Structural conservation in parallel beta/alpha-barrel enzymes that catalyze three sequential reactions in the pathway of tryptophan biosynthesis

Three successive steps in tryptophan biosynthesis are catalyzed by single-domain proteins, each folded as a parallel beta/alpha-barrel, as observed in the crystal structures of the bienzyme (phosphoribosyl)-anthranilate isomerase:indoleglycerolphosphate synthase from Escherichia coli [Priestle, J.P., Grütter, M. G., White, J. L., Vincent, M. G., Kania, M., Wilson, E., Jardetzky, T. S., Kirschner, K., & Jansonius, J. N. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5690-5694] and the alpha-subunit of the tetrameric bienzyme tryptophan synthase from Salmonella typhimurium [Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., & Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871]. Recent refinement of the crystal structures of these enzymes at atomic resolution revealed that they contain a common phosphate group binding site in the beta/alpha-barrel, created by residues of the loop between beta-strand 7 and alpha-helix 7 and the N-terminus of an additional helix 8'. The close similarities of their beta/alpha-barrel structures permitted the alignment of 50-75% of their respective amino acid sequences. Considerable sequence similarity was detected in the regions spanning the phosphate binding sites, whereas the percentage of identical residues was barely significant for the remaining parts of the enzymes. These observations suggest divergent evolution of these three beta/alpha-barrel enzymes involved in tryptophan biosynthesis. The same phosphate binding site was also observed in six other beta/alpha-barrel enzymes that are functionally unrelated to those involved in tryptophan biosynthesis: triosephosphate isomerase, ribulose-1,5-bisphosphate carboxylase/oxygenase, glycolate oxidase, flavocytochrome b2, trimethylamine dehydrogenase, and tentatively also fructosebisphosphate aldolase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome c binding affects the conformation of cytochrome a in cytochrome c oxidase.

Second derivative absorption spectroscopy has been used to assess the effects of complex formation between cytochrome c and cytochrome c oxidase on the conformation of the cytochrome a cofactor. When ferrocytochrome c is complexed to the cyanide-inhibited reduced or mixed valence enzyme, the conformation of ferrocytochrome a is affected. The second derivative spectrum of these enzyme forms displays two electronic transitions at 443 and 451 nm before complex formation, but only the 443-nm transition after cytochrome c is bound. This effect is not induced by poly-L-lysine, a homopolypeptide which is known to bind to the cytochrome c binding domain of cytochrome c oxidase. The effect is limited to cyanide-inhibited forms of the enzyme; no effect was observed for the fully reduced unliganded or fully reduced carbon monoxide-inhibited enzyme. The spectral signatures of these changes and the fact that they are exclusively associated with the cyanide-inhibited enzyme are both reminiscent of the effects of low pH on the conformation of cytochrome a (Ishibe, N., Lynch, S., and Copeland, R. A. (1991) J. Biol. Chem. 266, 23916-23920). These results are discussed in terms of possible mechanisms of communication between the cytochrome c binding site, cytochrome a, and the oxygen binding site within the cytochrome c oxidase molecule.     

Structure of human epoxide hydrolase reveals mechanistic inferences on bifunctional catalysis in epoxide and phosphate ester hydrolysis

The X-ray crystal structure of human soluble epoxide hydrolase (sEH) has been determined at 2.6 A resolution, revealing a domain-swapped quaternary structure identical to that observed for the murine enzyme [Argiriadi, M. A., Morisseau, C., Hammock, B. D., and Christianson, D. W. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10637-10642]. As with the murine enzyme, the epoxide hydrolytic mechanism of the human enzyme proceeds through an alkyl-enzyme intermediate with Asp-333 in the C-terminal domain. The structure of the human sEH complex with N-cyclohexyl-N'-(iodophenyl)urea (CIU) has been determined at 2.35 A resolution. Tyr-381 and Tyr-465 donate hydrogen bonds to the alkylurea carbonyl group of CIU, consistent with the proposed roles of these residues as proton donors in the first step of catalysis. The N-terminal domain of mammalian sEH contains a 15 A deep cleft, but its biological function is unclear. Recent experiments demonstrate that the N-terminal domain of human sEH catalyzes the metal-dependent hydrolysis of phosphate esters [Cronin, A., Mowbray, S., Dürk, H., Homburg, S., Fleming, I., Fisslthaler, B., Oesch, F., and Arand, M. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 1552-1557; Newman, J. W., Morisseau, C., Harris, T. R., and Hammock, B. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 1558-1563]. The binding of Mg(2+)-HPO4(2-) to the N-terminal domain of human sEH in its CIU complex reveals structural features relevant to those of the enzyme-substrate complex in the phosphatase reaction.     

Methods for aligning and for averaging 3D volumes with missing data

The visibility and resolution of a tomographic reconstruction containing multiple copies of discrete particles can be enhanced by averaging subtomograms after they are corrected aligned. However, the “missing wedge” in electron tomography can easily lead to erroneous alignment. We have explored a Fourier space cross-correlation method with a proper weighting scheme to align and average different sets of volumetric data, each of which has different missing data due to the limited specimen tilts. This approach depends neither on a preexisting template, nor an exact knowledge of the geometry, orientation, or amount of the missing data. This paper introduces a procedure where the missing data might be gradually “filled in” by consecutively aligning and averaging volumes with different orientations of their missing data. We have validated these techniques by a set of simulated data with various symmetries and extent of missing data. We have also successfully applied these procedures to experimental cryo-electron tomographic data [Chang, J.T., Schmid, M.F., Rixon, F.J., and Chiu, W., 2007. Electron cryotomography reveals the portal in the herpesvirus capsid. J. Virol. 81, 2065–2068; Schmid, M.F., Paredes, A.M., Khant, H.A., Soyer, F., Aldrich, H.C., Chiu, W., and Shively, J.M., 2006. Structure of Halothiobacillus neapolitanus carboxysomes by cryo-electron tomography. J. Mol. Biol. 364, 526–535].     

Effect of polar carotenoids on the oxygen diffusion-concentration product in lipid bilayers. An EPR spin label study.

The oxygen diffusion-concentration product was determined in phosphatidylcholine (PC) bilayers from oxygen broadening of the spin label EPR spectra. The use of fatty acid spin labels makes it possible to do structural and oximetric measurements with the same sample. We find that polar carotenoids, zeaxanthin and violaxanthin, increase ordering of hydrocarbon chains in saturated (dimyristoyl-PC) and unsaturated (egg yolk PC) membranes and also significantly decrease the oxygen diffusion-concentration product in the hydrocarbon region of these membranes. At 25 degrees C in the presence of 10 mol% of carotenoids, the product is about 30% smaller than in pure PC membranes. Intercalation of carotenoids decreases the oxygen diffusion-concentration product in the central part of the bilayer and has little effect on the product in the polar head group region. In contrast, cholesterol molecules significantly reduce the product on and near the membrane surface, and do not change it (saturated PC) or increase it (unsaturated PC) in the middle of the bilayer (Subczynski, W.K., Hyde, J.S. and Kusumi, A. (1989) Proc. Natl. Acad. Sci. USA 86, 4474-4478). The decrease of oxygen diffusion-concentration product may be a mechanism of carotenoid protective activity, which should be effective in plant and animal cells in the light as well as in the dark.     

Milk, revealed "silent" chemistry: new mode of cycloretinal synthesis

Bovine milk is by far the most commonly consumed milk in the western world. The protein composition in milk consists of casein and whey proteins, of which β-lactoglobulin (BLG) is the principal constituent of the latter. Here we provide biochemical evidence that this milk protein, in purified form and in pasteurized store-bought milk, promotes the formation of cycloretinal (all-trans retinal dimer), and a variety of other cycloterpenals of biological relevance [Fishkin et al., Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 7091-7096; Fishkin et al., Chirality, 2004, 16, 637-641; Kim et al., Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 19273-19278]. Cycloretinal is an eye metabolite and among several toxic byproducts of the visual cycle firmly established to cause age-related macular degeneration. Experiments in rabbits further demonstrate that BLG/milk can survive the digestive system and promote this reaction in vivo [Caillard et al., Am. J. Physiol., 1994, 266(6), G1053-G1059]. Proteomic studies on age-related macular degeneration patients have detected BLG in the eye of these patients further suggesting that this milk protein could contribute to disease progression [Crabb et al., Proc. Natl. Acad. Sci. U. S. A., 2002, 99(23), 14682-14687].     

Tuna cytochrome c at 2.0 A resolution. II. Ferrocytochrome structure analysis.

The x-ray crystal structure analysis of tuna ferrocytochrome c has been extended from 2.45 to 2.0 A resolution. The overall folding is unchanged and is the same as has been reported for tuna ferricytochrome c (Swanson R., Trus, B.L., Mandel, N., Mandel, G., Kallai, O.B., and Dickerson, R.E. (1977) J. Biol. Chem. 252, 759-755). No significant structural differences are observed between oxidation states. Difference map studies using reoxidized crystals of ferrocytochrome c confirm the absence of a conformation change. A detailed analysis of hydrogen bonding shows the presence of six beta or 310 bends of type II with obligatory glycines in the 3rd residue position. This explains 6 of the 10 nearly invariant glycines in the molecule. Close packing contacts account for three more, and only the invariant glycine 1 remains a mystery.     

The crystal and molecular structure of the third domain of silver pheasant ovomucoid (OMSVP3)

OMSVP3 and OMTKY3 (third domains of silver pheasant and turkey ovomucoid inhibitor) are Kazal-type serine proteinase inhibitors. They have been isomorphously crystallized in the monoclinic space group C2 with cell dimensions of a = 4.429 nm, b = 2.115 nm, c = 4.405 nm, beta = 107 degrees. The asymmetric unit contains one molecule corresponding to an extremely low volume per unit molecular mass of 0.0017 nm3/Da. Data collection was only possible for the OMSVP3 crystals. Orientation and position of the OMSVP3 molecules in the monoclinic unit cells were determined using Patterson search methods and the known structure of the third domain of Japanese quail ovomucoid (OMJPQ3) [Papamokos, E., Weber, E., Bode, W., Huber, R., Empie, M. W., Kato, I. and Laskowski, M., Jr (1982) J. Mol. Biol. 158, 515-537]. The OMSVP3 structure has been refined by restrained crystallographic refinement yielding a final R value of 0.199 for data to 0.15 nm resolution. Conformation and hydrogen-bonding pattern of OMSVP3 and OMJPQ3 are very similar. Large deviations occur at the NH2 terminus owing to different crystal packing, and at the C terminus of the central helix, representing an intrinsic property and resulting from amino acid substitutions far away from this site. The deviation of OMSVP3 from OMTKY3 complexed with the Streptomyces griseus protease B is very small [Fujinaga, M., Read, R. J., Sielecki, A., Ardelt, W., Laskowski, M., Jr and James, M. N. G. (1982) Proc. Natl Acad. Sci. USA, 79, 4868-4872].     

Selection of a cDNA clone which contains the complete coding sequence for the mature form of ornithine transcarbamylase from rat liver: expression of the cloned protein in Escherichia coli. Molecular cloning of rat ornithine transcarbamylase

A cDNA clone corresponding to the mature form of ornithine transcarbamylase (OTCase) was selected from a rat liver cDNA library constructed in bacteriophage lambda gt10. OTCase clones were selected using a synthetic DNA probe of 15 bases corresponding to the 3' end of the OTCase mRNA [Horwich, A. L., Kraus, J.P., Williams, K., Kalousek, F., K?nigsberg, W. & Rosenberg, L.E. (1983) Proc. Natl Acad. Sci. USA, 80, 4258-4262]. Putative OTCase clones were subcloned into the expression vector, pUC9, and the identity of inserts confirmed by colony immunoassay and by electrophoretic transfer of cloned proteins from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose filters followed by probing with monospecific anti-OTCase antibodies and 125I-labelled protein A. A clone corresponding to the full-length mature form of rat liver OTCase (plus 15 amino acids from Escherichia coli beta-galactosidase) was obtained and the identity of the clone was confirmed by comparison of the 5' sequence with a limited N-terminal amino acid sequence [Lusty, C., Jilka, R. L. & Nietsch, E. H. (1979) J. Biol. Chem. 254, 10030-10036]. A sequence discrepancy between the published sequence (Lusty et al.) and the sequence predicted from the cDNA structure is noted.     

Time-resolved infrared spectroscopy of electron transfer in bacterial photosynthetic reaction centers: dynamics of binding and interaction upon QA and QB reduction.

Light-induced forward electron transfer in the bacterial photosynthetic reaction center from Rhodobacter sphaeroides was investigated by time-resolved infrared spectroscopy. Using a highly sensitive kinetic photometer based on a tunable IR diode laser source [M?ntele, W., Hienerwadel, R., Lenz, F., Riedel, W. J., Grisar, R., & Tacke, M. (1990a) Spectrosc. Int. 2, 29-35], molecular processes concomitant with electron-transfer reactions were studied in the microsecond-to-second time scale. Infrared (IR) signals in the 1780-1430-cm-1 spectral region, appearing within the instrument time resolution of about 0.5 microseconds, could be assigned to molecular changes of the primary electron donor upon formation of a radical cation and to modes of the primary quinone electron acceptor QA and its environment upon formation of QA-. These IR signals are consistent with steady-state FTIR difference spectra of the P+Q- formation [M?ntele, W., Nabedryk, E., Tavitian, B. A., Kreutz, W., & Breton, J. (1985) FEBS Lett. 187, 227-232; M?ntele, W., Wollenweber, A., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472; Nabedryk, E., Bagley, K. A., Thibodeau, D. L., Bauscher, M., M?ntele, W., & Breton, J. (1990) FEBS Lett. 266, 59-62] and with time-resolved FTIR studies [Thibodeau, D. L., Nabedryk, E., Hienerwadel, R., Lenz, F., M?ntele, W., & Breton, J. (1990) Biochim. Biophys. Acta 1020, 253-259]. At given wavenumbers, kinetic components with a half-time of approximately 120 microseconds were observed and attributed to QA----QB electron transfer. The time-resolved IR signals, in contrast to steady-state experiments where full protein relaxation after electron transfer can occur, allow us to follow directly the modes of QA and QB and their protein environment under conditions of forward electron transfer. Apart from signals attributed to the primary electron donor, signals are proposed to arise not only from the C = O and C = C vibrational modes of the neutral quinones and from the C-O and C-C vibrations of their semiquinone anion form but also from amino acid groups forming their binding sites. Some of the signals appearing with the instrument rise time as well as the transient 120-microseconds signals are interpreted in terms of binding and interaction of the primary and secondary quinone electron acceptor in the Rb. sphaeroides reaction center and of the conformational changes in their binding site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and crystallographic analysis of two rhizopuspepsin inhibitor complexes.

The crystal structures of rhizopuspepsin complexed with two oligopeptide inhibitors have been determined. CP-69,799, an azahomostatine dipeptide isostere, had previously been associated with a displacement of the C-terminal subdomain of endothiapepsin [Sali, A., Veerapandian, B., Cooper, J. B., Foundling, S. I., Hoover, D. J., & Blundell, T. L. (1989) EMBO J. 8, 2179-2188]. Here, we report the measurement of two data sets, one from crystals soaked in the inhibitor and the other from protein crystallized in the presence of excess inhibitor. In neither case is there any significant movement of the C-terminal subdomain of the rhizopuspepsin. The data suggest that the energy associated with any conformational change is small and is overcome by the crystal packing forces. The second inhibitor, a hydrated difluorostatone, was examined in a search for transition-state analogs that could cast further light on the mechanism of action [Suguna, K., Padlan, E. A., Smith, C. W., Carlson, W. D., & Davies, D. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7009-7013]. The gem-diol provides a set of contact distances with the enzyme that mimic the interactions with the tetrahedral intermediate of the substrate during catalysis. These data provide support for the suggestion that the polarization of the keto group of the peptide substrate is enhanced by a hydrogen bond from the OD1 of Asp 35 (Suguna et al., 1987).     

高产条件下不同小麦品种耗水特性和水分利用效率的差异

设置不灌水(W0)、底墒水+拔节水(W1)、底墒水+拔节水+开花水(W2)3个灌水处理,采用6个冬小麦(Triticum aestivum.L.)品种,研究了不同品种耗水特性和水分利用效率的差异.结果表明:(1)依据籽粒产量和水分利用效率2个因子,采用聚类分析的方法,将供试品种分为高水分利用效率组(Ⅰ组)、中水分利用效率组(Ⅱ组)和低水分利用效率组(Ⅲ组).同一灌水条件下的籽粒产量,Ⅰ组显著高于Ⅱ组和Ⅲ组;Ⅱ组和Ⅲ组在W0条件下无显著差异,在W1和W2条件下Ⅱ组显著高于Ⅲ组.(2)从Ⅰ组、Ⅱ组、Ⅲ组中分别取1个品种,泰山23、潍麦8号、山农12进一步分析表明,在W0 和W1条件下,泰山23和潍麦8号的阶段耗水量和耗水模系数为开花至成熟>播种至拔节>拔节至开花,山农12为播种至拔节>开花至成熟>拔节至开花.W2条件下,3个品种的阶段耗水量和耗水模系数为开花至成熟>播种至拔节>拔节至开花;播种至拔节和拔节至开花的耗水模系数为泰山23>山农12>潍麦8号,此阶段的耗水量和耗水强度为泰山23品种最高;开花至成熟的耗水模系数为潍麦8号>山农12 >泰山23,此阶段的耗水量和耗水强度为泰山23品种最低.(3) 在W0 和W1条件下,总耗水量和灌水量、降水量及土壤耗水量占总耗水量的百分率为泰山23品种居中;W2条件下,灌水量和降水量占总耗水量的百分率为泰山23>潍麦8号>山农12,土壤耗水量及其占总耗水量的百分率反之,但泰山23的总耗水量最低.(4) 同一灌水条件下,泰山23品种100～200cm土层的土壤耗水量高于潍麦8号,表明该品种能充分利用深层土壤水;山农12品种在W0和W2条件下,100～200 cm土层的土壤耗水量高于泰山23和潍麦8号,但其籽粒产量和水分利用效率显著低于上述两品种.     

Is the mammalian cell plasma membrane a barrier to oxygen transport?

Oxygen transport in the Chinese hamster ovary (CHO) plasma membrane has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed in the lipid bilayer portion of the membrane at various distances from the membrane surface using the long-pulse saturation-recovery electron spin resonance (ESR) technique. The collision rate was estimated for 5-, 12-, and 16-doxylstearic acids from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameters across the membrane were obtained showing that the oxygen diffusion-concentration product is lower than in water for all locations at 37 degrees C. From oxygen transport parameter profiles, the membrane oxygen permeability coefficients were estimated according to the procedure developed earlier by Subczynski et al. (Subczynski, W. K., J. S. Hyde, and A. Kusumi. 1989. Proceedings of the National Academy of Sciences, USA. 86:4474-4478). At 37 degrees C, the oxygen permeability coefficient for the plasma membrane was found to be 42 cm/s, about two times lower than for a water layer of the same thickness as the membrane. The oxygen concentration difference across the CHO plasma membrane at physiological conditions is in the nanomolar range. It is concluded that oxygen permeation across the cell plasma membrane cannot be a rate-limiting step for cellular respiration. Correlations of the form PM = cKs between membrane permeabilities PM of small nonelectrolyte solutes of mol wt less than 50, including oxygen, and their partition coefficients K into hexadecane and olive oil are reported. Hexadecane: c = 26 cm/s, s = 0.95; olive oil: c = 23 cm/s, s = 1.56. These values of c and s differ from those reported in the literature for solutes of 50 less than mol wt less than 300 (Walter, A., and J. Gutknecht. 1986. Journal of Membrane Biology. 90:207-217). It is concluded that oxygen permeability through membranes can be reliably predicted from measurement of partition coefficients.     

Iron-sulfur cluster in aconitase. Crystallographic evidence for a three-iron center

Native x-ray diffraction data from single crystals of inactive aconitase from pig heart (Mr 80,000) have been collected on oscillation films to 2.7 A. Analysis shows that significant measurements of the anomalous scattering signal from the Fe-S cluster in the enzyme are available in the film data. The 5.0-A resolution anomalous difference Patterson function contains vectors for one Fe-S cluster (one aconitase molecule) per asymmetric unit in space group P2(1)2(1)2 with a = 173.6, b = 72.0, and c = 72.7 A. At 2.7-A resolution, the vector map is best interpreted by three Fe sites separated from each other by less than 3 A. The single-crystal diffraction data thus confirm the presence of a 3Fe center in the inactive form of aconitase. Furthermore, the data provide crystallographic evidence that 3Fe clusters exhibit structural heterogeneity. The Fe-Fe vectors cannot be interpreted in terms of 4-A distances as observed for the [3Fe-3S] cluster in Azotobacter ferrodoxin (Ghosh, D., O'Donnell, S., Furey, W., Robbins, A. H., and Stout, C. D. (1982) J. Mol. Biol. 158, 73-109). The results are therefore in agreement with a [3Fe-4S] cluster having 2.7-A Fe-Fe distances (Beinert, H., Emptage, M. H., Dreyer, J.-L., Scott, R. A., Hahn, J. E., Hodgson, K. O., and Thomson, A. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 393-396). However, the data do not unambiguously discriminate between this model and other 3Fe clusters having short Fe-Fe distances.     

CHEMISTRY OF ATTRACTION AND DEFENSE IN MACRO- AND MICROALGAE

Boland, W. & Pohnert, G. Max-Planck-Institut of Chemical Ecology, Carl-Zeiss-Promenade 10, 07745 Jena, Germany A diverse group of brown seaweeds produce bouquets of C₁₁ and C₈ metabolites, some of which act as pheromones that trigger gamete release or attract sperm to eggs following release [1]. The same compounds and, especially, their oxidative degradation products frequently and strongly deter feeding by mezograzers (Ampithoe longimana) which often consume seaweed spores, zygotes, and juveniles [2]. Besides macroalgae also several microalgae (ca. 20 Gomphonema spp., Asterionella formosa; Diatomophyceae) produce C11 and C8 hydrocarbons along with a toxic polar compound from the pool of highly unsaturated fatty acids (eicosanoids) [3]. In biosynthetic studies with cell free extracts of the diatom G. parvulum (9S)-hydroperoxy-eicosatetraenoic (9S-HPETE) acid was shown to be converted by a novel type of a lyase into the defensive com-pound 9-oxo-nona-5Z,7E-dienoic acid along with cyclic and linear C₁₁ hydrocarbons [4]. If the eicosanoid pre-cursors are first con-verted into C-12 hydroperoxides, subsequent lyase activity produces C₈ trienes together with 12-oxo-dodeca-5Z,8Z,10E-dienoic acid (A. formosa). Both unsaturated acids serve as efficient chemi-cal defenses against attacking feeders and microorganisms. The reaction cascade is triggered by damage of the diatom, initiating rapid release of the free fatty acid from phospholipids followed by lipoxygenation and oxidative cleavage into the unsaturated hydrocarbon and the oxo acid [5]. 1. Boland W 1995 Proc. Natl. Acad. Sci. 92:31-43. 2. Hay ME, Piel J, Boland W, Schnitzler I 1998 Chemoecology 8:91-98 3. Pohnert G, Boland W 1997 Tetrahedron 53:13681-13694. 4. Hombeck M, Pohnert G, Boland W 1999 J. Chem. Soc., Chem. Commun., 243-244. 5. Pohnert G 2000 Angew. Chem. 112:4506-4508.     

Differences in thermal stability between reduced and oxidized cytochrome b562 from Escherichia coli

The thermal stabilities of ferri- and ferrocytochrome b562 were examined. Thermally induced spectral changes, monitored by absorption and second-derivative spectroscopies, followed the dissociation of the heme moiety and the increased solvation of tyrosine residue(s) located in close proximity to the heme binding site. All observed thermal transitions were independent of the rate of temperature increase (0.5-2 degrees C/min), and the denatured protein exhibited partial to near-complete reversibility upon return to ambient temperature. The extent of renaturation of cytochrome b562 is dependent on the amount of time the unfolded conformer is exposed to temperatures above the transition temperature, Tm. All thermally induced spectra changes fit a simple two-state model, and the thermal transition was assumed to be reversible. The thermal transition for ferrocytochrome b562 yielded Tm and van't Hoff enthalpy (delta HvH) values of 81.0 degrees C and 137 kcal/mol, respectively. In contrast, Tm and delta HvH values obtained for the ferricytochrome were 66.7 degrees C and 110 kcal/mol, respectively. The estimated increase in the stabilization free energy at the Tm of ferricytochrome b562 following the one-electron reduction to the ferrous form, where delta delta G = delta Tm delta Sm [delta Sm = 324 cal/(K.mol), delta Tm = 14.3 degrees C] [Becktel, W. J., & Schellman, J. A. (1987) Biopolymers 26, 1859-1877], is 4.6 kcal/mol.     

The complete amino-acid sequence of a trout-testis non-histone protein, H6, localized in a subset of nucleosomes and its similarity to calf-thymus non-histone proteins HMG-14 and HMG-17.

The complete amino acid sequence of a basic non-histone protein, H6, isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells, has been determined. Protein H6, first described by D. T. Wigle and G. H. Dixon [J. Biol. Chem. 246, 5636--5644 (1971)] was extracted with 5% trichloracetic acid and purified by ion-exchange chromatography on carboxymethyl-cellulose (CM-52). Sequence analysis was performed by automatic Edman degradation of the amino terminus of the intact protein and a series of large fragments derived by cleavage with chymotrypsin, staphylococcal protease and with mild acid to cleave at aspartic acid residues. Protein H6 possesses 69 residues and shows considerable similarities to the 89-residue calf thymus HMG-17 protein previously sequenced [Walker, J. M., Hastings, J. R. B. & Johns, E. W. (1977) Eur. J. Biochem. 76, 461--468]. B. Levy W. and G. H. Dixon [Proc. Natl Acad. Sci. U.S.A. 74, 2810--2814 (1977)] have shown that H6 is selectively solubilized when trout testis nuclei (or chromatin) are digested with DNase I under conditions which preferentially hydrolyze that portion of DNA enriched in transcribed sequences [Levy, W. B. & Dixon, G. H. (1977) Nucleic Acids Res. 4, 883--898]. Recently H6 has been located as a stoichiometric component of a distinct subset of trout testis nucleosomes that are complexed with a core nucleosome comprising 140 base pairs of DNA and the inner histones H2A, H2B, H3 and H4 [Levy, W. B., Connor, W. & Dixon, G. H. (1979) J. Biol. Chem., in the press].     

Tryptophan-191----phenylalanine, a proximal-side mutation in yeast cytochrome c peroxidase that strongly affects the kinetics of ferrocytochrome c oxidation

On the basis of X-ray structural information, it was previously proposed that tryptophan-191 of yeast cytochrome c peroxidase (CCP) may be important in determining the spectroscopic and catalytic properties of the enzyme [Edwards, S. L., Xuong, Ng. H., Hamlin, R. C., & Kraut, J. (1987) Biochemistry 26, 1503-1511]. By use of site-directed mutagenesis and an Escherichia coli expression system, a mutant phenylalanine-191 (F191) CCP was prepared in order to examine the effects of altering the H-bonding and pi-pi interactions that occur between Trp-191 and the iron-coordinated proximal His-175 in the parent enzyme. The F191 mutant enzyme exhibits a dramatic decrease (approximately 3000-fold at pH 7) in V0/e for catalysis of peroxide-dependent ferrocytochrome c oxidation, while V0/e for oxidation of ferrocyanide is decreased only 4.6-fold compared to that of the parent. The Fe3+/Fe2+ Em,7 and the stability of the oxyferryl center in the H2O2-oxidized mutant enzyme are relatively unaffected by the mutation, but the species responsible for a radical-like signal centered at g = 2.00 has been destabilized approximately 100-fold with respect to spontaneous decay. Steady-state kinetic assays as well as transient-state laser flash photolysis experiments utilizing flavin semiquinones as reductants indicate that the mutant CCP forms a complex with cytochrome c but the oxyferryl center in the oxidized enzyme is no longer able to be rapidly reduced by ferrocytochrome c. The most likely reasons for this kinetic behavior are either that new steric constraints exist in the mutant which impede relaxation of the iron center to the resting ferric state or that the indole ring of Trp-191 is important in a specific interprotein electron-transfer pathway that exists between the heme centers of CCP and cytochrome c.     

Pitch detection of dynamic iterated rippled noise by humans and a modified auditory model

Iterated ripple noise (IRN) is a broadband noise with temporal regularities, which can give rise to a perceptible pitch. Since the perceptual pitch to noise ratio of these stimuli can be altered without substantially altering their spectral content, they have been useful in exploring the role of temporal processing in pitch perception [Yost, W.A., 1996. Pitch strength of iterated rippled noise, J. Acoust. Soc. Am. 100 (5), 3329-3335; Patterson, R.D., Handel, S.,Yost, W.A., Datta, A.J., 1996. The relative strength of the tone and noise components in iterated rippled noise, J. Acoust. Soc. Am. 100 (5), 3286-3294]. A generalised IRN algorithm is presented, in which multiple time varying temporal correlations can be defined. The resulting time varying pitches are perceptually very salient. It is also possible to segregate and track multiple simultaneous time varying pitches in these stimuli. Temporal auditory models have previously been shown to account for the perception of IRNs with static delays [Patterson, R.D., Handel, S.,Yost, W.A., Datta, A.J., 1996. The relative strength of the tone and noise components in iterated rippled noise, J. Acoust. Soc. Am. 100 (5), 3286-3294]. Here we show that some simple modifications to one such model [Meddis R., Hewitt, M.J., 1991. Virtual pitch and phase sensitivity of a computer model of the auditory periphery I. Pitch identification, J. Acoust. Soc. Am. 89, 2866-2882] allow it to track moving correlations, and also improve its performance in response to static correlations.     

Effects of anti-thrombospondin monoclonal antibodies on the agglutination of erythrocytes and fixed, activated platelets by purified thrombospondin

A monoclonal antibody (Mab) has been raised against native thrombospondin (TSP), the endogenous lectin of human platelets, that inhibits the hemagglutination of trypsinized, glutaraldehyde-fixed human erythrocytes by purified TSP. This Mab, designated A2.5, also inhibits the agglutination of fixed, activated platelets by TSP. Mab A2.5 immunoprecipitates a 25-kilodalton (kDa) peptide from chymotryptic digests of TSP that is not disulfide bonded to any other region of the TSP molecule. This fragment represents the previously characterized heparin binding domain of TSP [Dixit, V.M., Grant, G.A., Santoro, S.A., & Frazier, W.A. (1984) J. Biol. Chem. 259, 10100-10105]. In agreement with this assignment, heparin inhibits the binding of Mab A2.5 to TSP. Another Mab, designated C6.7, also blocks TSP-mediated hemagglutination, yet has no effect on the agglutination of fixed, activated platelets by TSP. This Mab has been shown to inhibit the thrombin-stimulated aggregation of live platelets and to immunoprecipitate an 18-kDa fragment from chymotryptic digests, which is distinct from the heparin binding domain [Dixit, V.M., Haverstick, D.M., O'Rourke, K.M., Hennessy, S.W., Grant, G.A., Santoro, S.A., & Frazier, W.A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3472-3476].