Purification and properties of membrane-bound coupling factor-latent ATPase from Mycobacterium phlei.

The membrane bound coupling factor-latent ATPase was solubilized from the membrane vesicles of Mycobacterium phlei by using 0.25 M sucrose or low ionic strength buffer. Purification of the solubilized enzyme by use of Sepharose-ADP conjugate gel yielded a homogenous preparation of latent ATPase which was purified about 216-fold in a single step with an 84% yield. The enzyme exhibits a specific activity of 39 mumoles of ATP hydrolyzed per min per mg protein. The purified enzyme exhibits coupling factor activity. Electrophoresis in two dissociating solvent systems indicates that the enzyme contains at least three major polypeptides of molecular weights 56,000, 51,000, and 46,000 daltons, and two minor polypeptides of 30,000 and 17,000 daltons. Equilibrium binding studies of ADP with purified coupling factor-latent ATPase reveal the presence of two nucleotide binding sites per molecule with an apparent Ka of 8.1 X 10(-5) M. By use of affinity chromatography, another latent ATPase has been isolated from the solubilized enzyme, which does not exhibit coupling factor activity.     

Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate.

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, KD = 1-2 muM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (KI = 240-300 muM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This "tightly bound" ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetics studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, Pi can maintain the active form of the enzyme.     

The role of the Mg2+ cation in ATPsynthase studied by electron paramagnetic resonance using VO2+ and Mn2+ paramagnetic probes

The electron paramagnetic resonance (EPR), electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectra of Mg2+-depleted chloroplast F1-ATPase substituted with stoichiometric VO2+ are reported. The ESEEM and HYSCORE spectra of the complex are dominated by the hyperfine and quadrupole interactions between the VO2+ paramagnet and two different nitrogen ligands with isotropic hyperfine couplings /A1/ = 4.11 MHz and /A2/ = 6.46 MHz and nuclear quadrupole couplings e2qQ1 approximately 3.89-4.49 MHz and e2qQ2 approximately 1.91-2.20 MHz, respectively. Aminoacid functional groups compatible with these magnetic couplings include a histidine imidazole, the epsilon-NH2 of a lysine residue, and the guanidinium group of an arginine. Consistent with this interpretation, very characteristic correlations are detected in the HYSCORE spectra between the 14N deltaM1 = 2 transitions in the negative quadrant, and also between some of the deltaM1 = 1 transitions in the positive quadrant. The interaction of the substrate and product ADP and ATP nucleotides with the enzyme has been studied in protein complexes where Mg2+ is substituted for Mn2+. Stoichiometric complexes of Mn x ADP and Mn x ATP with the whole enzyme show distinct and specific hyperfine couplings with the 31P atoms of the bonding phosphates in the HYSCORE (ADP, A(31Pbeta) = 5.20 MHz: ATP, A(31Pbeta) = 4.60 MHz and A(31Pgamma) = 5.90 MHz) demonstrating the role of the enzyme active site in positioning the di- or triphosphate chain of the nucleotide for efficient catalysis. When the complexes are formed with the isolated alpha or beta subunits of the enzyme, the HYSCORE spectra are substantially modified, suggesting that in these cases the nucleotide binding site is only partially structured.     

Globin chain separation by SDS polyacrylamide gel electrophoresis. Simple screening method for elongated hemoglobin chains

A simple method for the separation of hemoglobin chains from hemolysate or globin, by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is described. The alpha, beta, and gamma chains can be clearly separated from each other. The alpha chain has the highest mobility, the beta chain has a slower mobility than the gamma chain, while the delta chain has about the same mobility as the beta chain. Hemoglobins with elongated chains can easily be detected by this method. Tak-beta, elongated by 11 residues, moves much more slowly than betaA but is much faster than alpha Constant Spring which is elongated by 31 residues. Screening of several individuals with slow-moving hemoglobins using this method led to the finding of a case with Hb Tak-beta thalassemia and other carriers of Hb Tak.     

PROTOPORPHYRIN-SENSITIZED PHOTODYNAMIC MODIFICATION OF PROTEINS IN ISOLATED HUMAN RED BLOOD CELL MEMBRANES

Abstract— The photodynamic action of protoporphyrin on red cell ghosts is reflected by extensive cross-linking of membrane proteins to very high molecular weight protein aggregates. This process was studied with sepharose gel chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Most sensitive to this photodynamic effect are spectrin and band 2. 1, 2. 2, 2.3 and 4.1. polypeptides, which are cross-linked after very brief illumination periods, with a concomitant loss of spectrin-associated ATPase activity. Band 6 protein, representing the monomeric form of glyceraldehyde-3-phosphate dehydrogenase, is also very sensitive to protoporphyrin-induced cross-linking. The enzymatic activity decreased even faster than the amount of band 6 polypeptides, suggesting that modification(s) of the enzyme other than cross-linking, possibly by rapid photooxidation of a thiol group, may be responsible for inactivation.
Extracted and purified spectrin was cross-linked with about the same velocity as membrane-bound spectrin, reinforcing our previously drawn conclusion that membrane lipids are not involved in the cross-linking reaction. Eluted band 6 polypeptides on the other hand exhibited a relatively fast photo-oxidative modification but a much slower cross-linking to dimers and tetramers. This suggests that the membrane structure, e.g. the spectrin matrix may play an essential role in the incorporation of membrane-bound band 6 polypeptides in the high molecular weight cross-linked complex.     

Partial purification of active delta and epsilon subunits of the membrane ATPase from escherichia coli.

We have partially purified active delta and epsilon subunits of the E. coli membrane-bound Mg2+-ATPase (ECF1). Treating purified ECF1 with 50% pyridine precipitates the major subunits (alpha, beta, and gamma) of the enzyme, but the two minor subunits (delta and epsilon), which are present in relatively small amounts, remain in solution. The delta and epsilon subunits were then resolved from one another by anion exchange chromatography. The partially purified epsilon strongly inhibits the hydrolytic activity of ECF1. The epsilon fraction inhibits both the highly purified five-subunit ATPase and the enzyme deficient in the delta subunit. The latter result indicates that the delta subunit is not required for inhibition by epsilon. By contrast, two-subunit enzyme, consisting chiefly of the alpha and beta subunits, was insensitive to the ATPase inhibitor, suggesting that the gamma subunit may be required for inhibition by epsilon. The partially purified delta subunit restored the capacity of ATPase deficient in delta to recombine with ATPase-depleted membranes and to reconstitute ATP-dependent transhydrogenase. Previously we reported (Biochem, Biophys. Res. Commun. 62:764 [1975]) that a fraction containing both the delta and epsilon subunits of ECF1 restored the capacity of ATPase missing delta to recombine with depleted membranes and to function as a coupling factor in oxidative phosphorylation and for the energized transhydrogenase. These reconstitution experiments using isolated subunits provide rather substantial evidence that the delta subunit is essential for attaching the ATPase to the membrane and that the epsilon subunit has a regulatory function as an inhibitor of the ATPase activity of ECF1.     

Structures and interactions of proteins involved in the coupling function of the protonmotive F(o)F(1)-ATP synthase

The mitochondrial F(1)F(o) ATP synthase complex has a key role in cellular energy metabolism. The general architecture of the enzyme is conserved among species and consists of a globular catalytic moiety F(1), protruding out of the inner side of the membrane, a membrane integral proton translocating moiety F(o), and a stalk connecting F(1) to F(o). The X-ray crystallographic analysis of the structure of the bovine mitochondrial F(1) ATPase has provided a structural basis for the binding-change rotary mechanism of the catalytic process in F(1), in which the gamma subunit rotates in the central cavity of the F(1) alpha3/beta3 hexamer. Rotation of gamma and eta subunits in the E. coli enzyme and of, gamma and delta subunits in the mitochondrial enzyme, is driven, during ATP synthesis, by proton motive rotation of an oligomer of c subunits (10-12 copies) within the F(o) base piece. Average analysis of electron microscopy images and cross-linking results have revealed that, in addition to a central stalk, contributed by gamma and delta/eta subunits, there is a second lateral one connecting the peripheries of F(o) and F(1). To gain deeper insight into the mechanism of coupling between proton translocation and catalytic activity (ATP synthesis and hydrolysis), studies have been undertaken on the role of F(1) and F(o) subunits which contribute to the structural and functional connection between the catalytic sector F(1) and the proton translocating moiety F(o). These studies, which employed limited proteolysis, chemical cross-linking and functional analysis of the native and reconstituted F(1)F(o) complex, as well as isolated F(1), have shown that the N-terminus of alpha subunits, located at the top of the F(1) hexamer is essential for energy coupling in the F(1)F(o) complex. The alpha N-terminus domain appears to be connected to F(o) by OSCP (F(o) subunit conferring sensitivity of the complex to oligomycin). In turn, OSCP contacts F(o)I-PVP(b) and d subunits, with which it constitutes a structure surrounding the central gamma and delta rotary shaft. Cross-linking of F(o)I-PVP(b) and gamma subunits causes a dramatic enhancement of downhill proton translocation decoupled from ATP synthesis but is without effect on ATP driven uphill proton transport. This would indicate the existence of different rate-limiting steps in the two directions of proton translocation through F(o). In mitochondria, futile ATP hydrolysis by the F(1)F(o) complex is inhibited by the ATPase inhibitor protein (IF(1)), which reversibly binds at one side of the F(1)F(o) connection. The trans-membrane deltapH component of the respiratory deltap displaces IF(1) from the complex; in particular the matrix pH is the critical factor for IF(1)association and its related inhibitory activity. The 42L-58K segment of the IF(1) has been shown to be the most active segment of the protein; it interacts with the surface of one alpha/beta pairs of F(1), thus inhibiting, with the same pH dependence as the natural IF(1), the conformational interconversions of the catalytic sites involved in ATP hydrolysis. IF(1) has a relevant physiopathological role for the conservation of the cellular ATP pool in ischemic tissues. Under these conditions IF(1), which appears to be over expressed, prevents dissipation of the glycolytic ATP.     

Electrophoresis at elevated hydrostatic pressure of the multiheme hydroxylamine oxidoreductase

The behavior of the multiheme protein hydroxylamine oxidoreductase (HAO) in polyacrylamide gel electrophoresis was studied at hydrostatic pressures up to 3 kbar at 25 degrees C. Due to the limited working volume of the high pressure vessel, the electrophoresis cells were miniaturized. A microcell which accommodates 6 capillary gel tubes is described. Between 1 bar and 1.5 kbar the enzyme did not undergo structural changes detectable in the gel system. At approximately 2 kbar the active form of the enzyme was partially dissociated. At higher pressures, the enzyme was converted to forms which were irreversibly inactive and had a higher apparent molecular mass, suggesting aggregation or denaturation.     

A rod-linker-contained R-phycoerythrin complex from the intact phycobilisome of the marine red alga Polysiphonia urceolata

A linker-contained R-phycoerythrin (R-PE) complex was obtained by the Sephadex G-150 column chromatography from the Polysiphonia urceolata phycobilisome (PBS) that was dis-associated at 37 degrees C for 6 h in the dilute phosphate buffer (pH 7.0) with 5% (m/v) sodium dodecyl sulfate (SDS). The R-PE complex showed three absorption peaks at 498, 538 and 567 nm, and a fluorescence emission maximum at 578 nm. Polypeptide analysis of the complex by the 8-25% (m/v) gradient SDS-polyacrylamide gel electrophoresis demonstrated that it contained three red subunits, alpha(PE)(17.6),beta(PE)(19.2) and gamma(PE)(31.0), and a colorless 35.3 kDa rod-linker L(R)(35.3). Polypeptide proportion of the complex demonstrated that it was a hexamer in aggregate form gamma(PE)(31.6), (alpha(PE)(17.6),beta(PE)(19.2))(3)L(R)(35.3)(alpha(PE)(17.6),beta(PE)(19.2)(3)gamma(PE)(31.6) which is proposed to originate from a rod assembly of hexamer-linker-hexamer the substructure alpha(PE)(17.6),beta(PE)(19.2)(3) of which was decomposed off from the ends of the assembly during the PBS dissociation. The distinctive stability of the prepared hexamer is attributed to a large extent to the electrostatic interaction among its polypeptides, but not to the hydrophobic interaction.     

Crosslinking studies on the Ca2+, Mg2+-activated ATPase of Escherichia coli.

Crosslinking of membrane proteins of Escherichia coli with dithiobis (succinimidyl propionate) (DSP) resulted in loss of several enzyme activities including the Ca2+, Mg2+-activated ATPase. This enzyme was crosslinked by DSP to the membrane and was not released by dialysis at low ionic strength in the absence of dithiothreitol which could cleave the crosslinking group. DSP inactivated both phosphohydrolase and coupling activities of the solubilized ATPase. Loss of hydrolytic activity could be correlated with the extent of reaction of the alpha and/or beta subunits of the enzyme. The loss of coupling activity appeared to be associated with modification of the gamma and/or delta subunits.     

A facile electrophoretic technique to monitor phosphoenolpyruvate-dependent kinases

Phosphoenolpyruvate (PEP)-dependent kinases are central to numerous metabolic processes and mediate the production of adenosine triphosphate (ATP) by substrate-level phosphorylation (SLP). While pyruvate kinase (PK, EC: 2.7.1.40), the final enzyme of the glycolytic pathway is critical in the anaerobic synthesis of ATP from ADP, pyruvate phosphate dikinase (PPDK, EC: 2.7.9.1), and phosphoenolpyruvate synthase (PEPS, EC: 2.7.9.2) help generate ATP from AMP coupled to PEP as a substrate. Here we demonstrate an inexpensive and effective electrophoretic technology to determine the activities of these enzymes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). The generation of pyruvate is linked to exogenous lactate dehydrogenase (LDH), and the oxidation of reduced nicotinamide adenine dinucleotide (NADH) coupled to 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium chloride (INT) results in a formazan precipitate which is easily quantifiable. The selectivity of the enzymes is ensured by including either AMP or ADP and pyrophosphate (PP(i) ) or inorganic phosphate (P(i) ). Activity bands were readily obtained after incubation in the respective reaction mixtures for 20-30 min. Cell-free extract concentrations as low as 20 μg protein equivalent yielded activity bands and substrate levels were manipulated to optimize sensitivity of this analytical technique. High-pressure liquid chromatography (HPLC), two-dimensional (2-D) SDS-PAGE (where SDS is sodium dodecyl sulfate), and immunoblot studies of the excised activity band help further characterize these PEP-dependent kinases. Furthermore, these enzymes were readily identified on the same gel by incubating it sequentially in the respective reaction mixtures. This technique provides a facile method to elucidate these kinases in biological systems.     

Simultaneous detection of molecular weight and activity of adenylate kinases after electrophoretic separation

Adenylate kinases (AKs) are ubiquitous monomeric phosphotransferases catalyzing the reversible reaction, AMP + MgATP = ADP + MgADP, which plays a pivotal role in the energetic metabolism. In vertebrates, six AK isoforms are known. In this work, we report the detection of many AK isoforms directly on gel or NC after separation by denaturing electrophoresis and electroblotting, by an optimized protocol for the enzyme detection. The method allows to clarify the apparent MW of most of those AK isozymes that follow the cited reaction, especially onto NC where bands are sharper due to the absence of protein diffusion. In contrast, GTP:AMP phosphotransferases are not detectable. AK activity from many sources can be detected in both its reaction courses; ATP production appears as dark-blue bands, while ADP formation appears as nonfluorescent bands over a fluorescent background, under long-wavelength UV light. We show that nondenaturing gel electrophoresis is not the first choice for AK activity detection. Our method is different from the preceding reports on AK activity detection in bacteria after native polyacrylamide gel separations, in the absence of SDS or methanol. The procedure is also quantitative, allowing to determine the amount of enzyme present in samples.     

Decomposition of dinuclear manganese complexes for the preparation of nanostructured oxide materials

The crystal structures of several dinuclear complexes of manganese are reported, and the decomposition and analysis of the nanostructured products derived from them are presented. 1,4,7,10-Tetraazacyclododecane (cyclen) forms dinuclear complexes 1-4 containing doubly oxo-bridged or oxo-acetato bridging ligands depending on the manganese salt used for the reaction. Doubly oxo-bridged 1 crystallizes in the orthorhombic space group Pnma, a = 22.3850(14) A, b = 9.1934(5) A, c = 13.2424(10) A, V = 2725.2(3) A(3). 2, containing [Mn(SCN)5](3-) conteranions, crystallizes in monoclinic space group I2/a with a = 18.2699(10) A, b = 11.2384(6) A, c = 18.6432(9) A, alpha = 90.00 degrees, beta = 114.510(6) degrees, gamma = 90.00 degrees, V = 3483.0(3) A(3). Oxo-acetato-bridged 3 crystallizes in orthorhombic space group Pca21, a = 13.9322(11) A, b = 16.2332(13) A, c = 14.6794(8) A, V = 3320.0(4) A(3). Compound 4 consists of a templated quasi-one-dimensional manganese oxalate crystallized in the triclinic space group P1, a = 9.5442(11) A, b = 10.3758(10) A, c = 21.851(2) A, alpha = 83.720(12) degrees, beta = 80.106(13) degrees, gamma = 85.457(13) degrees, V = 2114.9(4) A(3). Compounds 1, 3, and 4 decompose to nanostructured oxide materials, which may be isolated in bulk as lamellar-structured particles or microspheres or deposited on substrates.     

Solubilization and purification of galactosyltransferase from golgi membranes of rat ventral prostate

UDP-galactose ovomucoid galactosyltransferase, a membrane-bound enzyme involved in the biosynthetic pathways for formation of the nonreducing terminal oligosaccharide sequences on glycoproteins, has been solubilized and purified from rat ventral prostate Golgi membranes. Solubilization was effected by treatment of the particulate fraction with Triton X-100 (0.5% v/v) and MnCl₂ (25 mM). The solubilized enzyme was purified by affinity chromatography on hen ovomucoid-sepharose column. The purified galactosyltransferase showed three protein bands of approx. 74,000, 60,000, and 54,000 daltons on sodium dodecyl sulfate gel electrophoresis. On gel filtration, enzyme activity eluted at approx. 70,000 daltons with a broad shoulder between 60,000 and 50,000 daltons. Isoelectric focusing of the purified enzyme resolved at least five active bands with pHi of 9, 7.4, 6.75, 6.1, and 4.8.     

Oxidative addition of small molecules to a dinuclear Au(I) amidinate complex, Au2[(2,6-Me2Ph)2N2CH]2. Syntheses and characterization of Au(II) amidinate complexes including one which possesses Au(II)-oxygen bonds

The dinuclear Au(I) amidinate complex Au2(2,6-Me2Ph-form)2 (1) is isolated in quantitative yield by the reaction of (THT)AuCl and the potassium salt of 2,6-Me2Ph-form in a 1:1 stoichiometric ratio. Various reagents such as Cl2, Br2, I2, CH3I, and benzoyl peroxide add to the dinuclear Au(I)amidinate complex Au2(2,6-Me2Ph-form)2 to form oxidative-addition Au(II) metal-metal-bonded complexes 2, 3, 4, 5, and 6. The Au(II) amidinate complexes are stable as solids at room temperature. The structures of the dinuclear Au2(2,6-Me2Ph-form)2 and the Au(II) oxidative-addition products Au2(2,6-Me2Ph-form)2X2, X=Cl, Br, I, are reported. Crystalline products with an equal amount of oxidized and unoxidized complexes in the same unit cell, [Au2(2,6-Me2Ph-form)2X2][Au2(2,6-Me2Ph-form)2], X=Cl, 2m, or Br, 3m, are isolated and their structures are presented. The structure of [Au2(2,6-Me2Ph-form)2X2][Au2(2,6-Me2Ph-form)2], X=Cl has a Au(II)-Au(II) distance slightly longer, 0.05A, than that observed in the fully oxidized product Au2(2,6-Me2-form)2Cl2, 2. The gold-gold distance in the dinuclear complex decreases upon oxidative addition with halogens from 2.7 to 2.5 A, similar to observations made with the Au(I) dithiolates and ylides. The oxidative addition of benzoyl peroxide leads to the isolation of the first stable dinuclear Au(II) nitrogen complex possessing Au-O bonds, Au2(2,6-Me2Ph-form)2(PhCOO)2, 6, with the shortest Au-Au distance known for Au(II) amidinate complexes, 2.48 A. The structure consists of unidentate benzoate units linked through oxygen to the Au(II) centers. The replacement of the bromide in 3 by chloride, and the benzoate groups in 6 by chloride or bromide also occurs readily. The unit cell dimensions are, for 1, a=7.354(6) A, b=9.661(7) A, c=11.421(10) A, alpha=81.74(5) degrees, beta=71.23(5) degrees, and gamma=86.07(9) degrees (space group P, Z=1), for 2.1.5C6H12, a=11.012(2) A, b=18.464(4) A, c=19.467(4) A, alpha=90 degrees, beta=94.86(3) degrees, and gamma=90 degrees (space group P21/c, Z=4), for 2m.ClCH2CH2Cl, a=16.597(3) A, b=10.606(2) A, c=19.809(3) A, alpha=90 degrees, beta=94.155(6) degrees, and gamma=90 degrees (space group P21/n, Z=2), for 3m, a=16.967(3) A, b=10.783(2) A, c=20.060(4) A, alpha=90 degrees, beta=93.77(3) degrees, and gamma=90 degrees (space group P21/n, Z=2), for 4.THF, a=8.0611(12) A, b=10.956(16) A, c=11.352(17) A, alpha=84.815(2) degrees, beta=78.352(2) degrees, and gamma=88.577(2) degrees (space group P, Z=1), for 5, a=16.688 A, b=10.672(4) A, c=19.953(7) A, alpha=90.00 (6) degrees, beta=94.565(7) degrees, and gamma=90.00 degrees (space group P21/n, Z=4), for 6.0.5C7H8, a=11.160(3) A, b=12.112(3) A, c=12.364(3) A, alpha=115.168(4) degrees, beta=161.112(4) degrees, and gamma=106.253(5) degrees (space group P, Z=1).     

Purification and some properties of phospholipase C from Achromobacter xylosoxidans.

A non-haemolytic phospholipase C (EC 3.1.4.3) was purified from the culture medium of Achromobacter xylosoxidans with a 5% yield and a purification factor of 330. A combination of ultrafiltration, acetone precipitation and two subsequent affinity chromatographic steps was used. The affinity chromatography is a new application of 2-(4-aminophenylsulphonyl)ethyl-cellulose, a sorbent that has previously been used for the purification of phospholipase C from Bacillus cereus. The purified enzyme gave four distinct bands on polyacrylamide gel electrophoresis, and each band was catalytically active. Under our experimental conditions, the phospholipids examined were hydrolysed in the following order: phosphatidylcholine, phosphatidylethanolamine, sphingomyelin. Neither the synthetic substrate p-nitrophenylphosphorylcholine nor phosphatidylinositol was hydrolysed under different experimental conditions. For maximal hydrolytic activity toward phosphatidylcholine, the enzyme required Triton X-100 and Ca2+ ions. EDTA was inhibitory, but the enzyme activity was almost completely restored by Zn2+. The molecular mass of the phospholipase C, estimated by gel permeation, was 34,000 daltons.     

Cyanide-bridged lanthanide(III)-transition metal extended arrays: interconversion of one-dimensional arrays from single-strand (type A) to double-strand (type B) structures. Complexes of a new type of single-strand array (type C)

A series of one-dimensional arrays of lanthanide-transition metal complexes has been prepared and characterized. These complexes, [(DMF)(10)Ln(2)[Ni(CN)(4)](3)](infinity), crystallize as linear single-strand arrays (structural type A) (Ln = Sm, 1a; Eu, 2a) or double-strand arrays (structural type B) (Ln = Sm, 1b; Eu, 2b) depending upon the conditions chosen, and they are interconvertible. The single-strand type A structure can be converted to the double-strand type B structure. When the 1b and 2b type B crystals are completely dissolved in DMF, their infrared spectra are identical to the infrared spectra of 1a and 2a type A crystals dissolved in DMF. These solutions produce type A crystals initially. It is believed that formation of the type A structure is kinetically favored while the type B structure is thermodynamically favored for lanthanide-nickel complexes 1 and 2. On the other hand the complex [(DMF)(10)Y(2)[Pd(CN)(4)](3)](infinity), 3, appears to crystallize only as the double-strand array (type B). The complexes [(DMF)(12)Ce(2)[Ni(CN)(4)](3)](infinity), 4, and [(DMF)(12)Ce(2)[Pd(CN)(4)](3)](infinity), 5, crystallize as a new type of single-strand array (structural type C). This structural type is a zigzag chain array. Crystal data for 1a: triclinic space group P1, a = 10.442(5) A, b = 10.923(2) A, c = 15.168(3) A, alpha = 74.02(2) degrees, beta = 83.81(3) degrees, gamma = 82.91(4) degrees, Z = 2. Crystal data for 1b: triclinic space group P1, a = 9.129(2) A, b = 11.286(6) A, c = 16.276(7) A, alpha = 81.40(4) degrees, beta = 77.41(3) degrees, gamma = 83.02(3) degrees, Z = 2. Crystal data for 2a: triclinic space group P1, a = 10.467(1) A, b = 10.923(1) A, c = 15.123(1) A, alpha = 74.24(1) degrees, beta = 83.61(1) degrees, gamma = 83.13(1) degrees, Z = 2. Crystal data for 2b: triclinic space group P1, a = 9.128(1) A, b = 11.271(1) A, c = 16.227(6) A, alpha = 81.36(2) degrees, beta = 77.43(2) degrees, gamma = 82.99(1) degrees, Z = 2. Crystal data for 3: triclinic space group P1, a = 9.251(3) A, b = 11.193(4) A, c = 16.388(4) A, alpha = 81.46(2) degrees, beta = 77.18(2) degrees, gamma = 83.24(3) degrees, Z = 2. Crystal data for 4: triclinic space group P1, a = 11.279(1) A, b = 12.504(1) A, c = 13.887(1) A, alpha = 98.68(1) degrees, beta = 108.85(1) degrees, gamma = 101.75(1) degrees, Z = 2. Crystal data for 5: triclinic space group P1, a = 11.388(3) A, b = 12.614(5) A, c = 13.965(4) A, alpha = 97.67(3) degrees, beta = 109.01(2) degrees, gamma = 101.93(2) degrees, Z = 2.     

Sterically hindered and robust pnictogen ligands derived from carboranes: synthesis and X-ray structure determination of tris(1'-methyl(1,2-dicarba-closo-dodecaboran-1-yl))phosphine, tris(1'-methyl(1,2-dicarba-closo-dodecaboran-1-yl))arsine and chloro(tris(1'-methyl(1,2-dicarba-closo-dodecaboran-1-yl))phosphine)gold(I)

Sterically hindered phosphine and arsine ligands derived from ortho-carborane were synthesized and characterized by X-ray crystallography. Tris(1'-methyl(1,2-dicarba-closo-dodecaboran-1-yl))phosphine, 2 (crystal data, hexagonal, space group P6(3), a = b = 12.251(3) A, c = 11.514(4) A, alpha = beta = 90 degrees, gamma = 120 degrees, V = 1496.6(7) A(3), Z = 2, R(1) = 0.0568) and tris(1'-methyl(1,2-dicarba-closo-dodecaboran-1-yl))arsine, 3 (crystal data, hexagonal, space group P6(3), a = b = 12.330(3) A, c = 11.474(4) A, alpha = beta = 90 degrees, gamma = 120 degrees, V = 1510.7(7) A(3), Z = 2, R(1) = 0.0930) were prepared in 82% and 68% yield, respectively. The phosphine ligand is resistant to air-oxidation but was converted to corresponding oxide when heated with hydrogen peroxide. The tertiary carboranyl phosphine reacted with (Tht)AuCl to yield chloro(tris(1'-methyl(1,2-dicarba-closo-dodecaboran-1-yl))phosphine)gold(I), 4 (crystal data, monoclinic, space group P2(1)/c, a = 19.101(4) A, b = 12.167(2) A, c = 13.846(3) A, alpha = gamma = 90 degrees, beta = 91.13(3) degrees, V = 3217.2(11) A(3), Z = 4, R(1) = 0.0396) in 82% yield. From the X-ray structure of the gold complex, the cone angle of the phosphine was determined to be 213(2) degrees, which is among the largest values reported to date.     

Synthesis and properties of [Ru(2)(acac)(4)(bptz)](n+) (n=0,1) and crystal structure of [Ru(2)(acac)(4)(bptz)

The neutral complex [Ru(2)(acac)(4)(bptz)] (I) has been prepared by the reaction of Ru(acac)(2)(CH(3)CN)(2) with bptz (bptz = 3,6-bis(2-pyridyl)-1,2,4,5-tetrazine) in acetone. The diruthenium(II,II) complex (I) is green and exhibits an intense metal-ligand charge-transfer band at 700 nm. Complex I is diamagnetic and has been characterized by NMR, optical spectroscopy, IR, and single-crystal X-ray diffraction. Crystal structure data for I are as follows: triclinic, P1, a = 11.709(2) A, b = 13.487(3) A, c = 15.151(3) A, alpha = 65.701(14) degrees, beta = 70.610(14) degrees, gamma = 75.50(2) degrees, V = 2038.8(6) A(3), Z = 2, R = 0.0610, for 4397 reflections with F(o) > 4sigmaF(o). Complex I shows reversible Ru(2)(II,II)-Ru(2)(II,III) and Ru(2)(II,III)-Ru(2)(III,III) couples at 0.17 and 0.97 V, respectively; the 800 mV separation indicates considerable stabilization of the mixed-valence species (K(com) > 10(13)). The diruthenium(II,III) complex, [Ru(2)(acac)(4)(bptz)](PF(6)) (II) is prepared quantitatively by one-electron oxidation of I with cerium(IV) ammonium nitrate in methanol followed by precipitation with NH(4)PF(6). Complex II is blue and shows an intense MLCT band at 575 nm and a weak band at 1220 nm in CHCl(3), which is assigned as the intervalence CT band. The mixed valence complex is paramagnetic, and an isotropic EPR signal at g = 2.17 is observed at 77 and 4 K. The solvent independence and narrowness of the 1200 nm band show that complex II is a Robin and Day class III mixed-valence complex.     

Time-resolved fluorescence of tryptophans in yeast hexokinase-PI: effect of subunit dimerization and ligand binding

Time-resolved and steady-state fluorescence measurements have been performed on monomeric and dimeric forms of yeast hexokinase-PI. Observation of similar emission spectra and fluorescence decay parameters for both the forms of the enzyme suggests that tryptophan residue(s) are not likely to be present at the subunit-subunit interface and the process of dimerization does not perturb the local environment of tryptophan(s). The fluorescence decay of tryptophans in enzyme could be fitted to a bi-exponential function with two lifetime components, tau1 approximately 2.2 ns and tau2 approximately 3.9 ns. Binding of glucose, which is known to convert the 'open' conformation of the enzyme to a 'closed' active conformation, results in approximately 30% reduction in emission intensity and a selective decrease in tau1 from approximately 2.2 to approximately 1.1 ns. These effects can be reversed by the addition of trehalose 6-phosphate (an inhibitor of yeast hexokinase), suggesting that the trehalose 6-phosphate inhibits the enzyme by binding to its 'open' inactive conformation rather than competing with glucose to bind to the 'closed' active conformation. Binding of nucleotide ligands (ATP, ADP and adenyl-(beta,gamma-methylene)-diphosphate (AMPPCP)) to the monomeric or dimeric form of enzyme quenched the steady-state fluorescence by approximately 4-8%, but had no measurable effect on the distribution of lifetimes or on their magnitudes. Addition of nucleotides to the enzyme-glucose complex also did not produce any further change in fluorescence decay parameters. These results indicate that it is highly unlikely that the formation of a ternary enzyme-glucose-nucleotide complex from the binary enzyme-glucose complex is accompanied by a large conformational change in the enzyme, as has been surmised in some earlier studies.