) Biochem. J. 323, 159-165; Rodriguez-Crespo, I., Gerber, N. C., and Ortiz de Montellano, P. R. (1996) J. Biol. Chem. 271, 11462-11467), the human eNOS appears to be markedly stabilized by H(4)biopterin.     

Protein engineering of thromboxane synthase: conversion of membrane-bound to soluble form

Thromboxane A2 synthase (TXAS) binds to the endoplasmic reticulum membrane and catalyzes both an isomerization of prostaglandin H2 (PGH2) to form thromboxane A2 (TXA2) and a fragmentation of PGH2 to form 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and malondialdehyde (MDA). TXAS is a non-classic cytochrome P450 in that it does not require molecular oxygen or an external electron donor for catalysis. Difficulty in obtaining crystals from the membrane-bound TXAS prompted us to modify the protein to a soluble form. Results from site-directed mutagenesis, hydropathy analysis, and homology modeling led us to identify a putative membrane association segment near the end of helix F in TXAS. We report here the generation of a soluble form of TXAS by deletion of the amino-terminal membrane-anchoring domain and replacement of the helix F and F-G loop region with the corresponding region of the structurally characterized microsomal P450 2C5. The resultant TXAS/2C5 chimera is expressed in bacteria as a cytosolic and monomeric protein. Addition of an amino-terminal leader sequence to enhance expression and a tetra-histidine segment at the carboxyl-terminus to facilitate purification yielded approximately 4 mg of nearly homogeneous TXAS/2C5 per liter of bacterial culture. The TXAS/2C5 chimera contains heme at nearly a 1:1 molar ratio and catalyzes the formation of TXA2, MDA, and HHT at a 1:1:1 ratio, although with a reduced catalytic activity compared to wild type TXAS. TXAS/2C5 exhibits electronic absorption spectra similar to wild type TXAS and has similar affinities toward distal heme ligands such as imidazole and U44069. The chimera was mono-dispersive and thus is promising for crystallization trials.     

The second enzyme in pyrrolnitrin biosynthetic pathway is related to the heme-dependent dioxygenase superfamily

Pyrrolnitrin is a commonly used and clinically effective treatment for fungal infections and provides the structural basis for the more widely used fludioxinil. The pyrrolnitrin biosynthetic pathway consists of four chemical steps, the second of which is the rearrangement of 7-chloro-tryptophan by the enzyme PrnB, a reaction that is so far unprecedented in biochemistry. When expressed in Pseudomonas fluorescens, PrnB is red in color due to the fact that it contains 1 mol of heme b per mole of protein. The crystal structure unexpectedly establishes PrnB as a member of the heme-dependent dioxygenase superfamily with significant structural but not sequence homology to the two-domain indoleamine 2,3-dioxygenase enzyme (IDO). The heme-binding domain is also structurally similar to that of tryptophan 2,3-dioxygenase (TDO). Here we report the binary complex structures of PrnB with d- and l-tryptophan and d- and l-7-chloro-tryptophan. The structures identify a common hydrophobic pocket for the indole ring but exhibit unusual heme ligation and substrate binding when compared with that observed in the TDO crystal structures. Our solution studies support the heme ligation observed in the crystal structures. Purification of the hexahistidine-tagged PrnB yields homogeneous protein that only displays in vitro activity with 7-chloro-l-tryptophan after reactivation with crude extract from the host strain, suggesting that an as yet unknown cofactor is required for activity. Mutation of the proximal heme ligand results, not surprisingly, in inactive enzyme. Redox titrations show that PrnB displays a significantly different reduction potential to that of IDO or TDO, indicating possible differences in the PrnB catalytic cycle. This is confirmed by the absence of tryptophan dioxygenase activity in PrnB, although a stable oxyferrous adduct (which is the first intermediate in the TDO/IDO catalytic cycle) can be generated. We propose that PrnB shares a key catalytic step with TDO and IDO, generation of a tryptophan hydroperoxide intermediate, although this species suffers a different fate in PrnB, leading to the eventual formation of the product, monodechloroaminopyrrolnitrin.     

Purification and characterization of a novel thermo-alkali-stable catalase from Thermus brockianus

A novel thermo-alkali-stable catalase from Thermus brockianus was purified and characterized. The protein was purified from a T. brockianus cell extract in a three-step procedure that resulted in 65-fold purification to a specific activity of 5300 U/mg. The enzyme consisted of four identical subunits of 42.5 kDa as determined by SDS-PAGE and a total molecular mass measured by gel filtration of 178 kDa. The catalase was active over a temperature range from 30 to 94 degrees C and a pH range from 6 to 10, with optimum activity occurring at 90 degrees C and pH 8. At pH 8, the enzyme was extremely stable at elevated temperatures with half-lives of 330 h at 80 degrees C and 3 h at 90 degrees C. The enzyme also demonstrated excellent stability at 70 degrees C and alkaline pH with measured half-lives of 510 h and 360 h at pHs of 9 and 10, respectively. The enzyme had an unusual pyridine hemochrome spectrum and appears to utilize eight molecules of heme c per tetramer rather than protoheme IX present in the majority of catalases studied to date. The absorption spectrum suggested that the heme iron of the catalase was in a 6-coordinate low spin state rather than the typical 5-coordinate high spin state. A K(m) of 35.5 mM and a V(max) of 20.3 mM/min.mg protein for hydrogen peroxide was measured, and the enzyme was not inhibited by hydrogen peroxide at concentrations up to 450 mM. The enzyme was strongly inhibited by cyanide and the traditional catalase inhibitor 3-amino-1,2,4-triazole. The enzyme also showed no peroxidase activity to peroxidase substrates o-dianisidine and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), a trait of typical monofunctional catalases. However, unlike traditional monofunctional catalases, the T. brockianus catalase was easily reduced by dithionite, a characteristic of catalase-peroxidases. The above properties indicate that this catalase has potential for applications in industrial bleaching processes to remove residual hydrogen peroxide from process streams.     

Cloning,Expression, and Characterization of Siamese Crocodile (<Emphasis Type="Italic">Crocodylus siamensis</Emphasis>) Hemoglobin from <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Recombinant Crocodylus siamensis hemoglobin (cHb) has been constructed and expressed using Escherichia coli as the expression system in conjunction with a trigger factor from the Cold-shock system as the fusion protein. While successful processing as soluble protein in E. coli was achieved, the net yields of active protein from downstream purification processes remained still unsatisfactory. In this study, cHb was constructed and expressed in the eukaryotic expression system Pichia pastoris. The results showed that cHb was excreted from P. pastoris as a soluble protein after 72 h at 25 °C. The amino acid sequence of recombinant cHb was confirmed using LC–MS/MS. Indeed, the characteristic of Hb was investigated by external heme incorporation. The UV–Vis profile showed a specific pattern of the absorption at 415 nm, indicating the recombinant cHb was formed complex with heme, resulting in active oxyhemoglobin (OxyHb). This result suggests that the heme molecules were fully combined with heme binding site of the recombinant cHb, thus producing characteristic red color for the OxyHb at 540 and 580 nm. The results revealed that the recombinant cHb was prosperously produced in P. pastoris and exhibited a property as protein–ligand binding. Thus, our work described herein offers a great potential to be applied for further studies of heme-containing protein expression. It represents further pleasing option for protein production and purification on a large scale, which is important for determination and characterization of the authenticity features of cHb proteins.     

Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein

In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis. We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1. Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Deltaibp mutant strain lacking the small heat-shock proteins IbpA and IbpB. Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22 degrees C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) about 0.5. Under these conditions over 90% of the fusion protein was present in a soluble form. The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns. The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E. coli culture.     

Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase

Toluene 4-monooxygenase (T4MO) is a member of the bacterial multicomponent monooxygenases, an enzyme family that utilizes a soluble diiron hydroxylase to oxidize a variety of hydrocarbons as the initial step in their metabolism. The hydroxylases obtain reducing equivalents from NAD(P)H via an electron transfer chain that is initiated by an oxidoreductase containing an N-terminal ferredoxin domain and C-terminal flavin- and NAD-binding domains. T4moF, the NADH oxidoreductase of T4MO, was expressed as a soluble protein in Escherichia coli BL21(DE3) from the pUC-derived expression vector pRS205. This vector contains a lac promoter instead of a T7 promoter. A three step purification from the soluble cell lysate yielded approximately 1 mg of T4moF per gram of wet cell paste with greater than 90% purity. The purified protein contained 1 mol of FAD and 2 mol of Fe per mol of T4moF; quantitative EPR spectroscopy showed approximately 1 mol of the S=1/2 signal from the reduced [2Fe-2S] cluster per mol of T4moF. Steady state kinetic analysis of p-cresol formation activity treating T4moF as the variable substrate while all other proteins and substrates were held constant gave apparent K(M-) and apparent k(cat)-values of 0.15 microM and 3.0 s(-1), respectively. This expression system and purification allows for the recovery of the soluble oxidoreductase in yields that facilitate further biochemical and structural characterizations.     

Temperature adaptation of biological membranes. Compensation of the molar activity of cytochrome c oxidase in the mitochondrial energy-transducing membrane during thermal acclimation of the carp (Cyprinus carpio L.)

The acclimation temperature of carp does not affect the amount of cytochrome c oxidase per mg mitochondrial protein as revealed from the reduced-minus-oxidized difference spectra of red muscle mitochondria from cold- and warm-acclimated carp. There are no differences between cold- and warm-acclimated fish in the substrate binding properties of the enzyme as judged from the Km values for cytochrome c at 30 degrees C (3.34 +/- 0.ee microM, acclimation temperature 10 degrees C and 3.55 +/- 0.31 microM, acclimation temperature 30 degrees C). The molar activities of the enzyme, however, differ for both acclimation temperatures: when intercalated in the 10 degrees C-acclimated mitochondrial membrane, the enzyme can catalyze the oxidation of 117.6 +/- 17.2 mol ferrocytochrome c/s per mol heme a as compared with 85.6 +/- 17.2 in the 30 degrees C-acclimated membrane (experimental temperature 30 degrees C). Correspondingly, higher specific activities of the succinate oxidase system are observed in mitochondria from cold-acclimated carp as compared with those obtained from warm-acclimated carp. The results indicate that cold acclimation of the eurythermic carp is accompanied by a partial compensation of the acute effect of decreasing temperature on the activity of cytochrome c oxidase in red muscle mitochondria. Based on the temperature-induced lipid adaptation reported for carp red muscle mitochondria (Wodtke, E. (1980) Biochim. Biophys. Acta 640, 698--709), it is concluded that during thermal acclimation the molar activity of cytochrome c oxidase is controlled by viscotropic regulation. The results fit to the conception that cardiolipin constitutes a lipid shell (annulus) surrounding the oxidase within the native membrane, but that it is the bilayer fluidity and not the annular fluidity which determines the activity of cytochrome c oxidase.     

Intrinsic factor covalently bound to Sepharose as affinity medium for the purification of a soluble intrinsic factor receptor from human urine

We have identified a soluble receptor for intrinsic factor (IF) in human urine. The purification of this protein by affinity chromatography required a preliminary purification of IF from hog pyloric mucosal extract. This was achieved by thermolabile cobalamin-ethanol-aminohexane Sepharose affinity chromatography with a 133-fold purification, a yield of 45% and a specific binding activity of 15720 pmol/mg protein. The purified Cbl-IF complex was coupled to epoxy-Sepharose with a yield of 23.8% and a specific activity of 1.2 nmol per mol of gel. The soluble IF receptor was purified form 200 ml of urine concentrate of pregnant women. Desorption was performed at pH 5.0 and in the presence of 5 mM EDTA. The soluble IF receptor was purified 17 200-fold with a yield of 52% and a IF binding capacity of 3260 pmol per mg of protein. A single protein with a M_r of 70 000 was found in silver-stained SDS-PAGE.     

Activation parameters of the adenosine triphosphatase of Micrococcus lysodeikticus. A comparison of the soluble and membrane-bound forms of the enzyme.

The Arrhenius plots for the membrane-bound ATPase and its soluble form purified from Micrococcus lysodeikticus, presented discontinuities near 30 degrees C at pH 7.5. Glycerol-containing lipids were not responsible for these discontinuities. The values of the enthalpies of activation were 12 (soluble) and 22 (membrane-bound) kcal/mol (50.2 and 92.0 kJ/mol) above 30 degrees C and 42 (soluble) and 29 (membrane-bound) kcal/mol (175.7 and 121.3 kJ/mol) below that temperature. The results suggested that both molecular forms of the ATPase were able to adopt at least two different structures, above and below the critical temperature. Of the two, only the high-temperature structure seemed to be enzymically active. In the case of lipid-dependent ATPases, such as the Escherichia coli enzyme, the transition between both enzyme structures probably occurred with simultaneous "melting" of their lipid microenvironment.     

Direct hemin transfer from IsdA to IsdC in the iron-regulated surface determinant (Isd) heme acquisition system of Staphylococcus aureus

The iron-regulated surface determinants (Isd) of Staphylococcus aureus, including surface proteins IsdA, IsdB, IsdC, and IsdH and ATP-binding cassette transporter IsdDEF, constitute the machinery for acquiring heme as a preferred iron source. Here we report hemin transfer from hemin-containing IsdA (holo-IsdA) to hemin-free IsdC (apo-IsdC). The reaction has an equilibrium constant of 10 +/- 5 at 22 degrees C in favor of holo-IsdC formation. During the reaction, holo-IsdA binds to apo-IsdC and then transfers the cofactor to apo-IsdC with a rate constant of 54.3 +/- 1.8 s(-1) at 25 degrees C. The transfer rate is >70,000 times greater than the rate of simple hemin dissociation from holo-IsdA into solvent (k transfer = 54.3 s(-1) versus k -hemin = 0.00076 s(-1)). The standard free energy change, Delta G 0, is -27 kJ/mol for the formation of the holo-IsdA-apo-IsdC complex. IsdC has a higher affinity for hemin than IsdA. These results indicate that the IsdA-to-IsdC hemin transfer is through the activated holo-IsdA-apo-IsdC complex and is driven by the higher affinity of apo-IsdC for the cofactor. These findings demonstrate for the first time in the Isd system that heme transfer is rapid, direct, and affinity-driven from IsdA to IsdC. These results also provide the first example of heme transfer from one surface protein to another surface protein in Gram-positive bacteria and, perhaps most importantly, indicate that the mechanism of activated heme transfer, which we previously demonstrated between the streptococcal proteins Shp and HtsA, may apply in general to all bacterial heme transport systems.     

Purification and characterization of heme oxygenase from chick liver. Comparison of the avian and mammalian enzymes

A major inducible form of heme oxygenase (EC 1.14.99.3) was purified from liver microsomes of chicks pretreated with cadmium chloride. The purification involved solubilization of microsomes with Emulgen 913 and sodium cholate, followed by DEAE-Sephacel, carboxymethyl-cellulose (CM-52) and hydroxyapatite chromatography, and FPLC through Superose 6 and 12 columns operating in series. The final product gave a single band on silver-stained SDS/polyacrylamide gels (Mr = 33,000). Optimal conditions for measurement of activity of solubilized heme oxygenase were studied. In a reconstituted system containing purified heme oxygenase, NADPH-cytochrome reductase, biliverdin reductase and NADPH, the Km for free heme was 3.8 +/- 0.5 microM; for heme in the presence of bovine serum albumin (5 mol heme/3 mol albumin) the Km was 5.0 +/- 0.8 microM; and the Km for NADPH was 6.1 +/- 0.4 microM (all values mean +/- SD, n = 3). Oxygen concentration as low as 15 microM, with saturating concentrations of heme and NADPH, did not affect the reaction rate, indicating that the supply of oxygen is not involved in the physiological regulation of activity of the enzyme. The pH optimum of the reaction was 7.4; at 37 degrees C, the apparent Vmax was 580 +/- 44 nmol biliverdin.(mg protein)-1.min-1 and the molecular activity was 19.2 min-1. Biliverdin IXa was the sole biliverdin isomer formed. In the presence of purified biliverdin reductase, biliverdin was converted quantitatively to bilirubin. Addition of catalase to the reconstituted system decreased the breakdown of heme to non-biliverdin products and led to nearly stoichiometric conversion of heme to biliverdin. Activity of the enzyme in the reconstituted system was inhibited by metalloporphyrins in the following order of decreasing potency: tin mesoporphyrin greater than tin protoporphyrin greater than zinc protoporphyrin greater than manganese protoporphyrin greater than cobalt protoporphyrin. Protoporphyrin (3.3 or 6.6 microM) (and several other porphyrins) and metallic ions (100 microM) alone had little if any inhibitory effect, except for Hg2+ which inhibited by 67% at 10 microM and totally at 15 microM. Following partial cleavage, fragments of the purified enzyme were sequenced. Comparison of sequences to those derived from cDNA sequences for the major inducible rat and human heme oxygenase showed 69% and 76% similarities, respectively. The histidine residue at position 132 of rat heme oxygenase-1 and the residues (Lys128-Arg136) flanking His132 were conserved in all three enzymes, as well as in the corresponding portion of a fourth less highly similar rat enzyme, heme oxygenase-2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Post-translational regulation of human indoleamine 2,3-dioxygenase activity by nitric oxide

The heme protein indoleamine 2,3-dioxygenase (IDO) is induced by the proinflammatory cytokine interferon-gamma (IFNgamma) and plays an important role in the immune response by catalyzing the oxidative degradation of L-tryptophan (Trp) that contributes to immune suppression and tolerance. Here we examined the mechanism by which nitric oxide (NO) inhibits human IDO activity. Exposure of IFNgamma-stimulated human monocyte-derived macrophages (MDM) to NO donors had no material impact on IDO mRNA or protein expression, yet exposure of MDM or transfected COS-7 cells expressing active human IDO to NO donors resulted in reversible inhibition of IDO activity. NO also inhibited the activity of purified recombinant human IDO (rhIDO) in a reversible manner and this correlated with NO binding to the heme of rhIDO. Optical absorption and resonance Raman spectroscopy identified NO-inactivated rhIDO as a ferrous iron (Fe(II))-NO-Trp adduct. Stopped-flow kinetic studies revealed that NO reacted most rapidly with Fe(II) rhIDO in the presence of Trp. These findings demonstrate that NO inhibits rhIDO activity reversibly by binding to the active site heme to trap the enzyme as an inactive nitrosyl-Fe(II) enzyme adduct with Trp bound and O2 displaced. Reversible inhibition by NO may represent an important mechanism in controlling the immune regulatory actions of IDO.