固氮酶铁钼辅基在分离纯化中结构变化的新证据

根据Ｋｉｍ－Ｒｅｅｓ模型［１］,固氮酶铁钼辅基（即ＦｅＭｏｃｏ或Ｍ簇）,是由一个ＭｏＦｅ３Ｓ３簇和一个Ｆｅ４Ｓ３簇通过三个Ｓ－桥联接而成．然而,自Ｓｈａｈ等（１９７７）首次从结晶的钼铁蛋白中分离出具有生物重组活性的ＦｅＭｏｃｏ以来,固氮研究者们一直致...     

Reversible inhibition of ovine ceruloplasmin by thiomolybdates

The synthesis and purification of the sodium salts of di (MoO2S2(2-)), tri (MoOS3(2-)) and tetra (MoOS4(2-)) thiomolybdate is reported. All three compounds were reversible inhibitors of the ovine ceruloplasmin (Cp) catalysed oxidation of O-dianisidine. Na2MoO2S2 inhibited via a non-competitive mechanism whereas Na2MoOS3 showed mixed non competitive and Na2MoS4 competitive mechanisms. All showed upward curving slope replots. Molybdenum trisulfide (MoS3) was synthesized and displayed mixed type inhibition kinetics but with a linear slope replot. Preliminary evidence suggested that both MoOS3(2-) and MoS4(2-) may also be substrates for ovine Cp.     

A new method for extraction of iron-molybdenum cofactor (FeMoco) from nitrogenase adsorbed to DEAE-cellulose. 2. Solubilization of FeMoco in a wide range of organic solvents

While the iron-molybdenum cofactor (FeMoco) of nitrogenase, a constituent of the active site for nitrogen reduction, can be extracted into N-methylformamide (NMF) and pyrrollidinone, the inability to solubilize it in any other organic solvents has hampered further understanding of its structure and chemical properties. A method to solubilize FeMoco, prepared in N,N-dimethylformamide (DMF) with Bu4N+ as counterion [McLean, P. A., Wink, D. A., Chapman, S. K., Hickman, A. B., McKillop, D. M., & Orme-Johnson, W. H. (1989) Biochemistry (preceding paper in this issue)], in acetonitrile, acetone, methylene chloride, tetrahydrofuran, and benzene is reported. FeMoco evaporated to dryness in vacuo dissolves readily in good yield (55-100%) and with no significant loss in specific activity. In addition, FeMoco can be extracted directly into these solvents from MoFe protein bound to a DEAE-Sepharose column if the protein is pretreated with DMF. Methods have also been developed to extract fully active FeMoco into acetone and acetonitrile in the absence of any amide solvents (NMF or DMF). Extraction of FeMoco into acetone (30% yield) involves only pretreatment of column-bound protein with methanol, while extraction into acetonitrile (22% yield) requires pretreatment with methanol followed by THF. We conclude that the presence of a suitable soluble cation confers solubility to the cofactor in many common organic solvents and that the solubility of FeMoco in a given solvent may be independent of the ability of that solvent to extract the cofactor from column-bound protein.     

Thiomolybdates in rumen contents and rumen cultures

Examination of direct and (Cu)-difference spectra of i) the aqueous supernatants of in vitro cultures of bovine rumen contents incubated with MoO42- and potential sources of S2- and ii) samples drawn directly from the rumen of animals receiving high Mo diets yielded evidence of the presence of thiomolybdates. Only MoS42- was detected in the soluble phase of in vitro cultures. Although intense and variable background absorbance precluded full characterization of thiomolybdate species in samples drawn directly from the rumen, both spectral data and the biochemical and clinical responses of animals given high Mo diets were consistent with the conclusion that MoS42- rather than MoOS32- was the predominant thiomolybdate species present in the aqueous phase. Addition of Ca2+ either to rumen cultures before incubation or as a supplement to diets high in MoO42- content inhibited the appearance of MoS42- in the aqueous phase. Evidence of the sequestration of MoS42- and MoOS32- by particulate or microbial fractions of rumen contents is considered in relation to the inhibitory action of Mo upon Cu absorption by ruminants.     

Determination of ligand binding constants for the iron-molybdenum cofactor of nitrogenase: monomers, multimers, and cooperative behavior

Equilibrium titrations in N-methylformamide (NMF) of G-25 gel filtered (ox)-state FeMo cofactor [FeMoco(ox)] from Azotobacter vinelandii nitrogenase were carried out using sodium ethanethiolate and followed using UV/Vis absorption spectroscopy. For Fe-Moco(ox), a non-linear least squares (NLLSQ) fit to the data indicated a strong equilibrium thiolate-binding step with Keq = 1.3+/-0.2x10(6) M(-1). With 245 molar excess imidazole, cooperative binding of three ethanethiolates was observed. The best NLLSQ fit gave Keq=2.0+/-0.1x10(5) M(-2) and a Hill coefficient n=2.0+/-0.3. A Scatchard plot of these data was concave upward, indicating positive cooperativity. The fit to previously published data involving benzenethiol titration of the one-electron reduced (semi-reduced) cofactor, FeMoco(sr), as followed by EPR required a model that included both a sub-stoichiometric ratio of thiol to FeMoco(sr) and about five cooperative ligand binding sites. These constraints were met by modeling FeMoco(sr) as an aggregate, with fewer thiol binding sites than FeMoco(sr) units. The best fit model was that of FeMoco(sr) as a dodecamer with five cooperative benzenethiol binding sites, yielding a thiol binding constant of 3.32+/-0.09x10(4) M(-4.8) and a Hill coefficient n=4.8+/-0.6. The results of all the other published ligand titrations of FeMoco(sr) were similarly analyzed successfully in terms of equilibrium models that include both cooperative ligand binding and dimer-level aggregation. A possible structural model for FeMoco aggregation in NMF solution is proposed.     

光谱技术研究固氮酶铁硫簇的理化特性

Ｎ－甲基甲酰胺碱度是提取高质量固氮酶铁钼辅基的关键因素之一。过量的亚甲蓝能氧化并分解铁铜铺基为含双相铁硫簇和铁硫簇固氮酶铁钼辅基和在紫外可见光谱区中均无特征吸收峰,而在３２０ｎｍ处却呈弱吸收峰,棕色固氮菌固氮酶和该菌的突变菌侏ＵＷ４５固氮酶（缺铁钼辅基）中的非含钼的铁硫簇在紫外可见光谱区３２０ｎｍ和４０５ｎｍ处均含有特征吸收峰．     

A new method for extraction of iron-molybdenum cofactor (FeMoco) from nitrogenase adsorbed to DEAE-cellulose. 1. Effects of anions, cations, and preextraction treatments

A convenient and rapid method of obtaining the cofactor of nitrogenase (FeMoco) with a low and apparently limiting Fe/Mo ratio has been developed. FeMoco can be extracted from the MoFe protein bound to DEAE-cellulose. The cofactor is eluted in either N-methylformamide (NMF), N,N-dimethylformamide (DMF), or mixtures of these solvents by use of salts such as Et4NBr,Bu4NBr,Ph4PCl, and Ph4AsCl. The method is simple, is rapid (45 min), yields concentrated cofactor, and, unlike the original method [Shah, V. K., & Brill, W. J. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3249-3253] which requires anaerobic centrifugation, is easily scaled up. Furthermore, it gives yields of cofactor in excess of 70%. Its disadvantages are a high Fe:Mo ratio when DMF is the extracting solvent and a high salt concentration in the resultant FeMoco solution. These disadvantages are easily overcome by removing excess Fe by pretreating the cofactor with bipyridyl while still on the column. This gives Fe:Mo ratios of (6 +/- 1):1 (11 trials) with specific activities ranging from 170 to 220 nmol of C2H4/[min.(nmol of Mo)]. Chromatography on Sephadex LH-20 removes ca. 99% of the excess salt. The adsorption of MoFe protein to DEAE-cellulose seems to facilitate denaturation by organic solvents so that pretreatment of the protein with acid, used in earlier methods, is unnecessary. There is an apparent dependence on the charge density of the anion employed for elution of FeMoco bound to DEAE-cellulose, such that Cl- greater than Br- much greater than I-, PF6- is the order of effectiveness of the Bu4N+ salts of these anions.(ABSTRACT TRUNCATED AT 250 WORDS)     

M?ssbauer studies of aconitase. Substrate and inhibitor binding, reaction intermediates, and hyperfine interactions of reduced 3Fe and 4Fe clusters

Active beef heart aconitase contains a [4Fe-4S] cluster. One iron of the cluster, Fea, is labile and can be removed easily by oxidation in air to yield the [3Fe-4S]1+ cluster of inactive aconitase. We have previously shown that substrate binds to Fea. We have continued our M?ssbauer studies by further investigating the active and inactive forms of the enzyme. When active aconitase, [4Fe-4S]2+, is mixed with substrate, two species (substrates or intermediates bound to Fea) labeled S1 and S2 are obtained. With the nitroanalogs of citrate and isocitrate, thought to be transition state analogs, and fluorocitrate, species S2, but not S1, is observed, suggesting that S2 represents a carbanion transition state complex. We have prepared M?ssbauer samples by rapid mix/rapid freeze techniques. Using either citrate, isocitrate or cis-aconitate, the natural substrates, we have been able to detect at 0 degree C reaction intermediates in the 5-35 ms time range and, studying enzyme substrate interactions at subzero temperatures in a water/methanol/ethylene glycol solvent, we have observed new species when substrates were added at -60 degrees C. Details of these experiments are given, although in neither case can unique interpretations be offered at this time. We have also investigated reduced active aconitase ([4Fe-4S]1+; EPR at g = 1.94) in the presence of substrate with material selectively enriched with 57Fe in either Fea or the other three cluster sites. The spectra were analyzed with a spin Hamiltonian, and the results are discussed and interpreted in terms of three inequivalent Fe sites in the cluster. Finally, we have studied enzyme containing the reduced [3Fe-4S]0 cluster. There is no indication that citrate binds to the 3Fe cluster, and since no significant activity was observed, we conclude that aconitase containing a 3Fe cluster is not active in either oxidation state.     

Topography of oligomycin sensitivity conferring protein in the mitochondrial adenosinetriphosphatase-ATP synthase

The topographical organization of oligomycin sensitivity conferring protein (OSCP) in the mitochondrial adenosinetriphosphatase (ATPase)-ATP synthase complex has been studied. The accessibility of OSCP to monoclonal antibodies has been qualitatively visualized by using the protein A-gold electron microscopy immunocytochemistry or quantitatively estimated by immunotitration of OSCP in depolymerized or intact membranes. Besides, OSCP cannot be labeled by 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) which selectively labels the hydrophobic core of membrane proteins. These observations demonstrate an external location of OSCP on the inner face of the inner mitochondrial membrane. The position of OSCP relative to other peptides of the complex has been analyzed by cross-linking experiments using either zero length N-(ethoxycarbonyl)-2-ethoxydihydroquinoline or 11-A span dimethyl suberimidate cross-linkers in the ATPase-ATP synthase complex. The OSCP cross-linked products were identified either by immunocharacterization with anti-alpha, anti-beta, or anti-OSCP monoclonal antibodies or by their molecular weight. OSCP was cross-linked with either the alpha- or beta-subunits of F1 or to a subunit of Mr 24 000. Other types of cross-linking were obtained by the labeling of OSCP with [cysteamine-35S]-N-succinimidyl 3-[[2-((2-nitro-4-azidophenyl)amino)ethyl]dithio]propionate ([35S]SNAP) and reconstitution of SNAP-OSCP with F1 in urea-treated submitochondrial particles. Under these conditions, OSCP is found to be adjacent to two other peptides of molecular weight close to 30 000. A comparison is made between the topology and the organization of the b-subunit of Escherichia coli and OSCP, suggesting an analogy between OSCP and the hydrophilic part of the b-subunit.     

O2 reactions at the six-iron active site (H-cluster) in [FeFe]-hydrogenase

Irreversible inhibition by molecular oxygen (O(2)) complicates the use of [FeFe]-hydrogenases (HydA) for biotechnological hydrogen (H(2)) production. Modification by O(2) of the active site six-iron complex denoted as the H-cluster ([4Fe4S]-2Fe(H)) of HydA1 from the green alga Chlamydomonas reinhardtii was characterized by x-ray absorption spectroscopy at the iron K-edge. In a time-resolved approach, HydA1 protein samples were prepared after increasing O(2) exposure periods at 0 °C. A kinetic analysis of changes in their x-ray absorption near edge structure and extended X-ray absorption fine structure spectra revealed three phases of O(2) reactions. The first phase (τ(1) ≤ 4 s) is characterized by the formation of an increased number of Fe-O,C bonds, elongation of the Fe-Fe distance in the binuclear unit (2Fe(H)), and oxidation of one iron ion. The second phase (τ(2) ≈ 15 s) causes a ～50% decrease of the number of ～2.7-? Fe-Fe distances in the [4Fe4S] subcluster and the oxidation of one more iron ion. The final phase (τ(3) ≤ 1000 s) leads to the disappearance of most Fe-Fe and Fe-S interactions and further iron oxidation. These results favor a reaction sequence, which involves 1) oxygenation at 2Fe(H(+)) leading to the formation of a reactive oxygen species-like superoxide (O(2)(-)), followed by 2) H-cluster inactivation and destabilization due to ROS attack on the [4Fe4S] cluster to convert it into an apparent [3Fe4S](+) unit, leading to 3) complete O(2)-induced degradation of the remainders of the H-cluster. This mechanism suggests that blocking of ROS diffusion paths and/or altering the redox potential of the [4Fe4S] cubane by genetic engineering may yield improved O(2) tolerance in [FeFe]-hydrogenase.     

Selective stimulation of in situ intermediary metabolism by free calcium in permeabilized rat adipocytes.

The hypothesis that ionized calcium [Ca2+]i may stimulate in situ rat adipocyte intermediary metabolism distal to glucose transport was tested. A metabolically active porous adipocyte model was employed in which pathway metabolism is exclusively pore-dependent using glucose 6-phosphate (G6P) as substrate. Cellular [Ca2+]i was, furthermore, directly adjusted to between 0-2.5 microM via the membrane pores. Three metabolic fluxes were examined, (1) glycolysis-Krebs ([6-14C]G6P oxidation), (2) glycolysis to lactate ([U-14C]G6P to [14C]lactate) and (3) pentose pathway ([1-14C]G6P oxidation). Glycolysis-Krebs oxidation was was found to be selectively (33% above basal P less than 0.001) stimulated by 0.625 microM free calcium. In contrast, there was no effect of [Ca2+]i on the other, exclusively cytoplasmic, pathways. The stimulation of glycolysis-Krebs by [Ca2+]i was inhibited by a mitochondrial calcium channel blocker (Ruthenium red) and persisted over a range of ATP/ADP ratios. Separate studies demonstrated that 2-[1-14C]ketoglutarate oxidation was also calcium-stimulated in the porous adipocytes (160% over baseline at 1 microM [Ca2+]i). These studies thus demonstrate that physiologically relevant increments in porous adipocyte [Ca2+]i enhance overall in situ glycolytic-Krebs pathway oxidation by a mechanism which entails mitochondrial calcium uptake. Methodologically, this metabolically active porous adipocyte model presents a novel experimental approach to investigations regarding the effects of ionized calcium on intermediary metabolism beyond glucose transport.     

Mechanisms of transformation of the antioxidant kaempferol into depsides. Gamma-radiolysis study in methanol and ethanol

In this study, we irradiated the antioxidant kaempferol in ethanol and methanol solutions with gamma rays at doses ranging from 0.2-20 kGy. NMR and ES-MS spectroscopy were used to identify radiolysis products. Two depsides, [2-[(4'-hydroxybenzoyl)oxy]-4,6-dihydroxyphenyl](oxo) methyl acetate and [2-[(4'-hydroxybenzoyl)oxy]-4,6-dihydroxyphenyl](oxo) ethyl acetate, were the major compounds of kaempferol degradation in methanol and in ethanol, respectively. Other products formed in low concentrations were identified as [4-hydroxyphenyl](oxo) methyl acetate, [4-hydroxyphenyl](oxo) ethyl acetate, and depside [2-[(4'-hydroxybenzoyl)oxy]-4,6-dihydroxyphenyl](oxo) acetic acid. The formation of the latter was observed in both solvents. We propose degradation mechanisms that suggest that (.)CH(2)OH and CH(3)(.)CHOH, produced by solvent radiolysis, react with the 3-OH kaempferol group because of its high H-donor capacity. pi-Electron delocalization in the flavonoxy formed after the first H-transfer leads to C-ring opening and consequently to the formation of depsides. G calculation of the degradation products and of (.)CH(2)OH and CH(3)(.)CHOH radicals confirmed the proposed mechanism of kaempferol radiolysis. The rate constants for the reaction between kaempferol and these free radicals were also calculated. Formation of depside has also been observed in many studies of the oxidation of flavonoids; those studying human metabolism have suggested similar redox transformation of flavonols. The antioxidant activities of radiolysis products were evaluated and compared to those of kaempferol.     

A relation between (NAD+)/(NADH) potentials and glucose utilization in rat brain slices.

Changes in several parameters involved in the control of metabolism were correlated with changes in glucose utilization in rat brain slices incubated under conditions which reduced glucose oxidation by 40 to 70%. The parameters included: the concentrations of ATP, ADP, AMP, and the adenylate energy charge; the cytoplasmic oxidation-reduction state ([NAD+]/[NADH]), determined from the [pyruvate]/[lactate] equilibrium; the mitochondrial oxidation-reduction state, determined from the [NH4+] ]2-oxoglutarate]/[glutamate] Equilibrium; the cytoplasmic and mitochondrial oxidation-reduction potentials (in volts), calculated from the respective [NAD+]/ [NADH] ratios using the Nernst equation; and the difference between the cytoplasmic and mitochondrial [NAD+]/[NADH] potentials. The conversion of [3, 4-14C] glucose to 14CO2 and of [U-14C] glucose to acetylcholine and to lipids, proteins, and nucleic acids by the brain slices were also determined. The values obtained by subtracting the mitochondrial from the cytoplasmic [NAD+1/[NADH] potentials correlated more closely with glucose utilization than did other parameters, under the conditions studied. For the synthesis of acetylcholine, the correlation coefficient was 0.96, and for the production of 14CO2 from [3, 4-14C] glucose it was 0.82.     

Muscarinic Cholinergic Receptor-Mediated Phosphoinositide Metabolism in Peripheral Nerve

Few receptor-mediated phenomena have been detected in peripheral nerve. In this study, the ability of the muscarinic cholinergic receptor agonist carbamylcholine to enhance phosphoinositide (PPI) breakdown in sciatic nerve was investigated by measuring the accumulation of inositol phosphates. Rat sciatic nerve segments were prelabeled with myo-[3H]inositol and then incubated either with or without carbamylcholine in the presence of Li+. [3H]Inositol monophosphate ([3H]IP) accumulation contained most of the radioactivity in inositol phosphates, with [3H]inositol bisphosphate ([3H]IP2) and [3H]inositol trisphosphate ([3H]IP3) accounting for 7-8% and 1-2% of the total, respectively. In the presence of 100 microM carbamylcholine, [3H]IP accumulation increased by up to 150% after 60 min. The 50% effective concentration for the response was determined to be 20 microM carbamylcholine and stimulated IP generation was abolished by 1 microM atropine. Enhanced accumulation of IP2 and IP3 was also observed. Determination of the pA2 values for the muscarinic receptor antagonists atropine (8.9), pirenzepine (6.5), AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) (5.7), and 4-diphenylacetoxy-N-methylpiperidinemethiodide (4-DAMP) (8.6) strongly suggested that the M3 muscarinic receptor subtype was predominantly involved in mediating enhanced PPI degradation. Following treatment of nerve homogenates and myelin-rich fractions with pertussis toxin and [32P]NAD+, the presence of an ADP-ribosylated approximately 40-kDa protein could be demonstrated. The results indicate that peripheral nerve contains key elements of the molecular machinery needed for muscarinic receptor-mediated signal transduction via the phosphoinositide cycle.     

Characterization of Azotobacter vinelandii nifZ deletion strains. Indication of stepwise MoFe protein assembly

The nifZ gene product (NifZ) of Azotobacter vinelandii has been implicated in MoFe protein maturation. However, its exact function in this process remains largely unknown. Here, we report a detailed biochemical/biophysical characterization of His-tagged MoFe proteins purified from A. vinelandii nifZ and nifZ/nifB deletion strains DJ1182 and YM6A (Delta nifZ and Delta nifZ Delta nifB MoFe proteins, respectively). Our data from EPR, metal, activity, and stability analyses indicate that one alpha beta subunit pair of the Delta nifZ MoFe protein contains a P cluster ([8Fe-7S]) and an iron-molybdenum cofactor (FeMoco) ([Mo-7Fe-9S-X-homocitrate]), whereas the other contains a presumed P cluster precursor, possibly comprising a pair of [4Fe-4S]-like clusters, and a vacant FeMoco site. Likewise, the Delta nifZ Delta nifB MoFe protein has the same composition as the Delta nifZ MoFe protein except for the absence of FeMoco, an effect caused by the deletion of the nifB gene. These results suggest that the MoFe protein is likely assembled stepwise, i.e. one alpha beta subunit pair of the tetrameric MoFe protein is assembled prior to the other, and that NifZ might act as a chaperone in the assembly of the second alpha beta subunit pair by facilitating a conformational rearrangement that is required for the formation of the P cluster through the condensation of two [4Fe-4S]-like clusters. The possibility of NifZ exercising its effect through the Fe protein was ruled out because the Fe proteins from nifZ and nifZ/nifB deletion strains are not defective in their normal functions. However, the detailed mechanism of how NifZ carries out its exact function in MoFe protein maturation awaits further investigation.     

Novel selective antagonist radioligands for the pharmacological study of A2B adenosine receptors

The adenosine A_2B receptor is the least well characterized of the four adenosine subtypes due to the lack of potent and selective agonists and antagonists. Despite the widespread distribution of A_2B receptor mRNA, little information is available with regard to their function. The characterization of A_2B receptors, through radioligand binding studies, has been performed, until now, by using low-affinity and non-selective antagonists like 1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX),(4-(2-[7-amino-2-(2-furyl)-[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)-phenol ([³H]ZM 241385) and 3-(3,4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propyl-xanthine ([¹²⁵I]ABOPX). Recently, high-affinity radioligands for A_2B receptors, [N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide ([³H]MRS 1754), N-(2-(2-Phenyl-6-[4-(2,2,3,3-tetratritrio-3-phenylpropyl)-piperazine-1-carbonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl)-acetamide ([³H]OSIP339391) and N-benzo[1,3]dioxol-5-yl-2-[5-(1,3-dipropyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide] ([³H]MRE 2029F20), have been introduced. This minireview offers an overview of these recently developed radioligands and the most important applications of drugs towards A_2B receptors.     

Cooperativity and intermediates in the equilibrium reactions of Fe(II,III) with ethanethiolate in N-methylformamide solution

The reaction of FeCl(2) or FeCl(3) with sodium ethanethiolate (SEt) in N-methylformamide (NMF) has been reevaluated to rectify a previous Fe(II) oxidation artifact. On titrating Fe(II) with EtS(-) concentrations up to 12 mol Eq, new features in the UV/vis spectrum (epsilon(344)=(3.1+/-0.2)x10(3) M(-1) cm(-1); epsilon(486)=(4.5+/-0.1)x10(2) M(-1) cm(-1)) indicated that the first observable step was the formation of a single complex different from the known tetrahedral tetrathiolate, [Fe(SEt)(4)](2-) . As the EtS(-) concentration increased past 12.5 mol Eq the UV/vis spectrum gradually transformed to that of [Fe(SEt)(4)](2-) (lambda(max)=314 nm). A Hill-formalism fit to the titration data of the initially formed complex indicated cooperative ligation by three ethanethiolate ions, with K(coop)=(1.7+/-0.1)x10(3) M(-3) and Hill "n"=2.4+/-0.1 (r=0.997). The 3:1 EtS(-)-Fe(II) complex is proposed to be [Fe(2)(SEt)(6)](2-). Titration of Fe(III) with EtS(-) showed direct cooperative formation of [Fe(SEt)(4)](-) [epsilon(340)=(3.4+/-0.5)x10(3) M(-1) cm(-1)] with a Hill-formalism K(coop)=(4.3+/-0.1)x10(2) M(-4) and a Hill coefficient "n"=3.7+/-0.2 (r=0.996). Further ligation past [Fe(SEt)(4)](-) was observed at EtS(-) concentrations above 35 mol Eq. The Fe(III) Hill constants are at variance with our previous report. However, the UV/vis spectrum of Fe(III) in NMF solution was found to change systematically over time, consistent with a slow progressive deprotonation of [Fe(nmf)](3+). The observed time-to-time differences in the equilibrium chemistry of Fe(III) with ethanethiolate in NMF thus reflect variation in the microscopic solution composition of FeCl(3) in alkaline NMF solvent. These results are related to the chemistry of nitrogenase FeMo cofactor in alkaline NMF solution.     

Activation of phospholipase C associated with isolated rabbit platelet membranes by guanosine 5''-[gamma-thio]triphosphate and by thrombin in the presence of GTP.

Rabbit platelets were labelled with [3H]inositol and a membrane fraction was isolated in the presence of ATP, MgCl2 and EGTA. Incubation of samples for 10 min with 0.1 microM-Ca2+free released [3H]inositol phosphates equivalent to about 2.0% of the membrane [3H]phosphoinositides. Addition of 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) caused an additional formation of [3H]inositol phosphates equivalent to 6.6% of the [3H]phosphoinositides. A half-maximal effect was observed with 0.4 microM-GTP[S]. The [3H]inositol phosphates that accumulated consisted of 10% [3H]inositol monophosphate, 88% [3H]inositol bisphosphate ([3H]IP2) and 2% [3H]inositol trisphosphate ([3H]IP3). Omission of ATP and MgCl2 led to depletion of membrane [3H]polyphosphoinositides and marked decreases in the formation of [3H]inositol phosphates. Thrombin (2 units/ml) or GTP (4-100 microM) alone weakly stimulated [3H]IP2 formation, but together they acted synergistically to exert an effect comparable with that of 10 microM-GTP[S]. The action of thrombin was also potentiated by 0.1 microM-GTP[S]. Guanosine 5'-[beta-thio]diphosphate not only inhibited the effects of GTP[S], GTP and GTP with thrombin, but also blocked the action of thrombin alone, suggesting that this depended on residual GTP. Incubation with either GTP[S] or thrombin and GTP decreased membrane [3H]phosphatidylinositol 4-phosphate ([H]PIP) and prevented an increase in [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) observed in controls. Addition of unlabelled IP3 to trap [3H]IP3 before it was degraded to [3H]IP2 showed that only about 20% of the additional [3H]inositol phosphates that accumulated with GTP[S] or thrombin and GTP were derived from the action of phospholipase C on [3H]PIP2. The results provide further evidence that guanine-nucleotide-binding protein mediates signal transduction between the thrombin receptor and phospholipase C, and suggest that PIP may be a major substrate of this enzyme in the platelet.     

A coupled assay for UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1,6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc).

UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1,6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc) (i.e., core 2 GlcNAc-T) is a developmentally regulated enzyme of the O-linked oligosaccharide biosynthesis pathway. We have developed a coupled-enzyme assay for core 2 GlcNAc-T that is approximately 100 times more sensitive than the standard assay using UDP-[3H]GlcNAc as a sugar donor. Core 2 GlcNAc-T reactions were performed using unlabeled UDP-GlcNAc donor and Gal beta 1-3GalNAc alpha-paranitrophenyl (pNp) as acceptor. The product, Gal beta 1-3(GlcNAc beta 1-6)GalNAc alpha-pNp was then further reacted with purified bovine beta 1-4Gal-T and UDP-[3H]Gal to produce Gal beta 1-3([3H]Gal beta 1-4GlcNAc beta 1-6) GalNAc alpha-pNp, which was separated on an Ultrahydrogel HPLC column. Approximately 10% of the available GlcNAc-terminating acceptor was substituted in the Gal-T reaction, allowing 1 pmol of product to be readily detected. The increased sensitivity of the coupled assay should facilitate studies of core 2 GlcNAc-T activity where material is limiting or specific activity is low.     

Lithium inhibits muscarinic-receptor-stimulated inositol tetrakisphosphate accumulation in rat cerebral cortex.

The effects of Li+ on carbachol-stimulated phosphoinositide metabolism were examined in rat cerebral-cortex slices labelled with myo-[2-3H]inositol. The muscarinic agonist carbachol evoked an enhanced steady-state accumulation of [3H]inositol monophosphate ([3H]InsP1), [3H]inositol bisphosphate ([3H]InsP2), [3H]inositol 1,3,4-trisphosphate ([3H]Ins(1,3,4)P3), [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) and [3H]inositol tetrakisphosphate ([3H]InsP4). Li+ (5 mM), after a 10 min lag, severely attenuated carbachol-stimulated [3H]InsP4 accumulation while simultaneously potentiating accumulation of both [3H]InsP1 and [3H]InsP2 and, at least initially, of [3H]Ins(1,3,4)P3. These data are consistent with inhibition of inositol mono-, bis- and 1,3,4-tris-phosphate phosphatases to different degrees by Li+ in brain, but are not considered to be completely accounted for in this way. Potential direct and indirect mechanisms of the inhibitory action of Li+ on [3H]InsP4 accumulation are considered. The present results stress the complex action of Li+ on cerebral inositol metabolism and indicate that more complex mechanisms than are yet evident may regulate this process.