Proteomic analysis reveals successive aberrations in protein expression from healthy mucosa to invasive head and neck cancer.

Development of head and neck squamous cell carcinoma (HNSCC) is a multistep process and in many cases involves a phenomenon coined 'field cancerization'. In order to identify changes in protein expression occurring at different stages of tumorigenesis and field cancerization, we analysed 113 HNSCCs and 73 healthy, 99 tumor-distant and 18 tumor-adjacent squamous mucosae by SELDI-TOF-MS on IMAC30 ProteinChip Arrays. Forty-eight protein peaks were differentially expressed between healthy mucosa and HNSCC. Calgizarrin (S100A11), the Cystein proteinase inhibitor Cystatin A, Acyl-CoA-binding protein, Stratifin (14-3-3 sigma), Histone H4, alpha- and beta-Hemoglobin, a C-terminal fragment of beta-hemoglobin and the alpha-defensins 1-3 were identified by mass spectrometry. The alpha-defensins showed various alterations in expression as validated by immunohistochemistry (IHC). Supervised prediction analysis revealed excellent classification of healthy mucosa (94.5% correctly classified) and tumor samples (92.9% correctly classified). Application of this classifier to the tumor-adjacent and tumor-distant mucosa samples disclosed dramatic changes: only 59.6% of the tumor-distant biopsies were classified as normal, 27.3% were predicted as aberrant or HNSCC. Strikingly, 72% of the tumor-adjacent mucosae were predicted as aberrant. These data provide evidence for the existence of genetically altered fields with inconspicuous histology. Comparison of the protein profiles in the tumor-distant-samples with clinical outcome of 32 patients revealed a significant association between aberrant profiles with tumor relapse events (P=0.018; Fisher's exact test, two-tailed). We conclude that proteomic profiling in conjunction with protein identification greatly outperforms histopathological diagnosis and may have significant predictive power for clinical outcome and personalized risk assessment.

Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide. Evidence for a dithiol-disulfide equilibrium and implications for redox regulation.

Calcineurin (CaN) is a Ca2+-and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe-Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3-8 microM and the inactivation was reversed by 2, 3-dimercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration-dependent inactivation. Analysis of the free thiol content performed on the H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4-dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe3+-Zn2+ dinuclear centre. Upon H2O2-mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN.

Growth inhibitory factor and zinc affect neural cell cultures in a tissue specific manner.

Deficiency of neuronal growth inhibitory factor (GIF) and abnormalities in zinc homeostasis have been suggested to play a role in the neuropathogenesis of Alzheimer's disease. We report here that embryonic chick cerebral cell cultures zinc and copper containing GIF in the presence of marmoset hippocampal extract reduces significantly and concentration dependently mitochondrial succinate dehydrogenase activity (MTT) and cell mass. In contrast, no indications could be found that GIF affected neural retina cell cultures. Our results suggest that the observed effects of GIF are not elicited by zinc.

Structural characterization of Cu(I) and Zn(II) sites in neuronal-growth-inhibitory factor by extended X-ray absorption fine structure (EXAFS).

Neuronal-growth-inhibitory factor (GIF) is a metalloprotein specific to the central nervous system which has been linked to Alzheimer's disease. The high metal content, approximately seven metal atoms/protein molecule, and 70% sequence identity to mammalian metallothioneins (MT), including a preserved array of 20 cysteinyl residues, place GIF in the family of MT. In contrast to MT, native GIF isolated from human or bovine brain contains an unusual metal composition, viz. four Cu(I) and three Zn(II) per polypeptide chain. Cu and/or Zn K-edge X-ray absorption spectra have been recorded for native Cu, Zn-GIF, Zn-substituted GIF, and these metals bound to the 32-residue N-terminal domain, Cu4-, Cu6- or Zn3-GIF-(1-32) at 77 K. The results are consistent with the metals being bound to the protein by cysteinyl residues in every case. The Cu-S distance is approximately 2.25 A and the EXAFS is considered to be consistent with primarily trigonal coordination of the Cu(I); Cu...Cu backscattering is observed at approximately 2.67 A, indicative of the formation of Cu(x)(Scys)y clusters. Thus, the Cu(I) environment is similar to that observed in MT. This is also the case for Zn(II), with 4 S at approximately 2.34 A. However, in contrast to Zn-MT for Zn-substituted GIF and Zn3-GIF-(1-32), Zn...Zn backscattering is observed at approximately 3.28 A. The significance of these results are discussed with respect to the specific biological activity of GIF.

Modulation of DNA binding of a tramtrack zinc finger peptide by the metallothionein-thionein conjugate pair.

The ability of metallothionein (MT) to modulate DNA binding by a two-finger peptide of Tramtrack (TTK), a CCHH zinc transcription factor, was investigated using metal-bound and metal-deficient forms of rabbit MT-2 and the TTK peptide. Thionein inhibited DNA binding by zinc-bound TTK, and Zn-MT restored DNA-binding by zinc-deficient apo-TTK. "Free" zinc at low concentrations was as effective as Zn-MT in restoring DNA binding by apopeptide but was inhibitory at concentrations equal to zinc bound to 2 mol eq and higher of Zn-MT. Substitution of cadmium for zinc reduced the affinity of the peptide for its DNA binding site. This effect was reversed by incubation with Zn-MT. The circular dichroic spectra of the TTK peptide indicated that zinc removal resulted in loss of alpha-helical structures, which are sites of DNA contact points. Reconstitution with cadmium resulted in stoichiometric substitution of 2 mol of Cd/mol of peptide but not recovery of alpha-helical structures. Incubation of Cd-TTK with Zn-MT restored the secondary structure expected for zinc-bound TTK. The ability of Zn-MT and thionein to restore or inhibit DNA-binding by TTK was associated with effects on the metallation status of the peptide and related alterations in its secondary structure.

Structural and spectroscopic studies of azide complexes of horse heart myoglobin and the His-64-->Thr variant.

The high-resolution X-ray crystallographic structures of horse heart azidometmyoglobin complexes of the wild-type protein and the His-64-->Thr variant have been determined to 2.0 and 1.8 A respectively. Azide binds to wild-type metmyoglobin in a bent configuration with an Fe-N-1-N-3 angle of 119 degrees and is oriented into the distal crevice in the direction of Ile-107. The proximity of the His-64 NE2 atom to the N-1 atom of the bound azide indicates stabilization of the ligand by the His-64 side chain through hydrogen bonding. In addition, structural characterization of wild-type horse heart azidometmyoglobin establishes that the only structural change induced by ligand binding is a small movement of the Leu-29 side chain away from the azide ligand. EPR and Fourier transform infrared spectroscopy were used to characterize the myoglobin azide complexes further. EPR spectroscopy revealed that, in contrast with wild-type azidometmyoglobin, two slightly different low-spin species are formed by azide bound to the His-64-->Thr variant both in solution and in a polycrystalline sample. One of these low-spin species has a greater relative intensity, with g values very similar to those of the azide complex of the wild-type protein. These EPR results together with structural information on this variant indicate the presence of two distinct conformations of bound azide, with one form predominating. The major conformation is comparable to that formed by wild-type myoglobin in which azide is oriented into the distal crevice. In the minor conformation the azide is oriented towards the exterior of the protein.

Characterization of dimethylargininase from bovine brain: evidence for a zinc binding site.

Dimethylargininase (EC 3.5.3.18) is involved in the regulation of the levels of the natural occurring free arginine derivatives L-Nomega,Nomega-dimethylarginine and L-Nomega-methylarginine, which are reversible inhibitors of nitric oxide synthase. A dimethylargininase has been isolated from bovine brain tissue and was characterized by using immunological, kinetic, and spectroscopic techniques. Western blot analysis using polyclonal antibodies revealed that the enzyme is widely distributed in bovine with the highest relative concentrations found in brain and kidney tissue. A similar tissue distribution has also been reported for the other so far isolated dimethylargininase from rat kidney [Ogawa, T., Kimoto, M., and Sasaoka, K. (1989) J. Biol. Chem. 264, 10205-10209]. The bovine enzyme is a monomeric, globular protein (molecular mass approximately 31.2 kDa) containing one tightly bound Zn2+ ion, which can be removed by dialysis against 1,10-phenanthroline. The determination of kinetic constants for both the native (holo-protein) and the zinc-depleted (apo-protein) enzyme at 37 degrees ¿C established that the dimethylargininase is not a zinc hydrolase. The specific activity was 0.66 unit/mg for the holo-protein and 0.19 unit/mg for the apo-protein. The secondary structure determination of the native enzyme by circular dichroism revealed 41% alpha-helix and 32% beta-sheet and beta-turn structure. In the apo-enzyme, a small, but significant decrease in the alpha-helical content (5%) was observed, consistent with a marked decrease in enzymatic activity to 30%. Upon preincubation of both enzyme forms at 50 degrees C, only the holo-enzyme showed a residual enzymatic activity. In thermostability studies, a 7 degrees C lower apparent Tm value was observed for the apo-enzyme compared to the 66 degrees C for the holo-enzyme, suggesting that the zinc ion has a structure-stabilizing role. Besides the tightly bound zinc, additional Zn2+ ions inhibit the enzyme competitively with a Ki value of 2.0 microM. A possible interrelationship between dimethylargininase and nitric oxide synthase is discussed.

A myoglobin variant with a polar substitution in a conserved hydrophobic cluster in the heme binding pocket.

Well-ordered internal amino acids can contribute significantly to the stability of proteins. To investigate the importance of the hydrophobic packing interface between helices G and H in the proximal heme pocket of horse heart myoglobin, the highly conserved amino acid, Leu104, was substituted with asparagine, a polar amino acid of similar size. The Leu104Asn mutant protein and its recombinant wild-type horse heart myoglobin counterpart were expressed from synthetic genes in Escherichia coli. Thermal denaturation of these two recombinant myoglobins, as studied by measurement of circular dichroism ellipticity at 222 nm, revealed that the Leu104Asn mutant had a significantly lower t(m) (71.8 +/- 1 degree C, pH 7.0) than recombinant wild-type myoglobin (81.3 +/- 1 degree C, pH 7.0). To examine the extent to which this 10 degrees C decrease in thermal stability was associated with structural perturbations, X-ray diffraction techniques were used to determine the three-dimensional structures of both the recombinant wild-type and Leu104Asn myoglobins to 0.17 nm resolution. Refinement of these structures gave final crystallographic R-factors of 16.0% and 17.9%, respectively. Structural comparison of the natural and recombinant wild-type myoglobins, together with absorption spectroscopic and electron paramagnetic resonance (EPR) analyses, confirmed the proper expression and folding of the recombinant protein in E. coli. Surprisingly, despite the decreased thermal stability of the Leu104Asn mutant, there are no significant structural differences between the mutant and wild-type myoglobins. EPR and absorption spectroscopic analyses further confirmed the similar nature of the heme iron centres in both proteins. Thus, the introduction of an energetically unfavourable change in side chain polarity at position 104 into a hydrophobic environment that does not support the hydrogen bonding potential of the mutant asparagine appears to perturb important stabilizing helix-helix and heme-protein interactions. The induced structural destabilization is thereby reflected by a significant decrease in the t(m) of horse heart myoglobin.

Electron paramagnetic resonance of D-xylose isomerase: evidence for metal ion movement induced by binding of cyclic substrates and inhibitors.

The interactions of substrates and inhibitors with the Mn2+ ions in the binuclear active center of D-xylose isomerase (XylI) were investigated by EPR spectroscopy at X- and Q-band frequencies. The metal binding site 1 (A site) was specifically occupied with Mn2+ ions by blocking the high-affinity metal binding site 2 (B-site) either with Co2+ ions, resulting in a catalytically active enzyme, or with Cd2+ or Pb2+ ions yielding an inactive enzyme species. Incubation of both the Co2+/Mn2+- and the Cd2+/Mn2+-XylI with the acyclic inhibitor xylitol revealed EPR spectra with well-resolved hyperfine patterns, but with increased zero field splitting (zfs) parameter D compared to the spectra without inhibitor. D was estimated by spectral simulation of the central --1/2<-->1/2 fine structure transition. D values of 33 and 50 mT were obtained for the Co2+/Mn2+-XylI and the Cd2+/Mn2+-XylI samples, respectively. These results indicate direct interaction of the xylitol with the Mn2+ in the A-site. More drastic changes are observed with the substrates D-xylose and D-glucose and with the cyclic inhibitors 5-thio-alpha-D-glucose and 2-desoxy-D-glucose. For Cd2+/Mn2+-XylI, the EPR spectra with substrates and cyclic inhibitors are similar to each other but different from the spectra with the acylic inhibitor xylitol. They exhibit well-resolved line patterns with a relative large zero field splitting, which was estimated to be in the range of D = 65-85 mT in the various complexes. Binding of substrates or of cyclic inhibitors to the Co2+/ Mn2+-XylI yields EPR spectra without resolved hyperfine interactions, indicative of dipolar interaction between the two paramagnetic metal ions. This can be explained with a decrease in the metal-metal distance. Furthermore, the EPR data strongly suggest that the corresponding metal ion movement is induced by binding of the cyclic conformation of either substrates or cyclic inhibitors and not by binding of the extended form of the sugars.

Isolation and characterization of a novel monomeric zinc- and heme-containing protein from bovine brain.

An acidic zinc- and heme-containing protein was isolated from the soluble fraction of bovine brain and has been purified to homogeneity. The zinc-heme protein is a monomeric globular protein with a molecular mass of 31 200 Da as determined by electrospray mass spectrometry. The protein was isolated with 0.90 +/- 0.05 zinc per protein and with substoichiometric amounts of heme. Amino acid sequences of four peptides (ca. 20% of the protein) were determined and the comparison of these sequences with those of protein and gene sequence databases revealed no significant correlation with any known protein. Thus, it is concluded that it is a novel protein of currently unknown biological function.

Evidence for Cu(I) clusters and Zn(II) clusters in neuronal growth-inhibitory factor isolated from bovine brain.

Neuronal growth-inhibitory factor (GIF), a central-nervous-system-specific metallothionein-like protein, has been isolated by means of an improved isolation procedure from bovine brain. The native protein contains 4-5 Cu+ and 2-2.5 Zn2+, which results in an overall stoichiometry of 6-7 mol metal ions/mol protein. Native Cu, ZN-GIF and the Zn2+ -substituted and Cd2+-substituted metalloforms have been characterized by means of electronic-absorption, CD, magnetic-circular-dichroism (MCD) and low-temperature (77 K) Cu(I)-luminescence spectroscopy. Analysis of the metal-induced-charge-transfer transitions below 300 nm in the electronic-absorption and CD spectra of Cu, ZN-GIF revealed spectral features characteristic of metal-thiolate coordination. The presence of formally spin-forbidden 3d --> 4s Cu(I)-cluster-centered transitions, above 300 nm in the corresponding CD and MCD spectra indicate the existence of a Cu(I) cluster. The 77-K luminescence spectrum of Cu, ZN-GIF revealed two emissive bands at approximately 420 nm and 570 nm, which were reported also for CU4 clusters in mammalian Cu8-metallothionein. By analogy with Cu8-metallothionein, we propose the presence of a Cu4 cluster with similar electronic structure in native GIF. However, the determined Cys/Cu+ ratio of approximately 2:1 in Cu, Zn-GIF is higher than the ratio found in mammalian Cu(I)-metallothionein forms (approximately 1.6:1 ), which implies that the coordination geometry of CU+-binding sites is different in the CU4 Cluster. The spectroscopic characterization of Zn2+-substituted and Cd2+-substituted GIF (6-7 metal ions/protein) showed CD and MCD features at positions identical to those reported for the well-characterized mammalian Zn7-metallothionein and Cd7-metallothionein. Therefore, it is inferred that the cluster organization in GIF with divalent metal ions is comparable to that found in mammalian metallothioneins. The effect of metal ions on the protein structure with regard to the biological function of GIF is discussed.

Origin of the pH-dependent spectroscopic properties of pentacoordinate metmyoglobin variants.

The pH dependence of the electronic and EPR spectra of two variants of horse heart myoglobin (Mb) in which the distal His64 ligand has been replaced by either Thr or Ile has been studied. Both of these variants exhibit spectroscopic changes with pH that are indicative of a transition between two ferric high-spin forms that occurs with a pKa of 9.49 for the His64Thr variant and 9.26 for the His64Ile variant and that is distinctly different from the pH-dependent spectroscopic changes related to titration of the distal aquo ligand of wild-type Mb. The electronic and EPR spectra of both variants at all values of pH studied are consistent with the presence of a pentacoordinate heme iron center. For the His64Thr variant, a high-resolution (1.9 A) structure determination establishes the lack of the distal aquo ligand and demonstrates an out-of-plane movement of the ferric iron toward the proximal histidine together with a decrease of the Fe-His bond length. Investigation of this pH-linked equilibrium by EPR spectroscopy reveals rhombically split high-spin signals at both pH 7 and 11 with a greater degree of rhombicity exhibited by the alkaline species. We propose that the pH-linked spectroscopic transition exhibited by these distal histidine variants results from the deprotonation of the proximal His93 residue to produce imidazolate ligation at alkaline pH.

FTIR analysis of the interaction of azide with horse heart myoglobin variants.

The interaction of azide with variants of horse heart myoglobin (Mb) has been characterized by Fourier transform infrared (FTIR), electron paramagnetic resonance (EPR), and UV-VIS absorption spectroscopy and by molecular modeling calculations. Distal histidine variants (His64Thr, His64Ile, His64Lys) and charged surface variants (Val67Arg, Lys45Glu, Lys45Glu/Lys63Glu) were included in this study. All variants, with the exception of Val67Arg, have a lower azide affinity than the wild-type protein. Analysis of the temperature dependence of the FTIR spectra (277-313 K) revealed that the wild-type protein and all variants exhibit a high-spin/low-spin equilibrium. Introduction of positively charged amino acid residues shifts nu max for the low-spin form to higher energy while negatively charged residues shifted this maximum to lower energy. The low azide binding affinity exhibited by the His64Thr and His64Ile variants is accompanied by a shift of the nu max for the low-spin infrared band to lower energy and by a significant increase in the corresponding half-bandwidths. This observation indicates greater mobility of the bound azide ligand in these variants. The His64Lys variant exhibits two infrared bands attributable to low-spin forms that are assigned to two different conformations of the lysyl residue. In one conformation, the lysine is proposed to form a hydrogen bond with the bound azide similar to that proposed to occur between the distal histidine and bound azide, and in the other conformation no interaction occurs.

Structural characterization of heme ligation in the His64-->Tyr variant of myoglobin.

A site-specific mutant of horse heart myoglobin has been prepared in which the distal heme pocket residue, His64, is replaced by tyrosine. The structure of this myoglobin variant has been determined to 2.0-A resolution using x-ray diffraction techniques and refined to a final crystallographic R-factor of 16.9%. The polypeptide backbone conformation of the His64-->Tyr variant of myoglobin is very similar to that of the wild-type protein. However, in the variant the water normally found coordinated to the heme iron atom and hydrogen-bonded to His64 has been displaced by the hydroxyl oxygen of the Tyr64 side chain. The tyrosine oxygen atom is directly coordinated to the heme iron atom with a bond length of 2.18 A. Distortion of heme planarity and changes in the packing of the Leu29 and Leu104 side chains are related to this mutation. The ligand environment of the ferric iron has been studied by electron paramagnetic resonance (EPR) spectroscopy using crystalline material and protein in solution. The protein in solution exhibits a rhombically split ferric high spin EPR spectrum with g values of 6.64, 5.34, and 1.98. The EPR spectrum of the crystalline sample consists of two different ferric high spin signals. The main signal is similar to the signal observed in solution and is assigned to His93-Fe(III)-Tyr64 coordination. The relatively high rhombicity of this signal can be explained as arising from distortions of the heme plane seen in the crystal structure. The second, more axial high spin signal found in the crystalline state can be tentatively assigned to another form of iron ligation with a different iron-tyrosine bond length and a less distorted heme plane.

Comparative studies of phosvitin from chicken and salmon egg yolk.

1. A method developed for the isolation of phosvitin from chicken egg yolk was successfully applied to the isolation of phosvitin from salmon eggs. 2. Salmon roe phosvitin is smaller in molecular size than chicken egg phosvitin. 3. Circular dichroism spectra of all phosvitins investigated displayed good similarities with spectra showing characteristics of unordered and beta-sheet secondary structure. 4. The main component in the Fourier transform infrared spectra of chicken egg phosvitin is indicative of unordered conformation, whereas the Fourier infrared data of the salmon egg phosvitin are consistent with more of beta-sheet structure compared to the chicken egg phosvitin.

X- and Q-band EPR studies on the two Mn(2+)-substituted metal-binding sites of D-xylose isomerase.

The two metal-binding sites (A and B)/subunit of the homotetrameric D-xylose isomerase (Xyl isomerase) from Streptomyces rubiginosus have been studied with Mn(2+)-EPR spectroscopy at X-band and Q-band frequencies and with electronic spectroscopy. Displacement studies in the visible absorbance range showed that Mn2+ have a higher affinity for the B site. With the low-affinity A site unoccupied, the coordination sphere of Mn2+ in the B site is quite distorted giving rise to a highly anisotropic X-band EPR spectrum. Simulation of the Q-band spectrum reveals a zero field splitting (zfs) D of about 45-48 mT and a rhombicity parameter E/D between 0.2 and 0.3. Occupation of both binding sites with Mn2+ induces a significant shift towards a higher symmetry in the coordination sphere of the B site resulting in similar zfs parameters for both binding sites. The change in A-site environment caused by B-site occupation was analysed in mixed Xyl isomerase derivatives, in which the B site is loaded with Co2+, Cd2+ or Pb2+ and the A site with Mn2+. In the Co2+/Mn2+ Xyl isomerase the Mn2+ has a relatively symmetric ligand environment with small zfs parameters (D = 12 mT, E/D < 0.15). Substituting Co2+ with Cd2+ or Pb2+ in the B site leads to a drastic increase in the zfs parameters of Mn2+ in the A site. The distortions are directly linked to the ionic radii of the ions bound to the B site and may be mediated by the carboxylate group of Glu216 that bridges the metal-binding sites. The EPR spectra also reflect the catalytic activity of the mixed metal samples. With the larger Cd2+ or Pb2+ in the B site, which are strongly influencing the stereochemistry of the A site, the catalytic activity is lost, whereas Co2+ and Mn2+ render the enzyme in an active state, so that the mutual influence on catalysis depends on the complex geometry of both metal-binding sites.

Visible, EPR and electron nuclear double-resonance spectroscopic studies on the two metal-binding sites of oxovanadium (IV)-substituted D-xylose isomerase.

The two metal-binding sites of the D-xylose isomerase from Streptomyces rubiginosus were studied using VO2+ as a sensor for the ligand environment. Titration of the tetrameric enzyme with VO2+, followed by EPR spectroscopy and inhibition studies, show that the first four VO2+ equivalents occupy, in analogy to Co2+, Cd2+ and Pb2+, the binding site B. The visible absorption data and the EPR parameters indicate that a nitrogen ligand is involved in the ligand sphere of the high-affinity B site. The low-affinity A site could be studied selectively by blocking the B site with visible and EPR-silent Cd2+. The visible data and EPR parameters for this site are consistent with a ligand environment composed of oxygen donors without nitrogen ligation. The nitrogen coordination in the high-affinity site could be demonstrated by electron nuclear double-resonance (ENDOR) studies of the 4VO2+ enzyme, and was assigned to a histidine ligand. The 14N resonances are interpreted in terms of a quartet with a coupling value of 13.2 MHz. 1H-ENDOR coupling of 1.7 MHz, exchangeable in D2O, has been assigned to the N-H proton of the histidine. Additional proton ENDOR couplings, which are not exchangeable, are due to protons bound to the carbon atoms of the histidine. For the low-affinity binding site, a nitrogen coordination could be definitely excluded by the ENDOR measurements. Exchangeable 1H-ENDOR couplings observed in this sample were assigned to H2O ligands in the vicinity of VO2+. The results closely relate to what is known from X-ray structure. However, the relative affinities for the two binding sites seem not to be the same for different bivalent cations. In mixed metal samples with four VO2+ and four Co2+ equivalents, the VO2+ is distributed between both binding sites. Small changes in the complex geometry of the A site, indicated by different EPR features, seem to occur if the B site is occupied by Co2+ or by Cd2+.

Spectroscopic studies on the metal-ion-binding sites of Co2(+)-substituted D-xylose isomerase from Streptomyces rubiginosus.

The coordination sphere of the two metal-binding sites/subunit of the homotetrameric D-xylose isomerase from Streptomyces rubiginosus has been probed by the investigation of the Co2(+)-substituted enzyme using electronic absorption, CD and magnetic circular dichroic spectroscopies in the visible region. The spectrum of the high-affinity site (B site) has an absorption coefficient, epsilon 545, of 18 M-1 cm-1, indicating a distorted octahedral complex geometry. The spectrum of the low-affinity site (A site) shows two absorption maxima at 505 nm and 586 nm with epsilon values of 170 M-1 cm-1 and 240 M-1 cm-1, respectively, which indicates a distorted tetrahedral or pentacoordinated complex structure as also observed for the enzyme from Streptomyces violaceoruber [Callens et al. (1988) Biochem. J. 250, 285-290] having the same feature but lower epsilon values. The first 4 mol Co2+ added/mol apoenzyme occupy both sites nearly equally. Subsequently the Co2+ located in the A site slowly moves into the B site. After equilibrium is reached, the next 4 mol Co2+/mol again occupy the A site with its typical spectrum, restoring full activity. Addition of 4 mol Cd2+ or Pb2+/mol Co4-loaded derivative displaces the Co2+ from the B site to form the Pb4/Co4 derivative containing Co2+ in the A site, reducing activity fourfold while the Pb4/Pb4 species is completely inactive. In contrast, Eu3+ displaces Co2+ preferentially from the A site. Thus, the high- and low-affinity sites may be different for different cations. After addition of the substrates D-xylose, D-glucose and D-fructose and the inhibitor xylitol the intense Co2+ A-site spectrum of both the active Co4/Co4 derivative and the less active Pb4/PCo4 derivative decreases, indicating that these compounds are bound to the A site, changing the distorted tetrahedral or pentacoordinated symmetry there to a distorted octahedral complex geometry.

Studies of reproductive cyclicity: evaluation of computer modeling as a tool.

Assessment of toxic effects on human reproductive function using fetal health or teratogenic criteria presumes the conditions of parental fertility. Toxins that compromise fecundity through derangement of the menstrual cycle may require quite different models and criteria. Nonlinear dynamic interactions of the hormonal and morphological components of the menstrual cycle add to the difficulty of such studies. With appropriate mathematical models, computer simulation can provide a useful guide to the design and conduct of in vivo experiment.