The molecular pathway for the regulation of phosphoribulokinase by thioredoxin f

Phosphoribulokinase (PRK) is one of several plant enzymes that is regulated by thiol-disulfide exchange as mediated by thioredoxin, which contains spatially vicinal, redox-active cysteinyl residues. In an earlier study (Brandes, H. K., Larimer, F. W., Geck, M. K., Stringer, C. D., Schürmann, P., and Hartman, F. C. (1993) J. Biol. Chem. 268, 18411-18414), our laboratory identified Cys-46 of thioredoxin f (Trx), as opposed to the other candidate Cys-49, as the primary nucleophile that attacks the disulfide of target proteins. The goal of the present study was to identify which of the two redox-active cysteinyl residues of PRK (Cys-16 or Cys-55) is paired with Cys-46 of Trx in the interprotein disulfide intermediate of the overall oxidation-reduction pathway. Incubation of a mixture of the C16S mutant of PRK and the C49S mutant of Trx with Cu2+ results in covalent complex formation as detected by SDS-polyacrylamide gel electrophoresis. Complexation is fully reversible by dithiothreitol and is retarded by ligands for PRK. Under the same conditions, Cu2+ induces very little complex formation between the following pairs of mutants: C16S PRK/C46S Trx, C55S PRK/C49S Trx, and C55S PRK/C46S Trx. When either 5-thio-2-nitrobenzoate-derivatized C16S or C55S PRK, as mimics of the oxidized (disulfide) form of the enzyme, is mixed with C49S Trx, stable covalent complex formation occurs only with the C16S PRK. Thus, two independent approaches identify Cys-55 of PRK in the intermolecular disulfide pairing with Trx.     

Efficient expression of the gene for spinach phosphoribulokinase in Pichia pastoris and utilization of the recombinant enzyme to explore the role of regulatory cysteinyl residues by site-directed mutagenesis

Phosphoribulokinase (PRK), unique to photosynthetic organisms, is regulated in higher plants by thioredoxin-mediated thiol-disulfide exchange in a light-dependent manner. Prior attempts to overexpress the higher plant PRK gene in Escherichia coli for structure-function studies have been hampered by sensitivity of the recombinant protein to proteolysis as well as toxic effects of the protein on the host. To overcome these impediments, we have spliced the spinach PRK coding sequence immediately downstream from the AOX1 (alcohol oxidase) promoter of Pichia pastoris, displacing the chromosomal AOX1 gene. The PRK gene is now expressed, in response to methanol, at 4-6% of total soluble protein, without significant in vivo degradation of the recombinant enzyme. This recombinant spinach PRK is purified to homogeneity by successive anion-exchange and dye-affinity chromatography and is shown to be electrophoretically and kinetically indistinguishable from the authentic spinach counterpart. Site-specific replacement of all of PRK's cysteinyl residues (both individually and in combination) demonstrates a modest catalytically facilitative role for Cys-55 (one of the regulatory residues) and the lack of any catalytic role for Cys-16 (the other regulatory residue), Cys-244, or Cys-250. Mutants with seryl substitutions at position 55 display non-hyperbolic kinetics relative to the concentration of ribulose 5-phosphate. Sulfate restores hyperbolic kinetics and enhances kinase activity, presumably reflecting conformational differences between the position 55 mutants and wild-type enzyme. Catalytic competence of the C16S-C55S double mutant proves that mere loss of free sulfhydryl groups by oxidative regulation cannot account entirely for the accompanying total inactivation.     

Surface topography of phytochrome A deduced from specific chemical modification with iodoacetamide

Phytochromes are a photoreversible photochromic light switch for photomorphogenesis in plants. The molecular structure and functional mechanism of phytochromes are not fully understood. On the basis of complete mapping of total tryptic digest of the iodoacetamide-modified oat phytochrome A (phyA), the molecular surface topography of phyA was probed by specific chemical modification of cysteine residues with [14C]iodoacetamide. Under native conditions, only two cysteines (Cys-158 and Cys-311) of eleven half-cystines of the N-terminal chromophore binding domain were modified to a significant extent. In the C-terminal domain, six cysteine residues (Cys-715, Cys-774, Cys-809, Cys-869, Cys-961, Cys-995) were readily accessible to iodoacetamide. Among the reactive cysteine residues, only cysteine-311 displayed reactivity that was dependent on the photochromic form (Pr left arrow over right arrow Pfr) of the photoreceptor. Surprisingly, the modification of Cys-311 in the vicinity of the chromophore attachment site (Cys-321) did not have any detectable effect on spectral properties of phyA. Most of the cysteines of the N-terminal domain (Cys-83, Cys-175, Cys-291, Cys-370, Cys-386, Cys-445, Cys-506) are deeply buried in the core of the chromophore binding domain, as they can be modified only after denaturation of the chromoprotein. In the C-terminal domain, modification of only one cysteine residue (Cys-939) required protein denaturation. Since all 22 half-cystines can be modified with iodoacetamide without reduction of the chromoprotein, it follows that oat phyA does not have any disulfide bonds. We found that Cys-311, Cys-774, Cys-961, and Cys-995 could be easily partially oxidized under the conditions used for phytochrome isolation. The surface topography/conformation of oat phyA and its role in protein-protein recognition in phytochrome-mediated signal transduction are discussed in terms of the relative reactivity of cysteine residues.     

Reductase domain cysteines 1048 and 1114 are critical for catalytic activity of human endothelial cell nitric oxide synthase as probed by site-directed mutagenesis

We examined whether highly conserved cysteine residues in the reductase domain of the constitutive isoform of nitric oxide synthase in human endothelial cells (ecNOS) are crucial for catalytic activity of the enzyme. Substitution of alanine for cysteines 976 (Cys-976), 991 (Cys-991), 1048 (Cys-1048), or 1114 (Cys-1114), located in the reductase domain of human ecNOS, was achieved by oligonucleotide-directed mutagenesis and expression in COS-7 cells. The specific activity of ecNOS was > 7-fold increased in wild-type and in mutants Cys-976 and Cys-991, but not in mutants Cys-1048 and Cys-1114. However, Western blot analysis indicated that expression of ecNOS protein was comparable in wild-type and in all mutants. NADPH concentration-dependent L-citrulline formation and NADPH oxidation during L-arginine metabolism were reduced in mutants Cys-1048 and Cys-1114 compared to wild-type. Similarly, NADPH cytochrome c reductase activity was increased in a time-dependent fashion in wild-type but not in mutants Cys-1048 and Cys-1114. These results indicate that Cys-1048 and Cys-1114 residues in the NADPH binding site of the reductase domain are critical for human ecNOS activity. The lack of utilization of NADPH in L-arginine metabolism and in cytochrome c reduction suggests that these active site cysteine residues may be responsible for binding of NADPH and/or for electron transfer in human ecNOS.     

Photo-cross-linking of rabbit skeletal troponin I deletion mutants with troponin C and its thiol mutants: the inhibitory region enhances binding of troponin I fragments to troponin C

Contraction of vertebrate striated muscle is regulated by the strong Ca(2+)-dependent interaction between troponin I (TnI) and troponin C (TnC). To critically evaluate this interaction, we generated four recombinant deletion fragments of rabbit fast skeletal TnI: the NH2-terminal fragment (TnI1-94), the NH2 terminus and the inhibitory region (TnI1-120), the inhibitory region and the COOH terminus (TnI96-181), and the COOH-terminal fragment (TnI122-181) containing amino acid residues 1-94, 1-120, 96-181, and 122-181, respectively. Native TnC and seven thiol mutants, containing single cysteine residues in the two globular domains and in the central helix of TnC, e.g., Cys-12, Cys-21, Cys-57, Cys-89, Cys-122, Cys-133, and Cys-158, were labeled with 4-maleimidobenzophenone, and their interaction with the recombinant TnI fragments and the synthetic inhibitory peptide (TnI98-114, residues 98-114) was studied by photo-cross-linking. Extensive cross-linking occurred between various domains of TnC and TnI. The cross-linking patterns (a) showed that both NH2- and COOH-terminal fragments of TnI are accessible to both of the globular domains of TnC, (b) indicated that linkage of the NH2- and COOH-terminal sequences to the inhibitory region of TnI (TnIir) caused marked enhancement of cross-linking with native TnC and all seven thiol mutants, and (c) identified the region in TnC where TnIir binds as that containing residues 98, 133, 158, and 57. Thus, the results suggest that TnI and TnC may adopt flexible and dynamic conformations in which multiple interactions involving various domains of the two polypeptides occur and TnIir acting as a linker facilitates these interactions. The interaction of TnI and its fragments with actin, TnC, and TnT, considered together with the biological activity indicates that residues 96-120 represent a key structural and functional region of TnI. Whereas the NH2-terminal region of TnI stabilizes binding to TnC and TnT, the COOH-terminal region stabilizes TnC and actin binding.     

Inhibition of smooth muscle actomyosin ATPase by caldesmon is associated with caldesmon-induced conformational changes in tropomyosin bound to actin

The smooth muscle tropomyosin isoforms beta and gamma were isolated in pure form and labeled with N-(1-pyrenyl)iodoacetamide (PIA) on the cysteine residues at either the N- or the C-terminal region (Cys-36 and Cys-190 of beta- and gamma-isoforms, respectively). The effect of caldesmon (CaD) on local conformational changes in different regions of the tropomyosin molecule was determined on the basis of changes in the excimer fluorescence (excited dimer of pyrene) formed in homodimers of tropomyosin isoforms. In the absence of actin, excimer fluorescence from the pyrene at Cys-190 of gamma-tropomyosin homodimer decreased in a simple manner on the addition of CaD, whereas the excimer from the Cys-36 of beta-tropomyosin homodimer exhibited a biphasic change, suggesting that additional weak binding sites exist near Cys-36. In the presence of actin, CaD-induced changes in the excimer fluorescence of pyrene-tropomyosin were observed only with Cys-36, and this change was associated with an inhibition of actin-activated myosin ATPase. A competition study with unlabeled tropomyosin isoforms indicated that the different excimer changes exhibited by beta- and gamma-tropomyosin in the presence of CaD were due to conformational changes in different regions of the tropomyosin molecule and not to differences in their affinities for CaD. Experiments with recombinant CaD mutants derived using the baculovirus expression system showed that the inhibition of tropomyosin potentiation of actomyosin ATPase by CaD requires the regions between residues 728-756 and 718-727 on the CaD molecule, although the latter region was sufficient for direct interaction with tropomyosin.     

Functional analysis of disulfide linkages clustered within the amino terminus of human apolipoprotein B

We tested the involvement of N-terminal six disulfide bonds (Cys-1 through Cys-12) of human apolipoprotein (apo) B in the assembly and secretion of lipoproteins using two C-terminal-truncated apoB variants, namely B50 and B18. In transfected rat hepatoma McA-RH7777 cells, B50 could assemble very low density lipoproteins (VLDL), and B18 was secreted as high density lipoproteins. When all 12 cysteine residues were substituted with alanines in B50, the mutant protein (B50C1-12) lost its ability to assemble lipid and was degraded intracellularly. However, mutation had no effect on B50C1-12 translation or translocation across the microsomal membrane. Post-translational degradation of B50C1-12 was partially inhibited by the proteasome inhibitor MG132. To determine which cysteines were critical in VLDL assembly and secretion, we prepared three additional mutant B50s, each containing four selected Cys-to-Ala substitutions in tandem (i.e. Cys-1 to Cys-4, Cys-5 to Cys-8, and Cys-9 to Cys-12). Expression of these mutants showed that disruption of disulfide bond formation within Cys-5 to Cys-8 diminished apoB secretion, whereas within Cys-1 to Cys-4 or Cys-9 to Cys-12 had lesser or no effect. In another two mutants in which only one disulfide bond (i.e. between Cys-5 and Cys-6 or between Cys-7 and Cys-8) was eliminated, only secretion of B50 with mutations at Cys-7 and Cys-8 was decreased. Thus, the disulfide bond involving Cys-7 and Cys-8 is most important for VLDL assembly and secretion. In addition, assembly and secretion of VLDL containing endogenous B100 or B48 were impaired in cells transfected with B50s containing Cys-7 and Cys-8 mutation. The Cys-to-Ala substitution abolished recognition of B50 by MB19, a conformational antibody with an epitope at the N terminus of human apoB. The Cys-to-Ala substitution also attenuated secretion of B18, but the effect of the mutation on B18 secretion was less evident than on B50.     

Thiolation of the gammaB-crystallins in intact bovine lens exposed to hydrogen peroxide

Oxidative damage of the lens causes disulfide bonds between cysteinyl residues of lens proteins and thiols such as glutathione and cysteine, which may lead to cataract. The effect of H2O2 oxidation was determined by comparing bovine lenses incubated with and without 30 mM H2O2. The H2O2 treatment decreased the glutathione and increased the protein-glutathione and protein-cysteine disulfides in the lens. The molecular mass of the gammaB-crystallin isolated from lenses, not treated with H2O2, agreed with the published sequence (Mr 20,966). Some lenses also had a less abundant gammaB-crystallin component 305 Da higher (Mr 21,270), suggesting the presence of a glutathione adduct. The gammaB-crystallins from H2O2 treated lenses had three components, the major one with one GSH adduct, another one with the mass of unmodified gammaB-crystallin, and a third with a mass consistent with addition of two GSH adducts. Mass spectrometric analysis of tryptic peptides of gammaB-crystallins from different lenses indicated that the +305 Da modifications were not at a specific cysteine. For the lenses incubated without H2O2, there was evidence of adducts at Cys-41 and in peptide 10-31, which includes 3 cysteines. Analysis of modified peptide 10-31 by tandem mass spectrometry showed GSH adducts at Cys-15, Cys-18, and Cys-22. In addition, gammaB-crystallins from H2O2-treated lenses had an adduct at Cys-109, partial oxidation at all 7 Met residues, and evidence for two disulfide bonds.     

Regulation of the cyanide-resistant alternative oxidase of plant mitochondria. Identification of the cysteine residue involved in alpha-keto acid stimulation and intersubunit disulfide bond formation

The cyanide-resistant alternative oxidase of plant mitochondria is a homodimeric protein whose activity can be regulated by a redox-sensitive intersubunit sulfhydryl/disulfide system and by alpha-keto acids. After determining that the Arabidopsis alternative oxidase possesses the redox-sensitive sulfhydryl/disulfide system, site-directed mutagenesis of an Arabidopsis cDNA clone was used to individually change the two conserved Cys residues, Cys-128 and Cys-78, to Ala. Using diamide oxidation and chemical cross-linking of the protein expressed in Escherichia coli, Cys-78 was shown to be: 1) the Cys residue involved in the sulfhydryl/disulfide system; and 2) not required for subunit dimerization. The C128A mutant was stimulated by pyruvate, while the C78A mutant protein had little activity and displayed no stimulation by pyruvate. Mutating Cys-78 to Glu produced an active enzyme which was insensitive to pyruvate, consistent with alpha-keto acid activation occurring through a thiohemiacetal. These results indicate that Cys-78 serves as both the regulatory sulfhydryl/disulfide and the site of activation by alpha-keto acids. In light of these results, the previously observed effects of sulfhydryl reagents on the alternative oxidase of isolated soybean mitochondria were re-examined and were found to be in agreement with a single sulfhydryl residue being the site both of alpha-keto acid activation and of the regulatory sulfhydryl/disulfide system.     

Molecular interaction of Agouti protein and Agouti-related protein with human melanocortin receptors

Agouti protein and the Agouti-related protein (AGRP) are antagonists of the melanocortin-3 receptor and melanocortin-4 receptor. Both proteins contain 10 cysteines in the C-terminal domain arranged in five disulfide bonds. One possible arrangement of the disulfide bonds predicts an octapeptide loop, and the chemical properties of four residues within this loop (residues 111-114 in human AGRP) bear striking resemblance to those of several melanocortin peptides, including alpha-MSH, MT-II, and SHU-9119. We showed that cyclic synthetic octapeptides based on the sequence of this loop from Agouti protein or human AGRP are functional antagonists of the human melanocortin-4 receptor. All peptides had a lower affinity for the melanocortin-3 receptor than for the melanocortin-4 receptor. Substitution of serines for cysteines resulted in linear peptides which had reduced binding affinities for both receptors. Mutational analysis of human AGRP indicated that its C-terminal domain is functionally equivalent to the intact human AGRP. The RFF111-113 triplet appears to be the most critical portion of AGRP in determining the binding affinity for both melanocortin-3 and melanocortin-4 receptors. These data strongly suggest that the loop defined by Cys-110 and Cys-117 is critical in determining the antagonist activity of human AGRP. Our data provide indirect evidence for the suggestion that the Cys-110 to Cys-117 octapeptide loop of human AGRP mimics the conformation of alpha-MSH, MT-II, and SHU-9119.     

Protons activate brain Na+ channel 1 by inducing a conformational change that exposes a residue associated with neurodegeneration

BNC1 is a mammalian neuronal cation channel in the novel DEG/ENaC ion channel family. BNC1 channels are transiently activated by extracellular protons and are constitutively activated by insertion of large residues, such as valine, in place of Gly-430; residue 430 is a site where analogous mutations in some Caenorhabditis elegans family members cause a swelling neurodegeneration. Mutation of Gly-430 to a small amino acid, cysteine, neither generated constitutive currents nor allowed modification of this residue by sulfhydryl-reactive methanethiosulfonate (MTS) compounds. However, when protons activated the channel, Cys-430 became accessible to extracellular MTS reagents, which modified Cys-430 to generate constitutive currents. Fluorescent MTS reagents also labeled Cys-430 in activated channels. These data indicate that protons induce a reversible conformational change that activates BNC1 thereby exposing residue 430 to the extracellular solution. Once Cys-430 is modified with a large chemical group, the channel is prevented from relaxing back to the inactive state. These results link ligand-dependent activation and activation by mutations that cause neurodegeneration.     

Mutational analysis of the pea phytochrome A chromophore pocket: chromophore assembly with apophytochrome A and photoreversibility

Ten site-specific mutants of pea apophytochrome A were expressed in Saccharomyces cerevisiae and analyzed for chromophore assembly with apoprotein and photoreversible absorbance changes. The mutants constitute two specific changes for each of five conserved amino acid residues located in the microenvironment of the chromophore attachment residue, which is Cys-323 in pea phytochrome A. All mutant apophytochromes were autocatalytically able to covalently attach phycocyanobilin, indicating that there were no major structural perturbations in the apoproteins. However, the rate of chromophore ligation varied significantly among the mutants. Spectrally, the mutant holophytochromes are of three types: mutant phytochromes that are indistinguishable from the wild-type adduct, mutants with blue-shifted Pr and Pfr absorption maxima compared to the wild-type adduct, and mutants that are not photoreversible. From an analysis of the results, we concluded that the residues Asp-309, Arg-318, His-321, and Gln-326 are probably not catalytically involved in the chromophore ligation reaction, but some residues may play significant structural and stereochemical roles. Arg-318 might anchor the chromophore, as has been suggested [Partis, M. D., & Grimm, R. (1990) Z. Naturforsch, 45c, 987-998; Parker, W., et al. (1993) Bioconjugate Chem. (in press)]. The conserved Gln-326, three residues downstream from the chromophore attachment site, is not electrostatically critical for the spectral integrity and photoreversibility of phytochrome, but this residue is sterically important to the lyase activity. It appears that the role of the five amino acid residues in the N- and C-terminal vicinities of the chromophore binding Cys-323 is structural rather than catalytic for the ligation reaction.     

Studies on an affinity label for the sulfuryl acceptor binding site in an aryl sulfotransferase

Active site-directed affinity labeling was utilized to elucidate peptide sequences at the binding site for sulfuryl acceptors in rat hepatic aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase, EC 2.8.2.9). The affinity labeling reagent, N-bromoacetyl-4-hydroxyphenylamine, was designed on the basis of substrate specificity studies with para-substituted phenols, utilization of a bromoacetamido group for reactivity with active site amino acid residues and its similarity to acetaminophen, a known substrate for aryl (phenol) sulfotransferases. AST IV utilized N-bromoacetyl-4-hydroxyphenylamine as a substrate with kinetic constants that compared favorably to those obtained with acetaminophen. Incubation of AST IV with N-bromoacetyl-4-hydroxyphenylamine at pH 7.0 in the absence of PAPS and other substrates resulted in an irreversible inactivation of the enzyme that was both time- and concentration-dependent. [14C]-N-bromoacetyl-4-hydroxyphenylamine was synthesized and used to analyze the regions of protein sequence that were involved in the binding of the affinity label. AST IV was incubated with [14C]-N-bromoacetyl-4-hydroxyphenylamine, hydrolyzed with endoproteinase Lys-C and the labeled peptides were purified by HPLC. Control incubations of AST IV with the affinity label in the presence of 4-propylphenol and PAP were utilized to ascertain the specificity of the interaction. Sequence analysis of the labeled peptides, carried out by automated Edman degradation, revealed labeling sites on cysteine (Cys-232, Cys-283 and Cys-289) and lysine (Lys-286) residues near the C-terminus of the protein. The locations of these labeling sites were further evaluated both by sequence-alignment with other sulfotransferases and by theoretical calculations on predicted secondary structure.     

HAH1 is a copper-binding protein with distinct amino acid residues mediating copper homeostasis and antioxidant defense

HAH1 is a 68-amino acid protein originally identified as a human homologue of Atx1p, a multi-copy suppressor of oxidative injury in sod1 delta yeast. Molecular modeling of HAH1 predicts a protein structure of two alpha-helices overlaying a four-stranded antiparallel beta-sheet with a potential metal binding site involving two conserved cysteine residues. Consistent with this model, in vitro studies with recombinant HAH1 directly demonstrated binding of Cu(I), and site-directed mutagenesis identified these cysteine residues as copper ligands. Expression of wild type and mutant HAH1 in atx1 delta yeast revealed the essential role of these cysteine residues in copper trafficking to the secretory compartment in vivo, as expression of a Cys-12/Cys-15 double mutant abrogated copper incorporation into the multicopper oxidase Fet3p. In contrast, mutation of the highly conserved lysine residues in the carboxyl terminus of HAH1 had no effect on copper trafficking to the secretory pathway but eliminated the antioxidant function of HAH1 in sod1 delta yeast. Taken together, these data support the concept of a unique copper coordination environment in HAH1 that permits this protein to function as an intracellular copper chaperone mediating distinct biological processes in eucaryotic cells.     

His-8 lowers the pKa of the essential Cys-12 residue of the ArsC arsenate reductase of plasmid R773

The 141-residue ArsC arsenate reductase of plasmid R773 has an essential cysteine residue, Cys-12. The pKa of Cys-12 was determined to be 6.4, compared with a pKa of 8.3 for free cysteine. The possibility of the formation of an ion pair between Cys-12 and a basic residue was investigated. Enzymatic activity was rapidly inactivated by the histidine-modifying reagent diethylpyrocarbonate. The codons for the two histidine residues in ArsC, His-8 and His-88, were changed by site-directed mutagenesis. Cells expressing arsCH88R, arsCH88S, arsCH88W, or arsCH88V genes retained arsenate resistance, and the purified proteins had wild type level of reductase activity. Cells expressing arsCH8P, arsCH8S, arsCH8G, or arsCH8R genes were each sensitive to arsenate, and the purified H8P, H8G, and H8R proteins each lacked enzymatic activity. Using the single histidine proteins it was shown that both histidines react with diethylpyrocarbonate but that only reaction with His-8 resulted in inactivation. The pKa value of Cys-12 was determined to be 6.3 in the H8R enzyme and 8.3 in the H8G enzyme. These results indicate that His-8 is essential for catalytic activity and that a positively charged residue is required at position 8 to lower the pKa of the cysteine thiolate at position 12.     

Post-translational modifications of water-soluble human lens crystallins from young adults

Post-translational modifications of the water-soluble human lens crystallins from young adult donors were identified and located using electrospray ionization mass spectrometric analysis of the intact proteins and fast atom bombardment mass spectrometry of enzymatic digests. Peptides corresponding to all of the sequences of alpha A-, alpha B-, and beta B2-crystallins were found, permitting the entire sequences to be searched for modifications. The major portions of these three crystallins were not modified. Modifications of alpha A-crystallin that were detected included 2 phosphorylated Ser residues (1 of which appears to be unique to human lenses), deamidation at some Gln and Asn residues, a disulfide bond between Cys-131 and Cys-142, and loss of the COOH-terminal Ser residue. Three phosphorylated Ser residues, but no deamidation, were found in alpha B-crystallin. The molecular weights of neither the intact protein nor the peptides in the enzymatic digests indicated any post-translational modification of the principal beta-crystallin, beta B2. The molecular weights of the other beta- and gamma-crystallins for which sequences have been published suggested the presence of post-translational modifications or errors in the published sequences. Although enough peptides were found to establish the presence of specific proteins, peptides corresponding to all portions of these proteins were not found, and elucidation of these structures is not yet complete. This mass spectrometric characterization of the total water-soluble proteins from normal young adult lenses provides a reference data base for future investigations of the modifications present in aged and cataractous lenses.     

Fluorescence energy transfer of complexes of skeletal muscle troponin C and melittin derivatives

We studied Ca2+-dependent structural change of rabbit skeletal troponin C (TnC)-melittin (ME) complex as a model of TnC-troponin I complex. In previous study, we found that the distance between Met-25 and Cys-98 of TnC in TnC-ME complex increased upon binding of Ca2+ to TnC [H. Sano and T. Iio (1995) J. Biochem. 118, 996-1000]. In this study, we used a fluorescence energy transfer method. As a fluorescent donor, we used the tryptophan residue in four melittin derivatives, in which residue 2, 5, 8, or 13 was replaced with tryptophan. As acceptor, we used dansylaziridine (DANZ) bound to Met-25 of TnC, or N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS) bound to Cys-98 of TnC. For all TnCDANZ-ME complexes, the donor-acceptor distance (11.9-17.7 A) did not remarkably depend on Mg2+ or Ca2+ binding of TnC or on the position of tryptophan in ME derivatives. The same results were obtained for TnCAEDANS-ME complexes in the absence of Ca2+ (distance 15.2-21.7 A). But in the presence of Ca2+, tryptophan residues in the central region of ME were near to Cys-98 of TnC (distance much less than 10.4 A). Based on these results, we conclude that ME is enfolded by the N- and C-lobes of TnC, and the ME rod is almost perpendicular to a line connecting Met-25 and Cys-98 of TnC. The position of the ME rod shifts upon binding of Ca2+ to TnC.     

CD52 is the ''major maturation-associated'' sperm membrane antigen

A thromboxane A2 receptor cDNA was isolated from a human placenta library by polymerase chain reaction (PCR) and was expressed in insect (Sf21) cells using baculovirus system. The recombinant receptor exhibited [3H]-SQ29548 and [125I]-BOP binding activities with Kd values of 1.01 +/- 0.09 nM and 1.63 +/- 0.23 nM, respectively. The receptor binding activity was inhibited by dithiothreitol in a time- and concentration-dependent manner, indicating the involvement of disulfide linkage in ligand binding. The role of the four conserved cysteinyl residues in ligand binding was further examined by site-directed mutagenesis. Each of the four cysteinyl residues was respectively mutated to a serine residue. C102S, C105S, and C183S mutants exhibited no ligand binding activity although successful expression was achieved as revealed by immunoblot analysis, whereas C257S mutant retained most of the binding activity. Homology analysis of all prostanoid receptors indicates that Cys-105 (first extracellular loop) and Cys-183 (second extracellular loop) are conserved and are presumed to form a disulfide bond for receptor stability as suggested by the inhibition of ligand binding by dithiothreitol reduction. Loss of binding activity by C102S mutant revealed that the sulfhydryl group of Cys-102 must play an essential role in ligand binding. Molecular modeling proposed that the Ser-201 is involved in interacting with TXA2 by forming hydrogen bonding. Point mutations of both Ser-201 and a conserved Ser-255 did not affect the ligand binding specificity and affinity for [3H]-SQ29548, but have significantly altered Kd values for [125I]-BOP. These results indicate that various cysteinyl and serine residues of thromboxane A2 receptor may play different roles in ligand binding.     

Functional significance of the "signature cysteine" in helix 8 of the Escherichia coli 4-aminobutyrate transporter from the amine-polyamine-choline superfamily. Restoration of Cys-300 to the Cys-less Gabp

gab permease (GabP) is the exclusive mediator of 4-aminobutyrate (GABA) transport across the Escherichia coli plasma membrane. Helix 8 and a portion of the adjoining cytoplasmic region (loop 8-9) constitute the GabP "consensus amphipathic region" (CAR), a potential channel-forming domain that is found to be evolutionarily conserved within the APC (amine-polyamine-choline) transporter superfamily. Upon the polar surface of the CAR, all known gab permeases display a "signature cysteine" not found in other members of the APC superfamily, suggesting that discrete features within the CAR might play a role in imparting specificity (kcat/Km) to the translocation reaction. Here we show that among the five cysteine residues in the E. coli GabP, only Cys-300, the signature cysteine, can restore wild type properties to the Cys-less GabP mutant. We conclude (i) from partial reaction studies (equilibrium exchange, counterflow) that rapid translocation of the GABA binding site from one side of the membrane to the other is greatly facilitated by Cys-300 and (ii) from pharmacological studies that loss of Cys-300 has little effect on the affinity that GabP exhibits for a structurally diverse array (kojic amine, 5-aminovaleric acid, GABA, nipecotic acid, and cis-4-aminocrotonic acid) of competitive ligands. These results raise the possibility that other GABA transporters might rely analogously upon conserved cysteine residues positioned within the amphipathic helix 8 and loop 8-9 regions.     

Corneal reinnervation after photorefractive keratectomy and laser in situ keratomileusis: an in vivo study with a confocal videomicroscope

The purpose of this study was to compare the regeneration of corneal nerves after photorefractive keratectomy (PRK) versus laser in situ keratomileusis (LASIK) in vivo with a confocal videomicroscope. In all, 15 eyes that had undergone PRK and 15 eyes that had been subjected to LASIK were compared with a confocal in vivo slit-scanning video-microscope. The subepithelial nerves were observed preoperatively and at 3, 6, and 12 months postoperatively. In all eyes, good microscope images of the subepithelial nerve plexus could be obtained preoperatively. Because of postoperative light reflection and scattering in the treated area, subepithelial nerve-fiber regeneration could be followed satisfactorily only in seven eyes after PRK and in five eyes following LASIK. In the eyes treated with PRK, recovery of subepithelial reinnervation started from the margin of the ablation zone, being directed toward the center of the cornea. At 8 weeks postoperatively, rarefied subepithelial nerve fibers were visible at the edges, and after 3 months, single nonbranched nerve fibers could be visualized in the center of the ablation zone. At 6-8 months following PRK, subepithelial nerve regeneration seemed to be completed; however, abnormal branching and accessory thin nerve fibers were present without exception. After LASIK, corneal nerve-fiber regeneration followed the same course described for PRK except that regenerated subepithelial nerve fibers were barely visible in the center after 6 months. Further changes in nerve structure were visible for up to 12 months postoperatively. Recovery of corneal sensitivity in humans has been reported to start at 4-6 weeks after PRK and is said to be completed within 6-12 months of surgery. Slit-scanning videomicroscope findings were in accordance with these observations.