A ganglioside spin label: ganglioside head group interactions.

A general procedure has been developed for covalent attachment of a nitroxide spin label in the head group region of gangliosides. Gangliosides so labeled and incorporated into lipid bilayer vesicles give a sharp, 3-line spectrum characteristic of a highly mobile structure. The molecular basis of apparent ganglioside-ganglioside head group interaction is briefly discussed.     

Superoxide-dependent reduction of nitroxides by thiols

It was found that superoxide can reduce certain nitroxide free radicals to their corresponding hydroxylamines in the presence of most sulfhydryl-containing compounds. The stoichiometry of the reaction was found to be three nitroxides reduced per superoxide. Evidence is presented indicating that superoxide directly reacts with a nitroxide to yield a N-hydroxy-N-hydroperoxyl compound. This product rapidly decomposes, giving a hydroxylamine and an oxidized sulfhydryl compound, which is postulated to be a sulfenyl hydroperoxide. It is proposed that this sulfenyl hydroperoxide reduces two additional nitroxyl free radicals to account for the unusual stoichiometry.     

Electron spin resonance studies on the inorganic-anion-transport system of the human red blood cell Binding of a disulfonatostilbene spin label (NDS-tempo) and inhibition of anion transport

The disulfonatostilbene spin label, NDS-TEMPO, was synthesized (purity over 96%) and the binding of the spin label to human red-cell ghosts was studied. NDS-TEMPO is readily adsorbed to the membrane surface. Both pretreatment of the ghosts with FDNB and DIDS and the presence of DNDS completely prevent the binding of NDS-TEMPO to red-cell ghosts. Chloride and sulfate competitively inhibit the binding of NDS-TEMPO. Conversely, NDS-TEMPO is a strong, competitive inhibitor of chloride and of sulfate transport. The dissociation constants of NDS-TEMPO from the ESR studies were in the range 1.0–2.0 μM (pH 7.6, 20°C). The inhibition constants of NDS-TEMPO as obtained from the flux experiments were in the range 0.5–2.5 μM (pH 7.3, 25°C). The close accordance of the NDS-TEMPO dissociation constants from the ESR studies with the NDS-TEMPO inhibition constants from the flux measurements indicate a specific labeling of the inorganic-anion-transport system.     

Reversible dissociation of flavin mononucleotide from the mammalian membrane-bound NADH: ubiquinone oxidoreductase (complex I)

Conditions for the reversible dissociation of flavin mononucleotide (FMN) from the membrane-bound mitochondrial NADH:ubiquinone oxidoreductase (complex I) are described. The catalytic activities of the enzyme, i.e. rotenone-insensitive NADH:hexaammineruthenium III reductase and rotenone-sensitive NADH:quinone reductase decline when bovine heart submitochondrial particles are incubated with NADH in the presence of rotenone or cyanide at alkaline pH. FMN protects and fully restores the NADH-induced inactivation whereas riboflavin and flavin adenine dinucleotide do not. The data show that the reduction of complex I significantly weakens the binding of FMN to protein thus resulting in its dissociation when the concentration of holoenzyme is comparable with K(d ( approximately 10(-8)M at pH 10.0).     

Effects of cations on the plasma membrane of Blastocladiella emersonii zoospores. An ESR study

The physical properties of the plasma membrane of the aquatic phycomycete Blastocladiella emersonii were investigated, in particular the effects of cations on membrane structure. Intact zoospores and lipid extracts were labelled with the spin-labels 5-nitroxystearate (5-NS), 12-nitroxystearate (12-NS), and 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo). Electron spin resonance spectroscopy indicated a total of three breaks in plots of the hyperfine splitting parameter, 2T_|, order parameter, S, and the partition coefficient, f, vs. temperature. The first and third break points (T_L and T_H) were found to be independent of the external K⁺, Ca²⁺, or Mg²⁺ concentrations. They were similar to the break points found in aqueous dispersions of lipid extracts and correlate well with the temperature limits for zoospore viability. In contrast, the middle break point (T_M) was markedly influenced by the external Ca²⁺ concentration. Ca²⁺ increased T_M from 12°C (no Ca²⁺ added) to 22°C (10 mM Ca²⁺), i.e., growth temperature. K⁺ reversed this Ca²⁺ effect, downshifting T_M from 22°C to 10°C. A comparison of the physico-chemical effects of these ions on the membrane, as revealed by the cation-induced shift in T_M, is closely correlated with the temperature dependence and physiological effects of cations on zoospore differentiation. This suggests that cations may modify the physical state of the plasma membrane and be involved in regulating the initial changes during zoospore encystment.     

Guanosine 3',5'-monophosphate and the action of alkylating agents.

The intracellular level of guanosine 3',5'-monophosphate (cGMP) has been measured in Walker carcinoma cells in tissue culture after treatment with various alkylating agents. At concentrations which caused a rise in the level of adenosine 3',5'-monophosphate (cAMP) chlorambucil and 5-(1-aziridinyl)-2,4-dinitrobenzamide (CB 1954) produced only a small (35%) elevation of cGMP, while merophan had no such effect. This suggests that any effect of cAMP will not be outweighed by an equivalent rise in cGMP. Sepcific cytosolic binding of cGMP decreased with increasing resistance of Walker cells to alkylating agents, while the dissociation constant, KD, for binding increased. This was also observed with cAMP binding which suggests that the same protein in responsible for binding both nucleotides.     

Polychlorinated biphenyls as inducers of hepatic microsomal enzymes: Structure-activity rules

A number of highly purified polychlorinated biphenyl (PCB) isomers and congeners were synthesized and administered to male Wistar rats at dosage levels of 30 and 150 μmol · kg⁻¹. The effects of this in vivo treatment on the drug-metabolizing enzymes were determined by measuring the microsomal benzo[a]pyrene (B[a]P) hydroxylase, dimethylaminoantipyrine (DMAP) N-demethylase and NADPH-cytochrome c reductase enzyme activities, the cytochrome b₅ content and the relative peak intensities and spectral shifts of the reduced microsomal cytochrome P-450: CO and ethylisocyanide (EIC) binding difference spectra. The results were compared to the effects of administering phenobarbitone (PB), 3-methylcholanthrene (MC) and PB plus MC (coadministered) to the test animals. The synthetic PCB congeners used in this study included 3,4,4′,5-tetrachlorobiphenyl (TCBP-1), 2,3′,4,4′-tetrachlorobiphenyl (TCBP-2), 2,3′,4,4′,5′-pentachlorobiphenyl (PCBP-1), 2,3,4,4′,5-pentachlorobiphenyl (PCBP-2), 2,3,3′,4,4′,5-hexachlorobiphenyl (HCBP-1), 2,3,3′,4′,5,6-hexachlorobiphenyl (HCBP-2), 2,3,3′,5,5′,6-hexachlorobiphenyl (HCBP-3), 2,2′,3,5,5′,6-hexachlorobiphenyl (HCBP-4) and 2,3,3′,4,5,5′-hexachlorobiphenyl (HCBP-5) and were used to reappraise the structure-activity rules for PCBs as hepatic microsomal enzyme inducers. The results suggested that (a) PCBs which induce MC or mixed-type activity must be substituted at both para positions, at least two meta positions but not necessarily on the same phenyl ring and can also contain one ortho chloro substituent; (b) due to the considerable structural diversity of the PB-type inducers the rules for induction of this activity by PCB congeners are not readily defined.     

Inhibition of ADP-ribosylation of histone by diadenosine 5', 5"' -p (1), p(4)-tetraphosphate

Diadenosine 5′, 5?-p¹, p⁴-tetraphosphate (Ap4A) strongly inhibited ADP-ribosylation reaction of histone by purified bovine thymus poly(ADP-ribose) polymerase. This compound showed a relatively weak inhibitory effect on Mg²⁺-dependent, enzyme-bound poly(ADP-ribose) synthesis. Among various adenine nucleotides tested, including several diadenosine nucleotides with varying phosphate chain length, Ap4A was the most effective inhibitor of the histone-modification reaction. Ap5A and Ap6A showed slightly lower inhibitory effect than Ap4A. Kinetic analysis of the inhibitor (Ap4A) with varying concentration of substrate (NAD⁺) revealed that this compound is a “mixed type inhibitor”, with a Ki value of 5.1 μM.     

A comparison of the inhibitory potency of reversibly acting inhibitors of anion transport on chloride and sulfate movements across the human red cell membrane

The effects of a variety of chemically diverse, reversibly acting inhibitors have been measured on both Cl^? and SO₄^2? equilibrium exchange across the human red cell membrane. The measurements were carried out under the same conditions (pH 6.3, 8°C) and in the same medium for both the Cl^? and SO₂⁴ tracer fluxes. Under these conditions the rate constant for Cl^?-Cl^? exchange is about 20 000 times larger than that for SO₄^2?-SO₄^2? exchange. Despite this large difference in the rates of transport of the two anions, eight different reversibly acting inhibitors have virtually the same effect on the Cl^? and SO₄^2? transport. The proteolytic enzyme papain also has the same inhibitory effect on both the Cl^? and SO₄^2? self-exchange. In addition, the slowly penetrating disulfonate 2-(4′-aminophenyl)-6-methylbenzenethiazol-3′,7-disulfonic acid (APMB) is 5-fold more effective from the outer than from the inner membrane surface in inhibiting both Cl^? and SO₄^2? self-exchange. We interpret these results as evidence that the rapidly penetrating monovalent anion Cl^? and the slowly penetrating divalent anion SO₄^2? are transported by the same system.     

Activation of tryptophan 5-monooxygenase by calcium-dependent regulator protein.

Rat brainstem tryptophan 5-monooxygenase was activated about 2-fold by rat brain calcium-dependent regulator (CDR) protein. The activation required both ATP and Mg²⁺ in the presence of low concentrations of Ca²⁺.     

Structural dynamics of the actomyosin complex probed by a bifunctional spin label that cross-links SH1 and SH2

We have used a bifunctional spin label (BSL) to cross-link Cys⁷⁰⁷ (SH1) and Cys⁶⁹⁷ (SH2) in the catalytic domain of myosin subfragment 1 (S1). BSL induces the same weakened ATPase activity and actin-binding affinity that is observed when SH1 and SH2 are cross-linked with pPDM, which traps an analog of the post-hydrolysis state A·M·ADP·P. Electron paramagnetic resonance showed that BSL reports the global orientation and dynamics of S1. When bound to actin in oriented muscle fibers in the absence of ATP, BSL-S1 showed almost complete orientational disorder, as reported previously for the weakly bound A·M·ADP. In contrast, helical order is observed for the strongly bound state A·M. Saturation transfer electron paramagnetic resonance showed that the disorder of cross-linked S1 on actin is nearly static on the microsecond timescale, at least 30 times slower than that of A·M·ADP. We conclude that cross-linked S1 exhibits rotational disorder comparable to that of A·M·ADP, slow rotational mobility comparable to that of A·M, and intermediate actin affinity. These results support the hypothesis that the catalytic domain of myosin is orientationally disordered on actin in a post-hydrolysis state in the early stages of force generation.     

Involvement of Ceramide in the Mechanism of Cr(VI)-induced Apoptosis of CHO Cells

Mitochondria reduce Cr(VI) to Cr(V) with concomitant generation of reactive oxygen species, thereby exhibiting cytotoxic effects leading to apoptosis in various types of cells. To clarify the mechanism by which Cr(VI) induces apoptosis, we examined the effect of Cr(VI) on Chinese hamster ovary (CHO) cells. Cr(VI) increased cellular levels of ceramide by activating acid sphingomyelinase (ASMase) and inhibiting the phosphorylation of pleckstrin homology domain-containing protein kinase B (Akt). Cr(VI) also induced cyclosporin A- and trifluoperazine-sensitive depolarization of mitochondria and activated caspase-3, 8 and 9, thereby causing fragmentation of cellular DNA. The presence of desipramine, an inhibitor of ASMase, and membrane permeable pCPT-cAMP suppressed the Cr(VI)-induced activation of caspases and DNA fragmentation. These results suggested that accumulation of ceramide play an important role in the Cr(VI)-induced apoptosis of CHO cells through activation of mitochondrial membrane permeability transition.     

Inbition of steroidogenic response to corticotropin in mouse adrenal tumor cells (Y-1 by the ionophore A23187 Role of protein biosynthesis

Addition of the ionophore A123187 to Y-1 mouse adrenal tumor cells in monolayer culture inhibits steroidogenesis and the steroidogenic response to corticotropin (50% inhibition at 1 · 10^?7 M). inhibition is rapid in onset and is not overcome by addition of external Ca²⁺. The ionophore also inhibits stimulation of steroid synthesis by cyclic AMP. A23187 inhibits incorporation of the amino acid lysine into protein by Y-1 cells and the dose dependence of this inhibition closely resembles that of the inhibition of the steroidogenic response to corticotropin. Addition of A23187 to a subcellular system for protein synthesis prepared from Y-1 cells, inhibits incorporation of the amino acid phenylalanine into protein and this effects and this effect is not overcome by high concentrations of Ca²⁺. The inhibitory effect of A23187 on the response to corticotropin, like that response itself, takes place at some part of steriod synthesis after entry of cholesteriol into the cells and before the side-chain cleavage of cholesterol. These studies confirm the importance of protein synthesis in the response to corticotropin and demonstrate that the effect of protein synthesized under the influence of corticotropin is exerted at some point in the events which bring substrate (cholesterol) to the mitochondrial side-chain cleavage enzyme system. It is also shown that A23187 inhibits protein synthesis, and hence the response to corticotropin, by a mechanism which is independent of the concentration of available Ca²⁺.     

Backbone dynamics of alamethicin bound to lipid membranes: spin-echo electron paramagnetic resonance of TOAC-spin labels

Alamethicin F50/5 is a hydrophobic peptide that is devoid of charged residues and that induces voltage-dependent ion channels in lipid membranes. The peptide backbone is likely to be involved in the ion conduction pathway. Electron spin-echo spectroscopy of alamethicin F50/5 analogs in which a selected Aib residue (at position n = 1, 8, or 16) is replaced by the TOAC amino-acid spin label was used to study torsional dynamics of the peptide backbone in association with phosphatidylcholine bilayer membranes. Rapid librational motions of limited angular amplitude were observed at each of the three TOAC sites by recording echo-detected spectra as a function of echo delay time, 2τ. Simulation of the time-resolved spectra, combined with conventional EPR measurements of the librational amplitude, shows that torsional fluctuations of the peptide backbone take place on the subnanosecond to nanosecond timescale, with little temperature dependence. Associated fluctuations in polar fields from the peptide could facilitate ion permeation.     

NMR analysis of [methyl-13C]methionine UvrB from Bacillus caldotenax reveals UvrB-domain 4 heterodimer formation in solution

UvrB is a central DNA damage recognition protein involved in bacterial nucleotide excision repair. Structural information has been limited by the apparent disorder of the C-terminal domain 4 in crystal structures of intact UvrB; in solution, the isolated domain 4 is found to form a helix-loop-helix dimer. In order to gain insight into the behavior of UvrB in solution, we have performed NMR studies on [methyl-13C]methionine-labeled UvrB from Bacillus caldotenax (molecular mass=75 kDa). The 13 methyl resonances were assigned on the basis of site-directed mutagenesis and domain deletion. Solvent accessibility was assessed based on the relaxation and chemical shift responses of the probe methyl resonances to the stable nitroxide, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL). M632, located at the potential dimer interface of domain 4, provides an ideal probe for UvrB dimerization behavior. The M632 resonance of UvrB is very broad, consistent with some degree of monomer-dimer exchange and/or conformational instability of the exposed dimer interface. Upon addition of unlabeled domain 4 peptide, the M632 resonance of UvrB sharpens and shifts to a position consistent with a UvrB-domain 4 heterodimer. A dissociation constant (KD) value of 3.3 microM for the binding constant of UvrB with the domain 4 peptide was derived from surface plasmon resonance studies. Due to the flexibility of the domain 3-4 linker, inferred from limited proteolysis data and from the relaxation behavior of linker residue M607, the position of domain 4 is constrained not by the stiffness of the linking segment but by direct interactions with domains 1-3 in UvrB. In summary, UvrB homodimerization is disfavored, while domain 4 homodimerization and UvrB-domain 4 heterodimerization are allowed.     

Determination of less polar heterocyclic aromatic amines in standardised beef extracts and cooked meat consumed in Austria by liquid chromatography and fluorescence detection

An analysis method was developed for the determination of trace levels of less polar heterocyclic aromatic amines (HAs) in food samples. The development started from a frequently used sample pre-treatment scheme which was slightly improved to make it applicable with high-performance liquid chromatography (HPLC) with fluorescence detection. The method was applied for the analysis of a standardised beef extract containing 5-15 ng/g of HAs and the results are compared with those of the other participants in the same European project. In addition, the method was used for the analysis of less polar HAs in cooked meat consumed in Austria.     

Analysis of the domain properties of the novel cytochrome P450 RhF

The properties of the heme, flavin mononucleotide (FMN) and FeS domains of P450 RhF, from Rhodococcus sp. NCIMB 9784, expressed separately and in combination are analysed. The nucleotide preference, imidazole binding and reduction potentials of the heme and FMN domains are unaltered by their separation. The intact enzyme is monomeric and the flavin is confirmed to be FMN. The two one-electron reduction potentials of the FMN are -240 and -270 mV. The spectroscopic and thermodynamic properties of the FeS domain, masked in the intact enzyme, are revealed for the first time, confirming it as a 2Fe-2S ferredoxin with a reduction potential of -214 mV.     

ATP-dependent spectral response of oxonol VI in an ATP-Pi exchange complex

(1) Energy transduction in an ATPase complex (complex V) has been studied in two reactions catalyzed by this system, i.e., ATP-dependent spectral shift of oxonol VI, and ATP-P_i exchange activity. (2) Aurovertin alone inhibits 50% of the oxonol shift at 2 μM, and no further inhibition occurs at up to 12 μM. In combination with even weakly effective uncouplers, 4 μM aurovertin fully abolishes the oxonol response. No such effects are observed in the presence of oligomycin and uncouplers. (3) No pH gradient is detectable by quenching of 9-amino-6-chloro-2-methoxyacridine; and nigericin is without effect on the oxonol response. Valinomycin is inhibitory even in the absence of added potassium, due to ammonium ions introduced during the purification steps. Thiocyanate inhibits the dye response by only 10–27%, depending on the preparation. The extent of the oxonol response depends on the ATP / ADP ratio rather than the phosphorylation potential. (4) The dye response in the ATPase complex is 4–7-times less sensitive to bile salts than in submitochondrial particles. The inhibition by cardiolipin can be reversed by the addition of phospholipids. (5) The possibility is discussed that the oxonol response in the ATPase complex reflects, at least in part, a more local, ATP-dependent and energy-related process.