In vitro evolution of horse heart myoglobin to increase peroxidase activity

Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, double, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly greater than that of the wild-type protein with k1 (for H2O2 oxidation of metmyoglobin) of 1. 34 x 10(4) M-1 s-1 ( approximately 25-fold that of wild-type myoglobin) and k3 [for reducing the substrate (2, 2'-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 x 10(6) M-1 s-1 (1.6-fold that of wild-type myoglobin). Thermal stability of these variants as measured with circular dichroism spectroscopy demonstrated that the Tm of the quadruple variant is decreased only slightly compared with wild-type (74.1 degreesC vs. 76.5 degreesC). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin (kon, 22.2 x 10(-6) M-1 s-1 vs. 22.3 x 10(-6) M-1 s-1; koff, 24.3 s-1 vs. 24.2 s-1; KO2, 0.91 x 10(-6) M-1 vs. 0.92 x 10(-6) M-1). The affinity of the quadruple variant for CO is increased slightly (kon, 0.90 x 10(-6) M-1s-1 vs. 0.51 x 10(-6) M-1s-1; koff, 5.08 s-1 vs. 3.51 s-1; KCO, 1.77 x 10(-7) M-1 vs. 1.45 x 10(-7) M-1). All four substitutions are in the heme pocket and within 5 A of the heme group.     

Kinetic study of the reaction of glutathione peroxidase with peroxynitrite

Glutathione peroxidases and their mimics, e.g., ebselen or diaryl tellurides, efficiently reduce peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) to nitrite and protect against oxidation and nitration reactions. Here, we report the second-order rate constant for the reaction of the reduced form of glutathione peroxidase (GPx) with peroxynitrite as (8.0 +/- 0.8) x 10(6) M-1 s-1 (per GPx tetramer) at pH 7.4 and 25 degreesC. The rate constant for oxidized GPx is about 10 times lower, (0.7 +/- 0.2) x 10(6) M-1 s-1. On a selenium basis, the rate constant for reduced GPx is similar to that obtained previously for ebselen. The data support the conclusion that GPx can exhibit a biological function by acting as a peroxynitrite reductase.     

Characterization of electrogenic Na/K pump in rat neostriatal neurons

The electrogenic Na/K pump current (Ip) was studied in the dissociated neostriatal neurons of the rat by using the nystatin-perforated patch recording mode. The Ip was activated by external K+ in a concentration-dependent manner with an EC50 of 0.7 mM at a holding potential (VH) of -40 mV. Other monovalent cations also caused Ip and the order of potency was Tl+>K+, Rb+>NH4+, Cs+>Li+. The Ip decreased with membrane hyperpolarization in an external solution containing 150 mM Na+, while the Ip did not show such voltage dependency without external Na+. Ouabain showed a steady-state inhibition of Ip in a concentration- and temperature-dependent manner at a VH of -40 mV. The IC50 values at 20 and 30 degrees C were 7.1 x 10(-6) and 1.3 x 10(-6) M, respectively. The decay of Ip after adding ouabain well fitted with a single exponential function. At a VH of -40 Mv, the association (k+1) and dissociation (k-1) rate constants estimated from the time constant of the current decay at 20 degrees C were 4.0 x10(2) s-1 M-1 and 6.3 x 10(-3) s-1, respectively. At 30 degrees C, k+1 increased to 2.8 x 10(3) s-1 M-1 while k-1 showed no such change with a value of 1.8 x 10(-3) s-1. A continuous Na+ influx was demonstrated by both the Na+-dependent leakage current and tetrodotoxin-sensitive Na+ current, which resulted in the continuous activation of the Na/K pump. It was thus concluded that the Na/K pump activity was well-maintained in the dissociated rat neostriatal neurons with distinct functional properties and that the activity of the pump was tightly connected with Na+ influxes.     

Oligomerization and divalent ion binding properties of the S100P protein: a Ca2+/Mg2+-switch model

S100P is a 95 amino acid residue protein which belongs to the S100 family of proteins containing two putative EF-hand Ca2+-binding motifs. In order to characterize conformational properties of S100P in the presence and absence of divalent cations (Ca2+, Mg2+ and Zn2+) in solution, we have analyzed hydrodynamic and spectroscopic characteristics of wild-type and several variants (Y18F, Y88F and C85S) of S100P using equilibrium centrifugation, gel-filtration chromatography, circular dichroism and fluorescence spectroscopies. Analysis of the experimental data shows the following. (1) In agreement with the predictions there are two Ca2+-binding sites in the S100P molecule with different affinity; the high affinity binding site has an apparent binding constant of approximately 10(7) M-1 and the low affinity binding site has an apparent binding constant of approximately 10(4) M-1. (2) The high and low affinity Ca2+-binding sites are located in the C and N-terminal parts of the S100P molecule, respectively. (3) These C and N-terminal sites can also bind other divalent ions. The C-terminal site binds Zn2+ (with relatively low affinity approximately 10(3) M-1), but not Mg2+. The N-terminal site binds Mg2+ with the apparent binding constant approximately 10(2) M-1. (4) Binding of Ca2+ to the C-terminal site and binding of Mg2+ to the N-terminal site occur in the physiological concentration range of these ions (micromolar for Ca2+ and millimolar for Mg2+). (5) Oligomerization state of the S100P molecule appears to change upon addition of Ca2+. On the basis of these observations a plausible model for S100P as a Ca2+/Mg2+ switch has been proposed.     

General treatment of competitive binding as applied to the potentiometric ion probe technique: application to the interaction of nonsteroidal anti-inflammatory drugs with bovine serum albumin

The binding of naproxen, ketoprofen, phenylbutazone, salicylic acid, azapropazone, and indobufen to bovine serum albumin was studied by applying the potentiometric ion probe technique. An ion-selective electrode for the ion probe 1-anilino-8-naphthalene-sulfonate was utilized for the purposes of this study. A modified site-oriented competitive binding model was used for the estimation of the drugs' binding parameters, considering different number of binding sites on the competing binding class(es) for the probe and the drug. Calculations were based exclusively on the concentration data of the free probe. The model's ability for accurate estimations of binding parameters was evaluated by simulation studies. The following values of binding parameters were found at 25 degrees C for the drugs under study; naproxen, n1 = 9.1, k1 = 9.4 x 10(5) M-1; ketoprofen, n1 = 8.8, k1 = 10.8 x 10(5) M-1; phenylbutazone, n1 = 3.2, k1 = 1.4 x 10(5) M-1; salicylic acid, n1 = 2.6, k1 = 1.8 x 10(5) M-1, n2 = 21.5, k2 = 1.0 x 10(4) M-1; azapropazone, n1 = 0.5, k1 = 7.8 x 10(5) M-1, n2 = 26.3, k2 = 1.9 x 10(4) M-1; indobufen, n1 = 5.8, k1 = 5.8 x 10(5) M-1, n2 = 19.9, k2 = 3.8 x 10(5) M-1, where ni the number of binding sites of the i class and ki the corresponding association constant.     

Expression and characterization of a 159 amino acid, N-terminal fragment of human complement component C1s

A 159 residue, N-terminal fragment of the human C1s complement component, C1s alpha(159), was expressed in the baculovirus, insect cell system. The protein was abundantly produced 3 days after infection, reaching levels as high as 40 microg/ml in cell culture media. It had a molecular weight of 18,100 (+/-4.9) Da by laser desorption mass spectrometry, close to the theoretical value of 18,111 Da, confirmed by sequencing. Sedimentation equilibrium and gel filtration column chromatography showed that C1s alpha(159) was a monomer in the presence of EDTA, and a dimer in the presence of Ca2+. The C1s alpha(159)2 dimer had a sedimentation coefficient of 3.1 S. When the C1s alpha(159)2 was mixed with Clq, there was little or no interaction. Likewise, unactivated C1r2 dimer had a sedimentation coefficient of 6.8 S, and when mixed with C1q little or no interaction was observed. When C1s alpha(159)2 was mixed with the 6.8 S C1r2 in Ca2+, a 7.5 S complex was formed, presumably the C1s alpha(159) x C1r x C1r x C1s alpha(159) tetramer. When C1q, which migrated at 10.1 S was mixed with C1s alpha(159)2 and C1r2 in the presence of Ca2+, a C1-like complex, but containing C1s alpha(159) instead of C1s, was formed which migrated at 14.0 S. This C1-like molecule remained unactivated unless challenged with an ovalbumin-antiovalbumin immune complex. In the presence of immune complex, the C1r became activated. This suggested that the presence of the 159 amino acid C1s alpha domain, which held the C1r to the C1q, was sufficient to permit activation by an immune complex, even though the catalytic domains of C1s were not present.     

Mechanism of the oxidation of 3,5,3',5'-tetramethylbenzidine by myeloperoxidase determined by transient- and steady-state kinetics

Earlier investigations of the oxidation of 3,5,3',5'-tetramethylbenzidine (TMB) using horseradish peroxidase and prostaglandin H-synthase have shown the formation of a cation free radical of TMB in equilibrium with a charge-transfer complex, consistent with either a two- or a one-electron initial oxidation. In this work, we exploited the distinct spectroscopic properties of myeloperoxidase and its oxidized intermediates, compounds I and II, to establish two successive one-electron oxidations of TMB. By employing stopped-flow techniques under transient-state and steady-state conditions, we also determined the rate constants for the elementary steps of the myeloperoxidase-catalyzed oxidation of TMB at pH 5.4 and 20 degrees C. The second-order rate constant for compound I formation from the reaction of native enzyme with H2O2 is 2.6 x 10(7) M-1 s-1. Compound I undergoes a one-electron reduction to compound II in the presence of TMB, and the rate constant for this reaction was determined to be (3.6 +/- 0.1) x 10(6) M-1 s-1. The spectral scans show that compound II accumulates in the steady state. The rate constant for compound II reduction to native enzyme by TMB obtained under steady-state conditions is (9.4 +/- 0.6) x 10(5) M-1 s-1. The results are applied to a new, more accurate assay for myeloperoxidase based upon the formation of the charge-transfer complex between TMB and its diimine final product.     

An aquaporin-2 water channel mutant which causes autosomal dominant nephrogenic diabetes insipidus is retained in the Golgi complex

Calcyclin (CaCY) is a member of the S100 subfamily of helix-loop-helix (EF-hand) calcium-binding proteins. Human CaCY was overexpressed in Escherichia coli and purified with an overall yield of 40 mg/l culture. Ca2+ and Zn2+ binding properties of CaCY were examined with respect to the oxidation state of the single Cys residue at position 3. CaCY with the SH group either reduced, blocked or oxidized stays as a dimer as shown by analytical ultracentrifugation. Upon binding of Ca2+, CaCY exhibits 30% enhancement of the Tyr fluorescence, the apparent binding constant (Ka) being 2.8-5.8x10(4) M(-1). Oxidized CaCY binds Ca2+ approximately twice as weakly than its reduced form. The affinity for Ca2+ is increased in the presence of caldesmon, which could be a potential target molecule. Fully reduced CaCY binds Zn2+ with an affinity of at least 1.0x10(7) M(-1). As compared to Ca2+, Zn2+ binding results in a three times greater enhancement of the Tyr fluorescence. Saturation occurs at a Zn2+/CaCY ratio of 2:1. The reactivity of Cys3 is reduced by Zn2+ binding, although oxidized CaCY still binds Zn2+. On the basis of the effects of thiol-directed labels on the affinities for Ca2+ and Zn2+, the fluorescence changes accompanying the binding, and the CaCY reactivity with a hydrophobic probe, it was concluded that the two cations bind to CaCY at different sites: Ca2+ binds probably at the EF-hand type sites, whereas binding of at least one Zn2+ ion involves the Cys residue, and results in a different structural change.     

Transformation of industrial dyes by manganese peroxidases from Bjerkandera adusta and Pleurotus eryngii in a manganese-independent reaction

We investigated the transformation of six industrial azo and phthalocyanine dyes by ligninolytic peroxidases from Bjerkandera adusta and other white rot fungi. The dyes were not oxidized or were oxidized very little by Phanerochaete chrysosporium manganese peroxidase (MnP) or by a chemically generated Mn3+-lactate complex. Lignin peroxidase (LiP) from B. adusta also showed low activity with most of the dyes, but the specific activities increased 8- to 100-fold when veratryl alcohol was included in the reaction mixture, reaching levels of 3.9 to 9.6 U/mg. The B. adusta and Pleurotus eryngii MnP isoenzymes are unusual because of their ability to oxidize aromatic compounds like 2,6-dimethoxyphenol and veratryl alcohol in the absence of Mn2+. These MnP isoenzymes also decolorized the azo dyes and the phthalocyanine complexes in an Mn2+-independent manner. The reactions with the dyes were characterized by apparent Km values ranging from 4 to 16 microM and specific activities ranging from 3.2 to 10.9 U/mg. Dye oxidation by these peroxidases was not increased by adding veratryl alcohol as it was in LiP reactions. Moreover, the reaction was inhibited by the presence of Mn2+, which in the case of Reactive Black 5, an azo dye which is not oxidized by the Mn3+-lactate complex, was found to act as a noncompetitive inhibitor of dye oxidation by B. adusta MnP1.     

Role of residue 99 at the S2 subsite of factor Xa and activated protein C in enzyme specificity

It is thought that only a limited number of residues in the extended binding pocket of coagulation proteases are critical for substrate and inhibitor specificity. A candidate residue from the crystal structures of thrombin and factor Xa (FXa) that may be critical for specificity at the S2 subsite is residue 99. Residue 99 is Tyr in FXa and Thr in activated protein C (APC). To determine the role of residue 99 in S2 specificity, a Gla-domainless mutant of protein C (GDPC) was prepared in which Thr99 was replaced with Tyr of FXa. GDPC T99Y bound Ca2+ and was activated by the thrombin-thrombomodulin complex normally. The T99Y mutant, similar to FXa, hydrolyzed the chromogenic substrates with a Gly at the P2 positions. This mutant was also inhibited by antithrombin (AT) (k2 = 4.2 +/- 0.2 x 10(1) M-1 s-1), and heparin accelerated the reaction >350-fold (k2 = 1.5 +/- 0.1 x 10(4) M-1 s-1). The T99Y mutant, however, did not activate prothrombin but inactivated factor Va approximately 2-fold better than wild type. To try to switch the specificity of FXa, both Tyr99 and Gln192 of FXa were replaced with those of APC in the Gla-domainless factor X (GDFX Y99T/Q192E). This mutant was folded correctly as it bound Ca2+ with a similar affinity as GDFX and was also activated by the Russell's viper venom at similar rate, but it cleaved the chromogenic substrates with a Gly at the P2 positions poorly. The mutant, instead, cleaved the APC-specific chromogenic substrates efficiently. The Y99T/Q192E mutant became resistant to inhibition by AT in the absence of heparin but was inhibited by AT almost normally in the presence of heparin (k2 = 3.4 +/- 0.5 x 10(5) M-1 s-1). The Y99T/Q192E mutant did not inactivate factor Va, and prothrombin activation by this mutant was impaired. These results indicate that 1) residue 99 is critical for enzyme specificity at the S2 subsite, 2) a role for heparin in acceleration of FXa inhibition by AT may involve the S2-P2 modulation, and 3) the exchange of residues 99 and 192 in FXa and APC may switch the enzyme specificity with the chromogenic substrates and inhibitors but not with the natural substrates.     

Structural and functional analysis of the metal-binding sites of Clostridium thermocellum endoglucanase CelD

Crystallographic analysis indicated that Clostridium thermocellum endoglucanase CelD contained three Ca(2+)-binding sites, termed A, B, and C, and one Zn(2+)-binding site. The protein contributed five, six, and three of the coordinating oxygen atoms present at sites A, B, and C, respectively. Proteins altered by mutation in site A (CelDD246A), B (CelDD361A), or C (CelDD523A) were compared with wild type CelD. The Ca(2+)-binding isotherm of wild type CelD was compatible with two high affinity sites (Ka = 2 x 10(6) M-1) and one low affinity site (Ka < 10(5) M-1). The Ca(2+)-binding isotherms of the mutated proteins showed that sites A and B were the two high affinity sites and that site C was the low affinity site. Atomic absorption spectrometry confirmed the presence of one tightly bound Zn2+ atom per CelD molecule. The inactivation rate of CelD at 75 degrees C was decreased 1.9-fold upon increasing the Ca2+ concentration from 2 x 10(-5) to 10(-3) M. The Km of CelD was decreased 1.8-fold upon increasing the Ca2+ concentration from 5 x 10(-6) to 10(-4) M. Over similar ranges of concentration, Ca2+ did not affect the thermostability nor the kinetic properties of CelDD523A. These findings suggest that Ca2+ binding to site C stabilizes the active conformation of CelD in agreement with the close vicinity of site C to the catalytic center.     

Coralyne binds tightly to both T.A.T- and C.G.C(+)-containing DNA triplexes

Coralyne is a DNA-binding antitumor antibiotic whose structure contains four fused aromatic rings. The interaction of coralyne with the DNA triplexes poly(dT).poly(dA).poly(dT) and poly[d(TC)].poly[d(GA)].poly[d(C+T)] was investigated by using three techniques. First, Tm values were measured by thermal denaturation analysis. Upon binding coralyne, both triplexes showed Tm values that were increased more than those of the corresponding duplexes. A related drug, berberinium, in which one of the aromatic rings is partially saturated, gave much smaller changes in Tm. Second, the fluorescence of coralyne is quenched in the presence of DNA, allowing the measurement of binding parameters by Scatchard analysis. The binding isotherms were biphasic, which was interpreted in terms of strong intercalative binding and much weaker stacking interactions. In the presence of 2 mM Mg2+, the binding constants to poly(dT).poly-(dA).poly(dT) and poly[d(TC)].poly[d(GA)].poly[(C+T)] were 3.5 x 10(6) M-1 and 1.5 x 10(6) M-1, respectively, while the affinity to the parent duplexes was at least 2 orders of magnitude lower. In the absence of 2 mM Mg2+, the binding constants to poly[d(TC)].poly[d(GA)].poly[d(C+T)] and poly-[d(TC)].poly[d(GA)] were 40 x 10(6) M-1 and 15 x 10(6) M-1, respectively. Thus coralyne shows considerable preference for the triplex structure but little sequence specificity, unlike ethidium, which will only bind to poly(dT).poly(dA).poly(dT). Further evidence for intercalation of coralyne was provided by an increase in the relative fluorescence quantum yield at 260 nm upon binding of coralyne to triplexes as well as an absence of quenching of fluorescence in the presence of Fe[(CN)6]4-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the competitive degradation of deoxyribose and other biomolecules by hydroxyl radicals produced by the Fenton reaction

The objective of this work is to reexamine the competitive degradation of deoxyribose by hydroxyl radicals (.OH) produced by the reaction between H2O2 and Fe(2+)-EDTA. The .OH radicals produced attack deoxyribose (D, rate constant kD) and eventually an .OH scavenger (S, rate constant kS). First, we examined the effect of [D], [H2O2], [Fe(2+)-EDTA], [EDTA]/[Fe2+] ratio and reaction time on the rate of D degradation, measured as the absorbance of the chromogen formed between the product of the reaction D + .OH (malondialdehyde) and thiobarbituric acid. In particular, it was showed that under our experimental conditions ([D] = 3 mM, [H2O2] = 0.85 mM, [Fe2+] = 0.13 mM), the rate of overall process is first order in Fe2+, zero order in H2O2 and is maximal for a ratio [EDTA]/[Fe2+] = 1.1. Second, the kinetics of .OH radical reaction in competition experiments between D and S (mannitol) was investigated. The results show that the ratio of the rates of D degradation in the absence (VD) and in the presence (VDS) of S should be represented by VD/VDS = 1 + ks[S]/(kD[D] + kx) where kx accounts for the rate of .OH reactions with other reagents such as Fe(2+)-EDTA, H2O2 etc . . . After having determined kx for each set of experimental conditions, we obtained the values of kS/kD by determining the variations of VD/VDS as a function of [S] and [D]. By taking kD = 1.9 x 10(9) M-1s-1 a value of kS = 1.9 x 10(9) M-1s-1 was obtained, very close to that obtained by pulse radiolysis. Finally, the validity of the established relation was confirmed for other biomolecules (methionine, k = 5.6 x 10(9)M-1s-1 and alanine, k = 3.3 x 10(8) M-1s-1). By contrast, it was not applicable to cysteine, thiourea and mercaptoethanol which was attributed to an interaction of the latter scavengers with Fe2+ and/or H2O2.     

Regeneration and luminescence of aequorin in Chinese hamster ovary cells transformed with cDNA for apoaequorin

Aequorin, a photoprotein which is regenerated from apoaequorin by incubation with coelenterazine, emits light when it binds Ca2+. The aim of this study was to determine if apoaequorin could be used in adherent mammalian cells for measuring cytosolic Ca2+, and imaging Ca2+, at the single cell level. Chinese hamster ovary (CHO-K1) cells were stably transformed with apoaequorin cDNA and expressed apoaequorin while attached to the culture dishes. Maximal luminescence intensity was obtained when 0.5 x 10(6) cells/ml were grown and incubated with 2.5 microM coelenterazine for 4 hr at 20 degrees C. Ca2+ mobilizing agents (ionomycin and maitotoxin) induced luminescence in CHO-K1 transformed cells. However, imaging of light emission from single cells proved to be unsuccessful. Ca2+ could be readily measured in the adherent CHO-K1 cells, but imaging was not possible at the single cell level.     

Spectral and kinetic properties of the Fet3 protein from Saccharomyces cerevisiae, a multinuclear copper ferroxidase enzyme

High affinity iron uptake in Saccharomyces cerevisiae requires Fet3p. Fet3p is proposed to facilitate iron uptake by catalyzing the oxidation of Fe(II) to Fe(III) by O2; in this model, Fe(III) is the substrate for the iron permease, encoded by FTR1. Here, a recombinant Fet3p has been produced in yeast that, lacking the C-terminal membrane-spanning domain, is secreted directly into the growth medium. Solutions of this Fet3p at >1 mg/ml have the characteristic blue color of a type 1 Cu(II)-containing protein, consistent with the sequence homology that placed this protein in the class of multinuclear copper oxidases that includes ceruloplasmin. Fet3p has an intense absorption at 607 nm (epsilon = 5500 M-1 cm-1) due to this type 1 Cu(II) and a shoulder in the near UV at 330 nm (epsilon = 5000 M-1 cm-1) characteristic of a type 3 binuclear Cu(II) cluster. The EPR spectrum of this Fet3p showed the presence of one type 1 Cu(II) and one type 2 Cu(II) (A parallel = 91 and 190 x 10(-4) cm-1, respectively). Copper analysis showed this protein to have 3.85 g atom copper/mol, consistent with the presence of one each of the three types of Cu(II) sites found in multinuclear copper oxidases. N-terminal analysis demonstrated that cleavage of a signal peptide occurred after Ala-21 in the primary translation product. Mass spectral and carbohydrate analysis of the protein following Endo H treatment indicated that the preparation was still 15% (w/w) carbohydrate, probably O-linked. Kinetic analysis of the in vitro ferroxidase reaction catalyzed by this soluble Fet3p yielded precise kinetic constants. The Km values for Fe(II) and O2 were 4.8 and 1.3 microM, respectively, while kcat values for Fe(II) and O2 turnover were 9.5 and 2.3 min-1, consistent with an Fe(II):O2 reaction stoichiometry of 4:1.     

Metal ion activation of galactosyltransferase

Galactosyltransferase, which functions as the catalytic component of lactose synthase and in the glycosylation of glycoproteins, has been previously reported to have an absolute dependence on Mn2+ for activity, with a Kd for Mn2+ (10(-3) M) 2 to 3 orders of magnitude greater than the physiological range of Mn2+ concentrations (v 10(-6) M). Reinvestigation of the metal ion dependence of this enzyme has shown that Zn2+, Cd2+, Fe2+, Co2+, and Pr3+ also produce activation, although with lower activities at saturation than that attained with Mn2+. Velocity against metal ion concentration curves for all metals, including Mn2+, are sigmoid, suggesting the presence of two or more activating metal binding sites on the enzyme. The presence of two sites is confirmed by studies using both Mn2+ and Ca2+. While galactosyltransferase is inactive in the presence of Ca2+ alone, at low concentrations of Mn2+ (10(-5) M), enzyme activity is stimulated by Ca2+. A more detailed investigation by steady state kinetics has revealed that there is a tight binding site for Mn2+ (site I: Kd of 2 X 10(-6) M) from which Ca2+ is excluded, and a site at which Ca2+ can replace Mn2+ (site II: Kd for Ca2+ of 1.76 X 10(-3) M), to which metal binding has a specific synergistic effect on UDP-galactose binding, possibly as a result of the formation of an enzyme-Ca2+-UDP-galactose bridge complex. The site I Mn2+, site II Ca2+-activated enzyme has a maximum velocity similar to that of the Mn2+-activated enzyme, and is the enzyme form that must act in lactose synthesis in vivo. A trypsin-degraded form of galactose transferase (galactosyltransferase-T) (Powell, J.T., and Brew, K. (1974) Eur. J. Biochem. 48, 217-228) appears to lack site I and is activated by Ca2+ in the absence of Mn2+.     

Activation of NMDA and non-NMDA receptors in the caudal ventrolateral medulla dilates the airways

Calpain is a ubiquitous calcium-dependent cysteine protease, whose cytoskeletal protein substrates suggest that it may be important in neuronal differentiation. Lead (Pb2+) is known to substitute for Ca2+ in a variety of intracellular processes, and interferes with the development of hippocampal neurons in vitro. We found that free Pb2+ at 1 nM does not activate calpain in the absence of Ca2+. Pb2+ inhibited the activity of calpain; the degree of calpain inhibition was dependent on an interaction between concentrations of both Ca2+ and Pb2+. In the presence of 1 microM free Ca2+, 10 pM free Pb2+ reduced calpain activity, but in the presence of 100 microM free Ca2+, 1 nM free Pb2+ failed to inhibit calpain. This provides evidence that Pb2+ competes for the Ca2+ binding sites on calpain. In the presence of 40 microM free Ca2+, 1 nM free Pb2+ significantly reduces Vmax without altering Km, suggesting that Pb2+ acts as a noncompetitive inhibitor of calpain. Inhibition of calpain is one mechanism by which Pb2+ may interfere with neuronal development.     

Ferulic acid dehydrodimers from wheat bran: isolation, purification and antioxidant properties of 8-O-4-diferulic acid

Wheat bran contains several ester-linked dehydrodimers of ferulic acid, which were detected and quantified after sequential alkaline hydrolysis. The major dimers released were: trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl)-7-methoxy-2, 3- dihydrobenzofuran-3-carboxylic acid (5-8-BendiFA), (Z)-beta-[4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy]-4-hydroxy-3-methox ycinnamic acid (8-O-4-diFA) and (E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid (5-5-diFA). trans-7-hydroxy-1-(4-hydroxy-3methoxyphenyl)-6-methoxy-1,2-dihydro - naphthalene-2,3-dicarboxylic acid (8-8-diFA cyclic form) and 4,4'-dihydroxy-3,3'-dimethoxy-beta,beta'-bicinnamic acid (8-8-diFA non cyclic form) were not detected. One of the most abundant dimers, 8-O-4-diFA, was purified from de-starched wheat bran after alkaline hydrolysis and preparative HPLC. The resultant product was identical to the chemically synthesised 8-O-4-dimer by TLC and HPLC as confirmed by 1H-NMR and mass spectrometry. The absorption maxima and absorption coefficients for the synthetic compound in ethanol were: lambda max: 323 nm, lambda min: 258 nm, epsilon lambda max (M-1 cm-1): 24,800 +/- 2100 and epsilon 280 (M-1 cm-1): 19,700 +/- 1100. The antioxidant properties of 8-O-4-diFA were assessed using: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes and; (b) scavenging of the radical cation of 2,2'-azinobis (3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue, Trolox C. The 8-O-4-diFA was a better antioxidant than ferulic acid in both lipid and aqueous phases. This is the first report of the antioxidant activity of a natural diferulate obtained from a plant.     

Characterization of the single tyrosine containing troponin C from lungfish white muscle. Comparison with several fast skeletal muscle troponin C's from fish species

Troponin C molecules from fast skeletal muscle of the following fish species (trout, whiting, lungfish, tilapia, and cod) have been purified to homogeneity. Upon binding of Ca2+ or Mg2+, lungfish troponin C is the only troponin C from fish white muscle to show the typical increase of tyrosine fluorescence emission quantum yield reported for rabbit fast skeletal muscle troponin C. The increase of tyrosine fluorescence signal occurring upon Ca2+ and Mg2+ titration of lungfish troponin C has been used to determine the corresponding affinity constants. With K(Ca) = 7.0 10(7) M-1 and K(Mg) = 3.6 10(3) M-1, the sites probed by the tyrosine residue of lungfish troponin C are typical of the COOH-terminal domain of fast skeletal troponin C's. The amino acid sequencing of the tyrosine containing tryptic peptides has allowed us to position the single tyrosine residue at position 7 in the Ca2+ binding loop of the third site, in identical position to Tyr109 of troponin C from rabbit fast skeletal muscle. Metal ion binding studies followed by intrinsic fluorescence or Tb3+ luminescence indicate that the conformation of the structural domain of lungfish troponin C with one metal ion bound is close to the physiological conformation of this domain.     

Assessment of the degree of disability in narcolepsy

The effect of free fatty acids (FFA) and non-enzymatic glycation on the binding kinetics of dansylsarcosine (DS) to human serum albumin (HSA) was studied using the stopped-flow technique. The influence of FFA on the binding parameters of 25% glycated HSA depended on the type of fatty acid. The addition of stearic, oleic and linoleic acids in a concentration of 0.3 mmol/l showed no inhibitory effects on the association rate constant (k2) value for DS binding to 25% glycated HSA (k2 without FFA: 385 +/- 10 s-1, k2 with FFA > or = 385 +/- 10 s-1). In contrast, shorter chain fatty acids (hexanoic, octanoic, decanoic, lauric and myristic acids) showed marked inhibitory effects for 0.3 mmol/l FFA (k2 range: 233 +/- 32 to 69 +/- 5 s-1) and for 0.6 mmol/l FFA (k2 range: 125 +/- 3 to 20 +/- 4 s-1). The association rate constant (k2) as well as the affinity constant (KA) of DS were markedly affected by glycation: k2 was 686 +/- 61 s-1 for 7% glycated HSA, 385 +/- 10 s-1 for 25% glycated HSA and 209 +/- 12 s-1 for 50% glycated HSA. KA decreased from 6.1 +/- 2.9 x 10(5) M-1 for 7% glycated HSA, to 5.1 +/- 0.1 x 10(5) M-1 for 25% glycated HSA and to 1.3 +/- 0.6 x 10(5) M-1 for 50% glycated HSA.(ABSTRACT TRUNCATED AT 250 WORDS)