Characterization of the interactions between the nucleoprotein and the phosphoprotein of Henipavirus

The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a previous study, we reported that in henipaviruses, the N-terminal domain of the phosphoprotein and the C-terminal domain of the nucleoprotein (N(TAIL)) are both intrinsically disordered. Here we show that Henipavirus N(TAIL) domains are also disordered in the context of full-length nucleoproteins. We also report the cloning, purification, and characterization of the C-terminal X domains (P(XD)) of Henipavirus phosphoproteins. Using isothermal titration calorimetry, we show that N(TAIL) and P(XD) form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 M and has a K(D) in the μM range. Using far-UV circular dichroism and nuclear magnetic resonance, we show that P(XD) triggers an increase in the α-helical content of N(TAIL). Using fluorescence spectroscopy, we show that P(XD) has no impact on the chemical environment of a Trp residue introduced at position 527 of the Henipavirus N(TAIL) domain, thus arguing for the lack of stable contacts between the C termini of N(TAIL) and P(XD). Finally, we present a tentative structural model of the N(TAIL)-P(XD) interaction in which a short, order-prone region of N(TAIL) (α-MoRE; amino acids 473-493) adopts an α-helical conformation and is embedded between helices α2 and α3 of P(XD), leading to a relatively small interface dominated by hydrophobic contacts. The present results provide the first detailed experimental characterization of the N-P interaction in henipaviruses and designate the N(TAIL)-P(XD) interaction as a valuable target for rational antiviral approaches.     

兔主动脉前庭自律细胞与窦房结电生理特性的比较

为进一步阐明左心室流出道(主动脉前庭)自律细胞的特性,及其与窦房结细胞的异同,本实验利用常规的玻璃微电极细胞内记录技术,观察了一些离子通道阻断剂分别对离体兔窦房结起搏细胞与左心室流出道慢反应自律细胞的电生理特性的影响,重点探讨了这两种自律细胞的0期、4期去极离子流的异同。结果表明:(1)用1μmol/L维拉帕米(verapamil,VER)灌流后,窦房结及主动脉前庭自律细胞的动作电位幅值(APA)、0相最大除极速率(V_(max))、最大舒张电位(MDP)绝对值、舒张期除极速率(VDD)、自发放电频率(RPF)均明显下降,复极90％时间(APD_(90))延长(P<0.05)。(2)用180μmol/L氯化镍(NiCl_2)灌流,两自律细胞的VDD均明显下降;APA、V_(max)和RPF也显著降低,且窦房结细胞的APD_(90)明显延长。(3)给予2 mmol/L 4-氨基吡啶(4-AP)后,窦房结及主动脉前庭自律细胞的VDD均明显增快,MDP绝对值、APA和V_(max)显著下降,APD_(90)明显延长(P<0.05)。(4)给予2 mmol/L氯化铯(CsCl),两自律细胞的VDD及RPF均明显变慢。结果提示:(1)主动脉前庭自发慢反应电位的0相、4相去极离子流及复极离子流均与窦房结优势起搏细胞相似。(2)主动脉前庭起搏细胞Ca~(2+)内流为其0相主要去极离子流,复极过程主要由K~+外流引起,4相自动除极以K~+外流衰减为主,另外     

Message from the President of the Society

The circadian rhythm of locomotor activity of the field mouse Mus booduga was studied and single animal phase response curves (PRCs) (n = 8) were constructed for 15-min daylight pulses of 1000 lux intensity. The light pulses, presented at different phases of the circadian cycle, evoked advancing and delaying phase shifts (ΔPHs) depending on the circadian time (CT) of light pulse application. ΔPHs by light pulses applied at the same phase are strongly correlated with the animals' circadian period (τ). The results indicate a significant correlation between (i) τ and the area under the advance zone of the PRC (A) (r = +0.72, p > 0.05), (ii) τ and the area under the delay zone of the PRC (D) (r = -0.98, p > 0.00001), (iii) τ and the difference between the area under delay and advance zone of PRC (D-A) (r = -0.97, p > 0.00001), and (iv) between τ and ΔpHs (at various phases of the circadian cycle) and further suggest that the waveform and time course of PRC depend on the animals' endogenous period (τ). (Chronobiology International, 13(6), 401–409, 1996)     

Mechanisms mediating the oxygen-induced vasoreactivity of the ductus arteriosus in the chicken embryo

The avian embryo provides a novel model for studying the ductus arteriosus (DA) during the transition from in ovo to ex ovo life. Here we examined the mechanisms regulating the vasoreactivity of the two morphologically distinct portions of the chicken DA (proximal and distal) in response to O(2). Oxygen-induced contraction is redox sensitive and reversed by the reducing agent dithiothreitol and the H(2)O(2) scavenger N-mercaptopropionylglycine. As in the mammalian DA, inhibiting mitochondrion-derived reactive oxygen species production with rotenone and antimycin A relaxed the O(2)-constricted DA. The contractile response to O(2) matures during hatching and is mimicked by the K(v) channel inhibitor 4-aminopyridine (4-AP) on day 19 and externally pipped (EP) embryos. Together, O(2) and 4-AP significantly increase DA tone above that observed with either alone. The O(2)-induced contraction is mediated by influx of extracellular Ca(2+) through l-type Ca(2+) and store-operated channels. Inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores play a minor role in the O(2)-induced contraction. The O(2)-induced contraction is mediated by the Rho kinase pathway, as fasudil and Y-27632 significantly relax the O(2) contracted DA. Prostaglandins E(2), F(2alpha), and D(2) produce significant contraction of the proximal DA. The O(2)-induced relaxation of the distal portion of the DA is mediated by an endothelial-derived nitric oxide/cGMP pathway. Both 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and endothelial cell removal inhibit O(2)-induced relaxation in the distal segment. Mechanisms regulating O(2)-induced contraction in chicken proximal DA are similar to those found in mammalian DA, making the chicken a useful model for studying development of this O(2)-sensitive vessel.     

The nature of the acid-volatile selenium in the liver of the male rat

1. The properties of rat liver acid-volatile selenium have been compared with those of H(2)Se and (CH(3))(2)Se. 2. In model experiments oxidation-sensitive H(2) (75)Se was trapped quantitatively under anaerobic conditions in 0.1m-AgNO(3), and (CH(3))(2) (75)Se was trapped quantitatively in 8m-HNO(3). The acid-labile selenium of a liver homogenate, and of a microsomal fraction, was found to behave quite unlike (CH(3))(2) (75)Se and in a manner indistinguishable from H(2) (75)Se. 3. It was concluded that the acid-volatile material is certainly not (CH(3))(2)Se and that it is probably H(2)Se. 4. The significance of these findings is discussed in relation to current knowledge about the metabolism and detoxication of selenium, and a scheme is proposed which incorporates this knowledge with recent observations on the interactions between trace amounts of selenium and tocopherol, and the production of acute selenium deficiency by Ag(+) in vitamin E-deficient rats.     

Studies on the molecular mechanism of the translocation of estrogen receptor from the cytoplasm into the nucleus

A detailed study of the molecular mechanism of the translocation of estrogen receptor (ER) from the cytoplasm into the nucleus was undertaken in an in vitro system of porcine uterus. The capabilities of vero-ER . E (basic ER molecular bound with estradiol) (sedimentation coefficient 4.5S; Stokes radius 44 A) and the complexes ["5S" ER . E, (vero-ER . E) . (component A); "6S" ER . E, (vero-ER . E) . (component B)6; "8S" ER . E, (vero-ER . E) . (component B)6 . (component A)] with ER-binding factors (ERBFs) to translocate into the isolated nuclei were estimated by subtracting the amounts of ER adsorbed by the nuclear envelopes from those of ER bound to the whole nuclei. The results strongly supported our previous assumption that vero-ER . E translocates into the nuclei, and the complexes with ERBFs do not. The results suggested also that the binding site of vero-ER to ERBFs is required to be unoccupied in the process of the translocation of ER from the cytoplasm into the nucleus. The presence of a cytoplasmic factor (component C) which binds specifically with "5S" ER . E under low salt conditions was indicated. The complex, ("5S" ER . E) . (component C), was shown to possess relatively high affinity towards nuclear envelopes, but not to translocate into the nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the origin of rhythmic calcium transients in the ICC-MP of the mouse small intestine

Interstitial cells of Cajal associated with the myenteric plexus (ICC-MP) are pacemaker cells of the small intestine, producing the characteristic omnipresent electrical slow waves, which orchestrate peristaltic motor activity and are associated with rhythmic intracellular calcium oscillations. Our objective was to elucidate the origins of the calcium transients. We hypothesized that calcium oscillations in the ICC-MP are primarily regulated by the sarcoplasmic reticulum (SR) calcium release system. With the use of calcium imaging, study of the effect of T-type calcium channel blocker mibefradil revealed that T-type channels did not play a major role in generating the calcium transients. 2-Aminoethoxydiphenyl borate, an inositol 1,4,5 trisphosphate receptor (IP(3)R) inhibitor, and U73122, a phospholipase C inhibitor, both drastically decreased the frequency of calcium oscillations, suggesting a major role of IP(3) and IP(3)-induced calcium release from the SR. Immunohistochemistry proved the expression of IP(3)R type I (IP(3)R-I), but not type II (IP(3)R-II) and type III (IP(3)R-III) in ICC-MP, indicating the involvement of the IP(3)R-I subtype in calcium release from the SR. Cyclopiazonic acid, a SR/endoplasmic reticulum calcium ATPase pump inhibitor, strongly reduced or abolished calcium oscillations. The Na-Ca exchanger (NCX) in reverse mode is likely involved in refilling the SR because the NCX inhibitor KB-R7943 markedly reduced the frequency of calcium oscillations. Immunohistochemistry revealed 100% colocalization of NCX and c-Kit in ICC-MP. Testing a mitochondrial NCX inhibitor, we were unable to show an essential role for mitochondria in regulating calcium oscillations in the ICC-MP. In summary, ongoing IP(3) synthesis and IP(3)-induced calcium release from the SR, via the IP(3)R-I, are the major drivers of the calcium transients associated with ICC pacemaker activity. This suggests that a biochemical clock intrinsic to ICC determines the pacemaker frequency, which is likely directly linked to kinetics of the IP(3)-activated SR calcium channel and IP(3) metabolism.     

A comparative study of the interaction of the cholinesterase from the brain of the cabbage fly, the acetylcholinesterase from bovine erythrocytes and the butyrylcholinesterase from horse blood serum with organophosphorus inhibitors

Studies have been made of the effect of several organophosphorus inhibitors, R1(R2)P(O) . SCH2CH2SR and R1(R2)P(O)SCH2CH2SRR . -O4SCH3 (or -I), which differ by the structure of split (R, P) and phosphoryl (R1, R2) parts of the molecule, on cholinesterase (ChE) from the brain of the fly Delia brassicae, acetylcholinesterase (AChE) of the bovine erythrocytes and butyrylcholinesterase (BuChE) from the blood serum of the horse. For fly ChE, higher values of a constant (kII) of the inhibition rate (at pH 7.5 and temperature 25 degrees C) were obtained both with thiophosphates and with thiophosphonates. This finding reveals higher reactivity of the active centre of this enzyme, as well as significantly lower selectivity of the latter to the structure of organophosphorus inhibitors. The data obtained suggest the existence of differences in the size of hydrophobic regions of anionic and esterase parts of the active centre in ChE of the fly and AChE of mammals, as well as the existence of some similarity between ChE of the fly and BuChE.     

Investigation of the mechanism of the methylmalonyl-CoA mutase reaction with the substrate analogue: ethylmalonyl-CoA.

1. Ethylmalonyl-CoA was found to be a substrate for methylmalonyl-CoA mutase from Propionibacterium shermanii, the product being mainly (2R)-methylsuccinyl-CoA along with some (2S)-diastereoisomer. 2. The relevant 1H-nuclear magnetic resonance signals of methylsuccinic acid and of its dimethyl ester were assigned to the diastereotopic methylene hydrogens using sterospecifically dideuterated specimens of known configuration. 3. [2(-2)H1]Ethylmalonyl-CoA was converted by methylmalonyl-CoA mutase in 2H2O mainly to (2R, 3S)-[3(-2)H1]methylsuccinyl-CoA. No dideuterated product was observed. 4. Starting from (1R)-[1(-2)H1]-ethathanol, (1S)-[1(-2)H1]ethanol and [2H6] ethanol the following deuterated specimens of ethylmalonic acid were synthesised and characterised: (3S)-[3(-2)H1], (3R)-[3(-2)H1] and [3(-2)H2, 4(-2)H3], respectively. 5. Conversion of (3S)-[3(-2)H1]-ethylmalonyl-CoA (70% 2H1 and 2% 2H2 species) on the mutase in water afforded mainly (2R)-[2(-2)H1]methylsuccinyl-CoA along with some (2S)-diastereoisomer. No deuterium loss was observed. 6. Methylmalonyl-CoA mutase converted (3R)-[3(-2)H1]ethylmalonyl-CoA (81% 2H1 and 2% 2H2 species) to the following methylsuccinyl-CoA species: 33% [3(-2)H1], the deuterium being in the threo position with respect to the methyl group; 21% [2(-2)H1]; 46% unlabelled. The ratio of the species with (2R) and (2S) configuration was about 60:40. 7. Reaction of [3(-2)H2, 4(-2)H3]ethylmalonyl-CoA (94.5% [2H5] species) with the mutase gave the following labelled methylsuccinyl-CoA species:53.4% [methyl-2H3, 2(-2)H1, 3(-2)H1], the 3-deuterium being in the threo position with respect to the methyl group; 37.6% [methyl-2H3, 2(-2)H1]; 5% [methyl(-2)H3, 2(-2)H1, 2(-2)H1, 3(-2)H1] the 3-deuterium being in erythro position with respect to the methyl group; 4% [methyl(-2)H3, 3(-2)H1]. The ratio of the species with (2R) and (2S) configuration was about 70:30. 8. Implications of these findings for the mechanism of the rearrangements catalysed by coenzyme B12 are discussed.     

Neurotransmitters in the thalamus relaying visceral input to the insular cortex in the rat

Neurotransmitters relaying ascending visceral information were examined by comparing the response of neurons in the insular cortex to vagal stimulation (0.8 Hz, 2 mA) before and after neurotransmitter antagonist injections (200 nl) in the ventroposterior parvocellular nucleus of the thalamus (VPpc). Cobalt (10 mM; presynaptic blocker) and kynurenate (100 microM; nonspecific excitatory amino acid antagonist) injections in the VPpc resulted in an attenuation (73-100 and 38-98%, respectively) of the evoked cortical response. Injections of the specific N-methyl-D-aspartate (NMDA) antagonist DL-2-amino-5-phosphonopentanoic acid (200 microM and 2 mM) did not affect the vagally evoked response, whereas the nonspecific non-NMDA antagonist L-glutamic acid diethylester (200 microM) attenuated the vagally evoked response by 66-100%. Three concentrations of the DL-alpha-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA)-specific antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 and 200 microM and 2 mM) attenuated the vagally evoked cortical response by 29 +/- 9, 31 +/- 10, and 59 +/- 8%, respectively. The more selective AMPA antagonist 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (200 microM and 2 mM) inhibited the vagally evoked cortical response by 53 +/- 8 and 52 +/- 3%, respectively. Phentolamine (0.1 and 1.0 microM), a general alpha-adrenergic antagonist, and picrotoxin (0.1 and 1.0 microM), a GABA(A) antagonist, did not affect the vagally evoked response. Atropine, a muscarinic cholinergic antagonist, decreased the vagally evoked response by 40 +/- 2% at a concentration of 0.1 microM, but a higher concentration of 1.0 microM had no effect. These results indicate that the non-NMDA excitatory amino acid receptor is necessary for the relay of visceral information in the VPpc. Muscarinic receptors may modulate visceral neuronal excitability in the VPpc, although the exact interaction between the inhibitory (m2) and excitatory (m3 or m5) muscarinic receptor types found in the thalamus is not known.     

Mixed municipal waste management in the Czech Republic from the point of view of the LCA method

Purpose  

This paper presents the results of a life-cycle assessment (LCA) study for integrated systems (IS) of mixed municipal waste (MMW) management in the Czech Republic. The seven IS categories assessed were: (a) incineration with slag recovery, (b) incineration without slag recovery, (c) landfills with incineration of the landfill gas by flaring, (d) landfills with recovery of the landfill gas, (e) mechanical–biological treatment (MBT) with aerobic treatment, (f) MBT biodrying with co-incineration of refuse-derived fuel, and (g) MBT biodrying with incineration of refuse-derived fuel from a monosource.     

Synthetic peptides as model substrates for the study of the specificity of the polycation-stimulated protein phosphatases

The substrate specificity of the different forms of the polycation-stimulated (PCS, type 2A) protein phosphatases and of the active catalytic subunit of the ATP, Mg-dependent (type 1) phosphatase (AMDC) was investigated, using synthetic peptides phosphorylated by either cyclic-AMP-dependent protein kinase or by casein kinase-2. The PCS phosphatases are very efficient toward the Thr(P) peptides RRAT(P)VA and RRREEET(P)EEE when compared with the Ser(P) analogues RRAS(P)VA and RRREEES(P)EEEAA. Despite their distinct sequence, both Thr(P) peptides are excellent substrates for the PCSM and PCSH1 phosphatases, being dephosphorylated faster than phosphorylase a. The slow dephosphorylation of RRAS(P)VA by the PCS phosphatases could be increased substantially by the insertion of N-terminal (Arg) basic residues. In contrast with the latter, the AMDC phosphatase shows very poor activity toward all the phosphopeptides tested, without preference for either Ser(P) or Thr(P) peptides. However, N-terminal basic residues also favor the dephosphorylation of otherwise almost inert substrates by the AMDC phosphatase. Hence, while the dephosphorylation of Thr(P) substrates by the PCS phosphatases is highly favored by the nature of the phosphorylated amino acid, phosphatase activity toward Ser(P)-containing peptides may require specific determinants in the primary structure of the phosphorylation site.     

Organization of the telencephalon in the channel catfish,Ictalurus punctatus

The cytoarchitectonics of the telencephalon of the channel catfish, Ictalurus punctatus, are described as a basis for experimental analysis of telencephalic afferents and efferents. The olfactory bulb comprises: (1) an outer layer of olfactory nerve fibers, (2) a glomerular layer, (3) an external cell layer, (4) an inner fiber layer, and (5) an internal cell layer. The telencephalic hemispheres comprise the areas ventralis and dorsalis telencephali. The area ventralis consists of: (1) a precommissural, periventricular zone including nucleus 'nother (Vn), the ventral nucleus (Vv), and the dorsal nucleus (Vd); (2) a precommissural, migrated zone of central (Vc) and lateral (VI) nuclei; (3) a supracommissural nucleus (Vs); (4) a caudal commissural zone of postcommissural (Vp) and intermediate (Vi) nuclei; and (5) a preoptic area (PP). The area dorsalis comprises: (1) medial (DM), (2) dorsal (Dd), (3) lateral [DL, containing dorsal (DLd), ventral (DLv), and posterior (DLp) regions], (4) posterior (DP), and (5) central (DC-1, -2, -3) areas. Nucleus taeniae (NT) is transitional between areas dorsalis and ventralis.     

On the chemiluminescence in the oxidation of tetravalent uranium to the uranyl ion by dimethyldioxirane

The reaction of the tetravalent uranium [U(IV)] with dimethyldioxirane (DMD) in strongly acidic water–acetone solutions is accompanied by chemiluminescence (CL) in the visible (Vis) and infra‐red (IR) regions. At least three independent reaction pathways are involved in the U(IV)–DMD oxidation: the first entails the non‐chemiluminescent oxidation of U(IV) to the uranyl ion (UO₂²⁺); the second involves the catalytic decomposition of DMD by U(IV) to afford singlet oxygen, as manifested by its characteristic IR‐CL; and in the third process, slow Vis‐CL (510–540 nm) is emitted, following DMD consumption. Copyright © 2002 John Wiley & Sons, Ltd.     

Vibrio vulnificus has the transmembrane transcription activator ToxRS stimulating the expression of the hemolysin gene vvhA

In an attempt to dissect the virulence regulatory mechanism in Vibrio vulnificus, we tried to identify the V. cholerae transmembrane virulence regulator toxRS (toxRS(Vc)) homologs in V. vulnificus. By comparing the sequences of toxRS of V. cholerae and V. parahaemolyticus (toxRS(Vp)), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kb BglII-HindIII fragment and a 1.2-kb HindIII fragment containing two complete open reading frames and one partial open reading frame attributable to toxR(Vv), toxS(Vv), and htpG(Vv) were cloned. ToxR(Vv) shared 55.0 and 63.0% sequence homology with ToxR(Vc) and ToxR(Vp), respectively. ToxS(Vv) was 71.5 and 65.7% homologous to ToxS(Vc) and ToxS(Vp), respectively. The amino acid sequences of ToxRS(Vv) showed transmembrane and activity domains similar to those observed in ToxRS(Vc) and ToxRS(Vp). Western blot analysis proved the expression of ToxR(Vv) in V. vulnificus. ToxRS(Vv) enhanced, in an Escherichia coli background, the expression of the V. vulnificus hemolysin gene (vvhA) fivefold. ToxRS(Vv) also activated the ToxR(Vc)-regulated ctx promoter incorporated into an E. coli chromosome. A toxR(Vv) null mutation decreased hemolysin production. The defect in hemolysin production could be complemented by a plasmid harboring the wild-type gene. The toxR(Vv) mutation also showed a reversed outer membrane protein expression profile in comparison to the isogenic wild-type strain. These results demonstrate that ToxR(Vv) may regulate the virulence expression of V. vulnificus.     

Independence of the (NAD+):(NADH) ratio from the adenylic system in the liver cytoplasm of the developing chick embryo]

No dependence was found between the index of the adenylic system phosphorylated state (ATP) : (ADP) (HPO2-4), the ratios (ATP) : (ADP) and (ATP : (ADP + AMP), on one hand, and the ratio (NAD+) : (NADH) in the cytoplasm, on the other one. The maximum value of the ratio (ATP) : (ADP) (HPO2-4) was observed on the 17th day of development and correlated with the maximum rate of gluconeogenesis. The ratio (NAD+) : (NADH) in the cytoplasm suffered no changes until hatching and decreased twice thereafter.     

Reactivity of the heme-dioxygen complex of the inducible nitric oxide synthase in the presence of alternative substrates

Single turnover reactions of the inducible nitric oxide synthase oxygenase domain (iNOSoxy) in the presence of several non alpha-amino acid N-hydroxyguanidines and guanidines were studied by stopped-flow visible spectroscopy, and compared with reactions using the native substrates L-arginine (L-arg) or N(omega)-hydroxy-L-arginine (NOHA). In experiments containing dihydrobiopterin, a catalytically incompetent pterin, and each of the studied substrates, L-arg, butylguanidine (BuGua), para-fluorophenylguanidine (FPhGua), NOHA, N-butyl- and N-(para-fluorophenyl)-N'-hydroxyguanidines (BuNOHG and FPhNOHG), the formation of a iron(II) heme-dioxygen intermediate (Fe(II)O2) was always observed. The Fe(II)O2 species then decayed to iron(III) iNOSoxy at rates that were dependent on the nature of the substrate. Identical reactions containing the catalytically competent cofactor tetrahydrobiopterin (BH4), iNOSoxy and the three N-hydroxyguanidines, all exhibited an initial formation of an Fe(II)O2 species that was successively converted to an Fe(III)NO complex and eventually to high-spin iron(III) iNOSoxy. The formation and decay kinetics of the Fe(III)NO complex did not vary greatly as a function of the N-hydroxyguanidine structure, but the formation of Fe(III)NO was substoichiometric in the cases of BuNOHG and FPhNOHG. Reactions between BH4-containing iNOSoxy and BuGua exhibited kinetics similar to those of the corresponding reaction with L-arginine, with formation of an Fe(II)O2 intermediate that was directly converted to high-spin iron(III) iNOSoxy. In contrast, no Fe(II)O2 intermediate was observed in the reaction of BH4-containing iNOSoxy and FPhGua. Multi-turnover reaction of iNOS with FPhGua did not lead to formation of NO or to hydroxylation of the substrate, contrary to reactions with BuGua or L-arg. Our results reveal how different structural and chemical properties of NOS substrate analogues can impact on the kinetics and reactivity of the Fe(II)O2 intermediate, and support an important role for substrate pKa during NOS oxygen activation.     

Participation of the C-terminal region of the D1-polypeptide in the first steps in the assembly of the Mn4Ca cluster of photosystem II

Amino acid residue D1-Asp(170) of the D1-polypeptide of photosystem II was previously shown to be implicated in the binding and oxidation of the first manganese to be assembled into the Mn(4)Ca cluster of the oxygen-evolving complex (OEC). According to recent x-ray crystallographic structures of photosystem II, D1-Glu(333) is proposed to participate with D1-Asp(170) in the coordination of Mn4 of the OEC. Other residues in the C-terminal region of the D1-polypeptide are proposed to coordinate nearby manganese of the cluster. Site-directed replacements in Synechocystis sp. PCC 6803 at D1-His(332), D1-Glu(333), D1-Asp(342), D1-Ala(344), and D1-Ser(345) were examined with regard to their ability to influence the binding and oxidation of the first manganese in manganese-depleted photosystem II core complexes. Direct and indirect measurements reveal in all mutants, but most marked in D1-Glu(333) replaced by His, an impaired ability of Mn(2+) to reduce Y(Z)., indicating a reduced ability (elevated K(m)) compared with WT to bind and oxidize the first manganese of the OEC. The effect on the K(m) of these mutations is, however, considerably weaker than some of those constructed at D1-Asp(170) (replacement by Asn, Ala, and Ser). These observations imply that the C-terminal residues ultimately involved in manganese coordination contribute to the high affinity binding at D1-Asp(170) likely through electrostatic interactions. That these residues are far from D1-Asp(170) in the primary structure of the D1-polypeptide, imply that the C terminus of the D1-polypeptide is already close to its mature conformation at the first stages of assembly of the Mn(4)Ca cluster.