Specific detection of cysteine and homocysteine in biological fluids by tuning the pH values of fluorosurfactant-stabilized gold colloidal solution

This study describes the use of 14 nm nonionic fluorosurfactant-capped gold nanoparticles (FSN-capped AuNPs) for the simultaneous detection of cysteine (Cys) and homocysteine (Hcy) using colorimetric method, requiring no use of separation techniques. It was found that the kinetics of Cys/Hcy-induced aggregation of the 14 nm FSN-capped AuNPs strongly depends on the pH value of gold colloidal solution. At a pH of 6.5, the Cys-induced aggregation kinetics of the FSN-capped AuNPs was almost identical to that induced by Hcy, facilitating simultaneous detection of total Cys and Hcy up to a concentration as low as 0.15 μM; while at pH 12.0, the kinetics of Cys-induced aggregation was much faster than that inducted by Hcy, leading to selective detection of Cys at concentration as low as 1.0 μM in the presence of Hcy. The applicability of the method was validated by spiking known amount of Cys and Hcy in human urine and plasma samples, obtaining a recovery of 95.4–105.5%. The present approach is simple, high selective and provides high reproducibility, and has a great potentiality in disease diagnosis.     

Reactive cysteine residues in the oxidative dimerization and Cu2+ induced aggregation of human γD-crystallin: Implications for age-related cataract

Cysteine (Cys) residues are major causes of crystallin disulfide formation and aggregation in aging and cataractous human lenses. We recently found that disulfide linkages are highly and partly conserved in β- and γ-crystallins, respectively, in human age-related nuclear cataract and glutathione depleted LEGSKO mouse lenses, and could be mimicked by in vitro oxidation. Here we determined which Cys residues are involved in disulfide-mediated crosslinking of recombinant human γD-crystallin (hγD). In vitro diamide oxidation revealed dimer formation by SDS-PAGE and LC-MS analysis with Cys 111-111 and C111-C19 as intermolecular disulfides and Cys 111-109 as intramolecular sites. Mutation of Cys111 to alanine completely abolished dimerization. Addition of αB-crystallin was unable to protect Cys 111 from dimerization. However, Cu²⁺-induced hγD-crystallin aggregation was suppressed up to 50% and 80% by mutants C109A and C111A, respectively, as well as by total glutathionylation. In contrast to our recently published results using ICAT-labeling method, manual mining of the same database confirmed the specific involvement of Cys111 in disulfides with no free Cys111 detectable in γD-crystallin from old and cataractous human lenses. Surface accessibility studies show that Cys111 in hγD is the most exposed Cys residue (29%), explaining thereby its high propensity toward oxidation and polymerization in the aging lens.     

Oxidation of human lens recombinant alphaA-crystallin and cysteine-deficient mutants

Disulfide cross-linking, one of the results of oxidative stress, has been thought to play an important role in cataractogenesis. High molecular mass (HMM) protein aggregation also contributes to cataract development, and a prevailing speculation is that disulfide cross-linking induces HMM aggregation. However, there is no direct evidence to support this speculation. Dimerization is an effect of disulfide cross-linking but cannot explain the size of HMM aggregates observed in the lens. alphaA-crystallin has two cysteine residues (Cys131 and Cys142) and we have prepared three Cys-deficient mutants, two single mutants (C131I and C142I) and one double mutant (C131I/C142I). They were subjected to H202 oxidation in an ascorbate-FeCl(3)-EDTA-H202 system. The effects of oxidation on the mutants, including changes in aggregate size and conformation, were compared with those of the wild-type alphaA-crystallin by FPLC gel filtration, absorption, fluorescence, and circular dichroism measurements. The results indicated that other amino acid residues besides Cys, such as Trp and Tyr, were also oxidized by H202. Disulfide dimerization alone seems to play a less important role in HMM aggregation than does the secondary conformational change resulting from the combined effect of the oxidation of Trp and Tyr as well as Cys.     

Evidence for intramolecular disulfide bond shuffling in the folding of mutant human lysozyme

Our previous results using the Saccharomyces cerevisiae secretion system suggest that intramolecular exchange of disulfide bonds occurs in the folding pathway of human lysozyme in vivo (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 265, 7570-7575). Here we report on the results of introducing an artificial disulfide bond in mutants with 2 cysteine residues substituting for Ala83 and Asp91. The mutant (C83/91) protein was not detected in the culture medium of the yeast, probably because of incorrect folding. Thereupon, 2 cysteine residues Cys77 and Cys95 were replaced with Ala in the mutant C83/91, because a native disulfide bond Cys77-Cys95 was found not necessary for correct folding in vivo (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). The resultant mutant (AC83/91) was secreted as two proteins (AC83/91-a and AC83/91-b) with different specific activities. Amino acid and peptide mapping analyses showed that two glutathiones appeared to be attached to the thiol groups of the cysteine residues introduced into AC83/91-a and that four disulfide bonds including an artificial disulfide bond existed in the AC83/91-b molecule. The presence of cysteine residues modified with glutathione may indicate that the non-native disulfide bond Cys83-Cys91 is not so easily formed as a native disulfide bond. These results suggest that the introduction of Cys83 and Cys91 may act to suppress the process of native disulfide bond formation through disulfide bond interchange in the folding of human lysozyme.     

Evidence for difference in the roles of two cysteine residues involved in disulfide bond formation in the folding of human lysozyme

Human lysozyme is made up of 130 amino acid residues and has four disulfide bonds at Cys6-Cys128, Cys30-Cys116, Cys65-Cys81, and Cys77-Cys95. Our previous results using the Saccharomyces cerevisiae secretion system indicate that the individual disulfide bonds of human lysozyme have different functions in the correct in vivo folding and enzymatic activity of the protein (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). In this paper, we report the results of experiments that were focused on the roles of Cys65 and Cys81 in the folding of human lysozyme protein in yeast. A mutant protein (C81A), in which Cys81 was replaced with Ala, had almost the same enzymatic activity and conformation as those of the native enzyme. On the other hand, another mutant (C65A), in which Cys65 was replaced with Ala, was not found to fold correctly. These results indicate that Cys81 is not a requisite for both correct folding and activity, whereas Cys65 is indispensable. The mutant protein C81A is seen to contain a new, non-native disulfide bond at Cys65-Cys77. The possible occurrence of disulfide bond interchange during our mapping experiments cannot be ruled out by the experimental techniques presently available, but characterization of other mutant proteins and computer analysis suggest that the intramolecular exchange of disulfide bonds is present in the folding pathway of human lysozyme in vivo.     

Requirement of Cysteines and Length of the Human Respiratory Syncytial Virus M2-1 Protein for Protein Function and Virus Viability

The M2-1 protein of human respiratory syncytial virus (hRSV) promotes processive RNA synthesis and readthrough at RSV gene junctions. It contains four highly conserved cysteines, three of which are located in the Cys(3)-His(1) motif at the N terminus of M2-1. Each of the four cysteines, at positions 7, 15, 21, and 96, in the M2-1 protein of hRSV A2 strain was individually replaced by glycines. When tested in an RSV minigenome replicon system using beta-galactosidase as a reporter gene, C7G, C15G, and C21G located in the Cys(3)-His(1) motif showed a significant reduction in processive RNA synthesis compared to wild-type (wt) M2-1. C96G, which lies outside the Cys(3)-His(1) motif, was fully functional in supporting processive RNA synthesis in vitro. Each of these cysteine substitutions was introduced into an infectious antigenomic cDNA clone derived from hRSV A2 strain. Except for C96G, which resulted in a viable virus, no viruses were recovered with mutations in the Cys(3)-His(1) motif. This indicates that the Cys(3)-His(1) motif is critical for M2-1 function and for RSV replication. The functional requirement of the C terminus of the M2-1 protein was examined by engineering premature stop codons that caused truncations of 17, 46, or 67 amino acids from the C terminus. A deletion of 46 or 67 amino acids abolished the synthesis of full-length beta-galactosidase mRNA and did not result in the recovery of viable viruses. However, a deletion of 17 amino acids from the C terminus of M2-1 reduced processive RNA synthesis in vitro and was well tolerated by RSV. Relocation of the M2-1 termination codon upstream of the M2-2 initiation codons did not significantly affect the expression of the M2-2 protein. Both rA2-Tr17 and rA2-C96G did not replicate as efficiently as wt rA2 in HEp-2 cells and was restricted in replication in the respiratory tracts of cotton rats.     

1.9 A x-ray study shows closed flap conformation in crystals of tethered HIV-1 PR

Three-dimensional structure of an asymmetrically mutated (C95M) tethered human immunodeficiency virus type 1 protease enzyme (HIV-1 PR) has been determined in an unliganded form using X-ray diffraction data to 1.9 A resolution. The structure, refined using X-PLOR to an R factor of 19.5%, is unexpectedly similar to the ligand-bound native enzyme, rather than to the ligand-free native enzyme. In particular, the two flaps in the tethered dimer are in a closed configuration. The environments around M95 and C1095 are identical, showing no structural effect of this asymmetric mutation at position 95. Oxidation of Cys1095 has been observed for the first time. There is one well-defined water molecule that hydrogen bonds to both carboxyl groups of the essential aspartic acids in the active site. Proteins 2001;43:57-64.     

Comparative studies on surface hydrophobicity of streptococcal strains of groups A, B, C, D and G

Cell surface hydrophobicity of group A, B, C, D and G streptococcal strains has been studied and compared in a new test based on the fact that the degree of bacterial aggregation in ammonium sulphate depends on amphiphilic surface antigens. M-positive group A strains showing good growth in normal human blood aggregated in the standard salt aggregation test at very low concentrations of ammonium sulphate, while M-negative strains, which were killed in normal human blood, usually aggregated at high salt concentrations. Agents such as 2 M-KSCN, 2 M-guanidine. HC1 or 2 M-urea decreased the aggregation of the M-positive strains in the salt aggregation test while non ionic detergents such as Tween 20 (1%, w/v) and ethylene glycol (2 M) did not affect cell aggregation. Binding of fibrinogen and albumin resulted in a decrease of surface hydrophobicity of the group A M-positive strains. Group B strains possess a hydrophilic surface character and did not aggregate, while group C and G strains behaved in the salt aggregation test like M-negative strains of group A streptococci. Group D strains did not aggregate even at high ammonium salt concentrations. The results are discussed in relation to the influence of lipoteichoic acid and other surface antigens on strains of the various groups, and it is suggested that M protein and possibly also other surface proteins contribute to the high surface hydrophobicity of group A strains.     

The iron-sulfur center of biotin synthase: site-directed mutants

Biotin synthase contains an essential [4Fe-4S]+ cluster that is thought to provide an electron for the cleavage of S-adenosylmethionine, a cofactor required for biotin formation. The conserved cysteine residues Cys53, Cys57 and Cys60 have been proposed as ligands to the [4Fe-4S] cluster. These residues belong to a C-X3-C-X2-C motif which is also found in pyruvate formate lyase-activating enzyme, lysine 2,3-aminomutase and the anaerobic ribonucleotide reductase-activating component. To investigate the role of the cysteine residues, Cys-->Ala mutants of the eight cysteine residues of Escherichia coli biotin synthase were prepared and assayed for activity. Our results show that six cysteines are important for biotin formation. Only two mutant proteins, C276A and C288A, closely resembled the wild-type protein, indicating that the corresponding cysteines are not involved in iron chelation and biotin formation. The six other mutant proteins, C53A, C57A, C60A, C97A, C128A and C188A, were inactive but capable of assembling a [4Fe-4S] cluster, as shown by M?ssbauer spectroscopy. The C53A, C57A and C60A mutant proteins are unique in that their cluster could not undergo reduction to the [4Fe-4S]+ state, as shown by EPR and M?ssbauer spectroscopy. On this basis and by analogy with pyruvate formate lyase-activating enzyme and the anaerobic ribonucleotide reductase-activating component, it is suggested that the corresponding cysteines coordinate the cluster even though one cannot fully exclude the possibility that other cysteines play that role as well. Therefore it appears that for activity biotin synthase absolutely requires cysteines that are not involved in iron chelation.     

The mechanism of high Mr thioredoxin reductase from Drosophila melanogaster

Drosophila melanogaster thioredoxin reductase-1 (DmTrxR-1) is a key flavoenzyme in dipteran insects, where it substitutes for glutathione reductase. DmTrxR-1 belongs to the family of dimeric, high Mr thioredoxin reductases, which catalyze reduction of thioredoxin by NADPH. Thioredoxin reductase has an N-terminal redox-active disulfide (Cys57-Cys62) adjacent to the flavin and a redox-active C-terminal cysteine pair (Cys489'-Cys490' in the other subunit) that transfer electrons from Cys57-Cys62 to the substrate thioredoxin. Cys489'-Cys490' functions similarly to Cys495-Sec496 (Sec = selenocysteine) and Cys535-XXXX-Cys540 in human and parasite Plasmodium falciparum enzymes, but a catalytic redox center formed by adjacent Cys residues, as observed in DmTrxR-1, is unprecedented. Our data show, for the first time in a high Mr TrxR, that DmTrxR-1 oscillates between the 2-electron reduced state, EH2, and the 4-electron state, EH4, in catalysis, after the initial priming reduction of the oxidized enzyme (Eox) to EH2. The reductive half-reaction consumes 2 eq of NADPH in two observable steps to produce EH4. The first equivalent yields a FADH--NADP+ charge-transfer complex that reduces the adjacent disulfide to form a thiolate-flavin charge-transfer complex. EH4 reacts with thioredoxin rapidly to produce EH2. In contrast, Eox formation is slow and incomplete; thus, EH2 of wild-type cannot reduce thioredoxin at catalytically competent rates. Mutants lacking the C-terminal redox center, C489S, C490S, and C489S/C490S, are incapable of reducing thioredoxin and can only be reduced to EH2 forms. Additional data suggest that Cys57 attacks Cys490' in the interchange reaction between the N-terminal dithiol and the C-terminal disulfide.     

Recombinant human [Cys281]insulin-like growth factor-binding protein 2 inhibits both basal and insulin-like growth factor I-stimulated proliferation and collagen synthesis in fetal rat calvariae.

It is recognized that insulin-like growth factors (IGFs) are bound to specific high-affinity insulin-like growth factor-binding proteins (IGFBPs). The role of IGFBPs in bone metabolism is not well established. The effect of recombinant human [Cys281]IGFBP-2 ([Cys281]rhIGFBP-2) on bone formation in 21-day-old fetal rat calvariae was investigated. [Cys281]rhIGFBP-2 was expressed in and purified from conditioned medium of a clonal Chinese hamster ovary cell line. IGF-I-stimulated cell proliferation was inhibited dose dependently by [Cys281]rhIGFBP-2, with half-maximal inhibition observed at 2 x 10(-8) M. Suppression of the IGF-I-stimulated DNA synthesis was observed at an apparent dose ratio of 1:10. [Cys281]rhIGFBP-2 (10(-6) M) also inhibited the basal incorporation of [3H]thymidine into DNA by up to 45%. Insulin-stimulated cell proliferation was not affected in the presence of the binding protein. In addition, [Cys281]rhIGFBP-2 inhibited bone collagen synthesis under basal and IGF-I-stimulated conditions. In contrast, [Cys281]rhIGFBP-2 did not alter the parathyroid hormone-stimulated bone cell proliferation rate. In conclusion, binding of hIGF-I to rhIGFBP-2 results in an inhibition of the actions of free IGF-I on bone cell replication and matrix synthesis. Parathyroid hormone-stimulated cell proliferation is not mediated by an increase in free IGFs.     

Loss of ectodomain shedding due to mutations in the metalloprotease and cysteine-rich/disintegrin domains of the tumor necrosis factor-alpha converting enzyme (TACE)

Tumor necrosis factor-alpha converting enzyme (TACE), a multidomain protease essential for development and disease, releases the ectodomains from many transmembrane proteins in a regulated fashion. To understand the mechanism underlying the regulation of TACE activity, we sought to identify the cause of ectodomain shedding deficiencies in two mutated CHO sublines designated M1 and M2. Transfection of expression vectors for human and mouse TACE restored ectodomain shedding of TNF-alpha and TGF-alpha, suggesting that defects in the TACE gene contribute to the phenotype of M1 and M2 cells. The overall levels of endogenous TACE forms in M1 cells were significantly lower than those found in their parental cells, whereas only TACE zymogen, but not its mature form, was detectable in M2 cells. Molecular analyses suggested that M1 cells contained only one expressible TACE allele encoding an M435I point mutation in the catalytic center of the protease, and M2 cells produced two TACE variants with distinct point mutations in the catalytic domain (C225Y) and the cysteinerich/disintegrin domain (C600Y). Overexpression of the C225Y and C600Y TACE by transient transfection largely compensated for maturation defects in the variants but failed to restore TNF-alpha and TGF-alpha release in the shedding-defective CHO cell lines and fibroblasts derived from TACE-null mouse embryo. Further mutagenesis and functional analyses demonstrated that Cys(600) was absolutely essential for ectodomain shedding, suggesting that Cys(600), similar to Cys(225), participates in disulfide bonding, which is critical for both the processing and catalysis of TACE.     

Disulfide bond rearrangement during formation of the chorionic gonadotropin beta-subunit cystine knot in vivo

The intracellular kinetic folding pathway of the human chorionic gonadotropin beta-subunit (hCG-beta) reveals the presence of a disulfide between Cys residues 38-57 that is not detected by X-ray analysis of secreted hCG-beta. This led us to propose that disulfide rearrangement is an essential feature of cystine knot formation during CG-beta folding. To test this, we used disulfide bond formation to monitor progression of intracellular folding intermediates of a previously uncharacterized protein, the CG-beta subunit of cynomolgous macaque (Macaca fascicularis). Like its human counterpart hCG-beta with which it shares 81% identity, macaque (m)CG-beta is a cystine knot-containing subunit that assembles with an alpha-subunit common to all glycoprotein hormone members of its species to form a biologically active heterodimer, mCG, which, like hCG, is required for pregnancy maintenance. An early mCG-beta folding intermediate, mpbeta1, contained two disulfide bonds, one between Cys34 and Cys88 and the other between Cys38 and Cys57. The subsequent folding intermediate, mpbeta2-early, was represented by an ensemble of folding forms that, in addition to the two disulfides mentioned above, included disulfide linkages between Cys9 and Cys57 and between Cys38 and Cys90. These latter two disulfides are those contained within the beta-subunit cystine knot and reveal that a disulfide exchange occurred during the mpbeta2-early folding step leading to formation of the mCG-beta knot. Thus, while defining the intracellular kinetic protein folding pathway of a monkey homologue of CG-beta, we detected the previously predicted disulfide exchange event crucial for CG-beta cystine knot formation and attainment of CG-beta assembly competence.     

NM23-H1 tumor suppressor physically interacts with serine-threonine kinase receptor-associated protein, a transforming growth factor-beta (TGF-beta) receptor-interacting protein, and negatively regulates TGF-beta signaling

NM23-H1 is a member of the NM23/NDP kinase gene family and a putative metastasis suppressor. Previously, a screen for NM23-H1-interacting proteins that could potentially modulate its activity identified serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor (TGF)-beta receptor-interacting protein. Through the use of cysteine to serine amino acid substitution mutants of NM23-H1 (C4S, C109S, and C145S) and STRAP (C152S, C270S, and C152S/C270S), we demonstrated that the association between these two proteins is dependent on Cys(145) of NM23-H1 and Cys(152) and Cys(270) of STRAP but did not appear to involve Cys(4) and Cys(109) of NM23-H1, suggesting that a disulfide linkage involving Cys(145) of NM23-H1 and Cys(152) or Cys(270) of STRAP mediates complex formation. The interaction was dependent on the presence of dithiothreitol or beta-mercaptoethanol but not H(2)O(2). Ectopic expression of wild-type NM23-H1, but not NM23-H1(C145S), negatively regulated TGF-beta signaling in a dose-dependent manner, enhanced stable association between the TGF-beta receptor and Smad7, and prevented nuclear translocation of Smad3. Similarly, wild-type NM23-H1 inhibited TGF-beta-induced apoptosis and growth inhibition, whereas NM23-H1(C145S) had no effect. Knockdown of NM23-H1 by small interfering RNA stimulated TGF-beta signaling. Coexpression of wild-type STRAP, but not STRAP(C152S/C270S), significantly stimulated NM23-H1-induced growth of HaCaT cells. These results suggest that the direct interaction of NM23-H1 and STRAP is important for the regulation of TGF-beta-dependent biological activity as well as NM23-H1 activity.     

血清RBP,Hcy,CysC及尿m ALB水平在糖尿病肾病早期诊断中的临床应用

目的:研究血清视黄醇结合蛋白(RBP)、同型半胱氨酸(Hcy)、胱抑素C(Cys C)及尿微量白蛋白(m ALB)水平在糖尿病肾病早期诊断中的临床价值,为临床诊疗提供依据。方法:选取2014年6月到2016年2月我院收治的2型糖尿病患者138例,根据24小时尿蛋白排泄率将患者分为单纯糖尿病(A组65例),早期糖尿病肾病(B组73例),另选取同期健康体检者65例为对照组,检测各组血清中RBP、Hcy、Cys C及尿m ALB水平。结果:B组RBP、Hcy、Cys C及尿m ALB水平均显著高于A组和对照组,比较差异具有统计学意义(P0.05),A组RBP、Hcy、Cys C显著高于对照组,比较差异具有统计学意义(P0.05);RBP、Hcy、Cys C及尿m ALB联合检测阳性率显著高于单独检测(P0.05)。结论:早期糖尿病肾病患者血清RBP、Hcy、Cys C及尿m ALB水平显著上升,联合检测能提高检测阳性率。     

Oligomerization of Cu,Zn-Superoxide Dismutase (SOD1) by Docosahexaenoic Acid and Its Hydroperoxides In Vitro: Aggregation Dependence on Fatty Acid Unsaturation and Thiols

Docosahexaenoic acid (C22:6, n-3, DHA) is a polyunsaturated fatty acid highly enriched in the brain. This fatty acid can be easily oxidized yielding hydroperoxides as primary products. Cu, Zn-Superoxide dismutase (SOD1) aggregation is a common hallmark of Amyotrophic Lateral Sclerosis (ALS) and the molecular mechanisms behind their formation are not completely understood. Here we investigated the effect of DHA and its hydroperoxides (DHAOOH) on human SOD1 oligomerization in vitro. DHA induced the formation of high-molecular-weight (HMW) SOD1 species (>700 kDa). Aggregation was dependent on free thiols and occurred primarily with the protein in its apo-form. SOD1 incubation with DHA was accompanied by changes in protein structure leading to exposure of protein hydrophobic patches and formation of non-amyloid aggregates. Site-directed mutagenesis studies demonstrated that Cys 6 and Cys 111 in wild-type and Cys 6 in ALS-linked G93A mutant are required for aggregation. In contrast, DHAOOH did not induce HMW species formation but promoted abnormal covalent dimerization of apo-SOD1 that was resistant to SDS and thiol reductants. Overall, our data demonstrate that DHA and DHAOOH induce distinct types of apo-SOD1 oligomerization leading to the formation of HMW and low-molecular-weight species, respectively.     

Reaction of oxidized dopamine with endogenous cysteine residues in the human dopamine transporter

There is evidence to suggest that dopamine (DA) oxidizes to form dopamine ortho-quinone (DAQ), which binds covalently to nucleophilic sulfhydryl groups on protein cysteinyl residues. This reaction has been shown to inhibit dopamine uptake, as well as other biological processes. We have identified specific cysteine residues in the human dopamine transporter (hDAT) that are modified by this electron-deficient substrate analog. DAQ reactivity was inferred from its effects on the binding of [(3)H]2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (beta-CFT) to hDAT cysteine mutant constructs. One construct, X5C, had four cysteines mutated to alanine and one to phenylalanine (Cys(90)A, Cys(135)A, C306A, C319F and Cys(342)A). In membrane preparations 1 mM DAQ did not affect [(3)H]beta-CFT binding to X5C hDAT, in contrast to its effect in wild-type hDAT in which it reduced the B:(max) value by more than half. Wild-type cysteines were substituted back into X5C, one at a time, and the ability of DAQ to inhibit [(3)H]beta-CFT binding was assessed. Reactivity of DAQ with Cys(90) increased the affinity of [(3)H]beta-CFT for the transporter, whereas reactivity with Cys(135) decreased the affinity of [(3)H]beta-CFT. DAQ did not change the K:(D) for [(3)H]beta-CFT binding to wild-type. The reactivity of DAQ at Cys(342) decreased B:(max) to the same degree as wild-type. The latter result suggests that Cys(342) is the wild-type residue most responsible for DAQ-induced inhibition of [(3)H]beta-CFT binding.     

Folding mechanism of mutant human lysozyme C77/95A with increased secretion efficiency in yeast.

A mutant human lysozyme C77/95A, in which Cys77 and Cys95 are replaced with alanine, has been characterized by 8-fold greater secretion in yeast (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967) and almost the same three-dimensional structure as wild-type human lysozyme (Inaka, K., Taniyama, Y., Kikuchi, M., Morikawa, K., and Matsushima, M. (1991) J. Biol. Chem. 266, 12599-12603). To clarify the molecular features of C77/95A and the reason for its increased secretion in yeast, the stabilities of the mutant C77/95A and the wild-type proteins were examined by guanidine hydrochloride denaturation, and the unfolding-refolding kinetics were determined from circular dichroism and fluorescence stopped-flow measurements. Equilibrium experiments showed that the delta G of unfolding of C77/95A in water was 5.8 kcal/mol less stable than that of the wild-type protein at pH 4.0 and 10 degrees C. The unfolding rate of C77/95A was 4 orders of magnitude faster than that of the wild-type protein whereas the two proteins shared similar refolding rates. The slowly refolding phase of the wild-type protein disappeared in C77/95A, indicating that the disulfide bond affects this phase. These observations show that the disulfide bond Cys77-Cys95 contributes to the stabilization of the folded form of human lysozyme by suppressing the unfolding rate and that the increase in the unfolding rate, or the disappearance of the slowly refolding phase in vitro, could correlate with the increase in secretion efficiency in vivo.     

Origin of the thiazole ring of camalexin, a phytoalexin from Arabidopsis thaliana.

The principal phytoalexin that accumulates in Arabidopsis thaliana after infection by fungi or bacteria is 3-thiazol-2'-yl-indole (camalexin). Detached noninoculated leaves of Arabidopsis and leaves inoculated with the fungus Cochliobolus carbonum were fed [35S]cysteine (Cys) and [35S]methionine. Inoculated leaves incorporated more than a 200-fold greater amount of radioactivity from [35S]Cys into camalexin, as compared with noninoculated leaves. The amount of radioactivity from [35S]Cys that was incorporated into camalexin from inoculated Arabidopsis leaves was 10-fold greater than the amount of radioactivity that was incorporated into camalexin from [35S]methionine. Additional labeling experiments were performed to determine whether other atoms of Cys are incorporated into camalexin. [14C]Cys and [35S]Cys were incorporated into camalexin with approximately the same efficiency. Cys labeled either with deuterium (D3-Cys[2,3,3]) or 13C and 15N ([U-13C,15N]Cys) was also fed to inoculated leaves of Arabidopsis; camalexin was analyzed by mass spectroscopic analysis. The average ratio of molecular ion intensities of 203/200 for [U-13C,15N]Cys-labeled camalexin was 4.22, as compared with 0.607 for the average 203/200 ratio for unlabeled camalexin. The mass fragment-ion intensity ratios of 60/58 (thiazole ring ion fragment) and 143/142 were also higher for [U-13C,15N]Cys-labeled camalexin, as compared with unlabeled camalexin. The 59/58 and 201/200 ratios were higher for D3-Cys-labeled camalexin as compared with unlabeled camalexin. These data are consistent with the predicted formation of the thiazole ring of camalexin from Cys.