Nα-Trinitrophenyl glucagon An inhibitor of glucagon-stimulated cyclic AMP production and its effects on glycogenolysis

N^α-Trinitrophenyl glucagon was prepared by reaction with trinitrobenzene sulfonic acid and purified by ion-exchange chromatography. This derivative has essentially no ability to activate adenylate cyclase from rat liver nor to increase the levels of cyclic AMP in isolated hepatocytes nor to stimulate protein kinase activity. This derivative also can act as a glucagon antagonist with regard to cyclic AMP production and can decrease the degree of stimulation of adenylate cyclase caused by glucagon, as well as lowering the glucagon-stimulated elevation of cyclic AMP levels in intact hepatocytes. Nevertheless, this derivative is capable of activating glycogenolysis.in isolated hepatocytes and in augmenting the effect of glucagon on glycogenolysis. This metabolic effect of the glucagon derivative thus appears to occur independent of changes in cyclic AMP levels. These results suggest that glucagon can also activate glycogenolysis by a cyclic AMP-independent process.     

Redox properties of b-type cytochromes in Escherichia coli and rat liver mitochondria and techniques for their analysis

We describe here apparatus and procedures for conducting potentiometric titrations and for analyzing the collected data in terms of the number of components present, their amounts and their midpoint potentials. Using these procedures we have determined the presence of three forms of cytochrome b₁ in Escherichia coli with midpoint potentials at pH 7.1 of about ?50, +110 and +220 mV. We were not able to demonstrate a change in any of these potentials by the addition of phosphate, ATP, or 2,4-dinitrophenol. We have been able to confirm the presence of two forms of cytochrome b in non-energized mitochondria and the apparent conversion of the low-potential component to a new high potential component upon energization of the mitochondria. However we cite further experimental data that question the actual conversion of one form of cytochrome b to another. An alternative interpretation based on our analysis suggests that the high voltage component may be present in a masked form in the non-energized mitochondria.     

A monolayer study of the reaction of trinitrobenzene sulphonic acid with amino phospholipids

The reaction of trinitrobenzene sulphonic acid with amino phospholipids, and in particular phosphatidylethanolamine has been studied by the monolayer technique. Injection of trinitrobenzene sulphonic acid under a monolayer of amino phospholipid results in an increase in surface pressure. The rate and extent of the pressure change is greatly affected by the initial surface pressure, the fatty acid composition of the lipid, and the presence of other non-reactive lipids, especially negatively charged phospholipids.The extent of the reaction was measured with ³²P-labelled phospholipids isolated from Bacillus subtilis. Only about 80% of the phosphatidylethanolamine in the monolayer could be converted to its trinitrophenyl derivative. In the presence of negatively charged phospholipids such as cardiolipin or phosphatidylglycerol, a further 20% decrease in the trinitrophenylation of phosphatidylethanolamine was found. The pressure increase occurring during trinitrophenylation could also be correlated with the extent of the reaction by comparison of the force-area curves of pure phosphatidylethanolamine, its trinitrophenyl derivative and mixtures of both compounds.The data may offer an explanation for the observation that incomplete labelling of amino phospholipids frequently occurs in natural membranes and furthermore indicate that the use of chemical labelling techniques in the study of lipid asymmetry in biological membranes must be approached with great caution.     

Spectrophotometric assay for ornithine decarboxylase

A rapid and sensitive spectrophotometric assay for ornithine decarboxylase is described. It is based on the observation that the product of ornithine decarboxylase, putrescine, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a colored product soluble in 1-pentanol whereas ornithine does not. The amount of putrescine produced by the enzyme was determined by measuring the absorbance of the 1-pentanol extract of the reaction mixture at 420 nm, and by comparing the results to those obtained by the trapping of 14CO2 and by HPLC assays. The three assays were found to be equivalent in sensitivity, with the spectrophotometric assay having the advantages of being relatively rapid, requiring only common laboratory equipment, and not requiring the use of radioactive isotopes.     

The involvement of sarcotubular membranes in genetic muscular dystrophy

Microsomal preparations from breast muscle of normal and dystrophic chickens are characterized with regard to ultrastructural features, protein composition, Ca²⁺ transport and ATPase activity.Dystrophic muscle yields a greater microsomal dry weight, with a reduced protein to lipid ratio. This is related to the presence of a considerable number of low density microsomes, in addition to seemingly normal microsomes. The low density microsomes display a reduced number of protein particles on freeze fracture faces.Electrophoretic analysis reveals nearly identical patterns in normal and dystrophic microsomes. Furthermore, normal and dystrophic microsomes sustain equal rates of Ca²⁺ transport and ATPase, demonstrating an identical protein specific activity. However, the dystrophic microsomes have a lower capacity to retain transported Ca²⁺.The high yield of low density microsomes with reduced capacity for Ca²⁺ uptake is attributed to the presence of membranes proliferated in the junctional and tubular sarcomere regions of the dystrophic muscle. It is suggested that proliferation of such membranes accounts for the altered excitation-contraction coupling and cable properties of genetically dystrophic muscle.     

CO-binding pigments and the functional terminal oxidase of the trypanosomatid hemoflagellate Crithidia fasciculata

CO-difference absorbance spectra of both intact cells and of mitochondrial preparations isolated from Crithidia fasciculata were obtained after anaerobiosis was attained either with substrates or with dithionite. Under both sets of conditions, the CO-difference spectrum of cytochrome a₃, with difference absorbance maxima at 430 and 589 nm and minima at 443 and 612 nm, was readily identified in both the intact cells and in the mitochondria. In addition to the difference absorbance bands of cytochrome a₃-CO, three difference absorbance maxima at 417, 538 and 570 nm and a minimum at 556 nm were observed. The magnitude of the maximum at 570 nm relative to the maximum of cytochrome a₃-CO at 589 nm was less for mitochondria rendered anaerobic with substrate than for mitochondria rendered anaerobic with dithionite. This difference was taken to define operationally two groups of mitochondrial CO-binding pigments: Group I is that group observed on anaerobiosis with substrate: Group II is the additional group observed on anaerobiosis with dithionite. The Group I CO-binding pigments were virtually absent from submitochondrial particles derived by sonication, but the Group II pigments remained.Photochemical action spectra were obtained with isolated mitochondria and intact cells to ascertain if cytochrome o was present. These action spectra, obtained in CO plus O₂ atmospheres, had maxima only at 432, 550 and 588 nm, attributable to the photodissociation of cytochrome a₃-CO. Even after suppression of cytochrome a₃ activity to 10% of the normal value, no contribution of cytochrome o activity to the photochemical action spectrum was observed. Cytochrome a₃ is therefore the only functional terminal oxidase present in the mitochondria of Crithidia fasciculata.     

Structural insights into enzyme-substrate interaction and characterization of enzymatic intermediates of organic hydroperoxide resistance protein from Xylella fastidiosa

Organic hydroperoxide resistance proteins (Ohr) belong to a family of proteins that possess thiol-dependent peroxidase activity endowed by reactive cysteine residues able to reduce peroxides. The crystal structure of Ohr from Xylella fastidiosa in complex with polyethylene glycol, providing insights into enzyme-substrate interactions is described herein. In addition, crystallographic studies, molecular modeling and biochemical assays also indicated that peroxides derived from long chain fatty acids could be the biological substrates of Ohr. Because different oxidation states of the reactive cysteine were present in the Ohr structures from X. fastidiosa, Pseudomonas aeruginosa and Deinococcus radiodurans it was possible to envisage a set of snapshots along the coordinate of the enzyme-catalyzed reaction. The redox intermediates of X. fastidiosa Ohr observed in the crystals were further characterized in solution by electrospray ionization mass spectrometry and by biochemical approaches. In this study, the formation of an intramolecular disulfide bond and oxidative inactivation through the formation of a sulfonic acid derivative was unequivocally demonstrated for the first time. Because Ohr proteins are exclusively present in bacteria, they may represent promising targets for therapeutical drugs. In this regard, the structural and functional analyses of Ohr presented here might be very useful.     

Function and properties of a soluble c-type cytochrome c-551 in secondary photosynthetic electron transport in whole cells of Chromatium vinosum as studied with flash spectroscopy

Changes in the absorption spectrum induced by 10-μs flashes and continuous light of various intensities were studied in whole cells of Chromatium vinosum.This paper describes the role and function of a soluble c-type cytochrome, c-551, which was surprisingly found to act in many ways similar to the cytochrome c-420 in Rhodospirillum rubrum, described in a previous paper [1].After the photooxidation of the membrane bound high potential cytochrome c-555 by a 10-μs flash, (the low potential cytochrome c-552 was kept permanently in the oxidized state) the oxidation of c-551 is observed ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.3}\text{ms}$). From a careful analysis of the absorbance difference spectrum and the kinetics it is concluded that there is approximately 0.6–0.7 c-551 per reaction center and that essentially all the c⁺-555 is reduced via the cytochrome c-551. The oxidized-reduced difference spectrum of c-551 shows peaks at 551 and 421.5 nm. The reduction of c⁺-551 following the flash-induced oxidation is strongly inhibited by HOQNO, but only slightly by antimycin A.Cytochrome c-551 reduces only the oxidized high potential cytochrome c-555, which is probably located on the outside of the membrane, on the opposite side of the primary acceptor. The low potential cytochrome c-552 does not show any detectable interaction with cytochrome c-551. After the cells have been sonicated, no c-551 is photooxidized and at least part of the cytochrome occurs in the solution.Analysis of the reduction kinetics of c⁺-551 in the absence and presence of external donors suggests that c⁺-551 is partly reduced via a cyclic pathway, which is blocked by addition of o-phenanthroline, and partly via a non-cyclic pathway. The non-cyclic reduction rate of c⁺-551 (k = 6 s^?1) is increased approximately 5–10 times upon thiosulphate addition, suggesting a role for c-551 between the final donor pool and the oxidized membrane bound c-type cytochromes.     

Modular construction of plasmids through ligation-free assembly of vector components with oligonucleotide linkers

We have developed a modular method of plasmid construction that can join multiple DNA components in a single reaction. A nicking enzyme is used to create 5' and 3' overhangs on PCR-generated DNA components. Without the use of ligase or restriction enzymes, components are joined using oligonucleotide linkers that recognize the overhangs. By specifying the sequences of the linkers, desired components can be assembled in any combination and order to generate different plasmid vectors.     

Images of divalent cations in unstained symmetric and asymmetric lipid bilayers

Divalent cations have been microscopically visualized in association with simple lipid bilayers. Symmetric and asymmetric oriented bilayers were constructed from fatty acid monolayers and were cut in thin transverse sections for examination by bright field electron microscopy in the absence of stains, fixatives or embedding materials. It has been found that bilayers formed of lipid molecules having alkaline earth head groups exhibit natural electron contrast. The intrinsic image has been linked to local variations in the bilayer absolute electron density profile determined by X-ray diffraction analysis of the same specimens (McIntosh, T.J., Waldbillig, R. C. and Robertson J. D. (1976) Biochim. Biophys. Acta 448, 15–33). By combining the microscopic, chemical and X-ray evidence it has been estimated that local increments of about 1 g/cm³ can produce detectable electron contrast in 500 Å transverse sections of bilayers.     

The role of the phospholipid phase transition in the regulation of glucagon binding to lecithin

Glucagon can interact rapidly with multilamellar vesicles of dimyristoyl glycerophosphocholine over a narrow temperature range around or above the phase transition temperature of the pure phospholipid. The temperature dependence of the rates arises, in large part, from glucagon-induced alterations in the phase transition properties of the phospholipid. Similar effects are observed with dilaury glycerophosphocholine but the rate of reaction of glucagon with multilamellar dipalmitoyl glycerophosphocholine is too slow to measure.The rate of reaction of glucagon with equimolar mixtures of two phospholipid molecules has also been studied. Mixtures of dilauryl glycerophosphocholine and distearoyl glycerophosphocholine are known to exhibit lateral phase separation in the gel state. The presence of distearoyl glycerophosphocholine has no effect on the rate of reaction with glucagon, despite the increased number of phase boundaries present. In the case of mixtures of dilauryl glycerophosphocholine and dimyristoyl glycerophosphocholine, glucagon appears to induce some lateral phase separation. This is demonstrated by the ability of glucagon to react rapidly with this lipid mixture, even at temperatures well below the phase transition temperature of the mixture and by differential scanning calorimetry.The thermodynamics of the binding of glucagon to dimyristoyl glycerophosphocholine and dilauryl glycerophosphocholine were analyzed with Scatchard plots calculated from measurements of the fluorescence enhancement caused by lipids. Equilibrium binding constants of glucagon to dimyristoyl glycerophosphocholine and dilauryl glycerophosphocholine are 1·10⁵ and 5·10⁴ M^?1, respectively. These values are relatively insensitive to temperature, indicating that the equilibrium being measured is between lipid-bound glucagon and free lipid which has had its phase transition properties altered. The number of moles of lipid bound per mole of glucagon decreases markedly above the phase transition temperature. In the water-soluble complex formed between glucagon and dimyristoyl glycerophosphocholine, the peptide binds directly to only 40% of the lipid molecules but, nevertheless, is able to modify the phase transition properties of all of the lipid in the particle.     

The influence of cellular amino acids and the Na+: K+ pump on the membrane potential of the Ehrlich ascites tumor cell

The membrane potential of the Ehrlich ascites tumor cell was shown to be influenced by its amino acid content and the activity of the Na⁺: K⁺ pump. The membrane potential (monitored by the fluorescent dye, 3,3′-dipropylthiodicarbocyanine iodide) varied with the size of the endogenous amino acid pool and with the concentration of accumulated 2-aminoisobutyrate. When cellular amino acid content was high, the cells were hyperpolarized; as the pool declined in size, the cells were depolarized. The hyperpolarization seen with cellular amino acid required cellular Na⁺ but not cellular ATP. Na⁺ efflux was more rapid from cells containing 2-aminoisobutyrate than from cells low in internal amino acids. These observations indicate that the hyperpolarization recorded in cells with high cellular amino acid content resulted from the electrogenic co-efflux of Na⁺ and amino acids.Cellular ATP levels were found to decline rapidly in the presence of the dye and hence the influence of the pump was seen only if glucose was added to the cells. When the cells contained normal Na⁺ (approx. 30 mM), the Na⁺: K⁺ pump was shown to have little effect on the membrane potential (the addition of ouabain had little effect on the potential). When cellular Na⁺ was raised to 60 mM, the activity of the pump changed the membrane potential from the range ?25 to ?30 mV to ?44 to ?63 mV. This hyperpolarization required external K⁺ and was inhibited by ouabain.     

Cytoplasmic changes in level and distribution of glucose-6-phosphatase activities from rat liver during diethylnitrosamine-induced carcinogenesis.

Glucose-6-phosphatase (EC 3.1.3.9) activities were determined in isolated microsomes, cytoplasmic smooth and rough membranes, ribosomes and free cytosol from rat liver undergoing carcinogenesis by diethylnitrosamine (DENA) and compared with cytoplasmic fractions isolated in parallel from healthy animals from the same age.With continuous administration of a low dose of DENA (2.6 mg/kg rat per day for 20 weeks in the drinking water) livers of carcinogen treated rats became heavier than the control livers but the body weight decreased. About 70% of total glucose-6-phosphatase activity could be detected in the microsomal fraction. While there was no significant difference in this activity in both animal groups up to the 4th week, glucose-6-phosphatase of cancerous liver showed a distinct decrease of activity compared with normal liver.During cancer induction this enzyme became more soluble, confirmed by the observation that it was detached from firmer structures of cytoplasm as rough membranes and polysomes and translocated to smooth membranes and the soluble cytoplasmic fraction successively. The corresponding increase in glucose-6-phosphatase activity in the 105 000 g supernatant appears to be due to the loss of enzyme activity in a distinct cytoplasmic membrane fraction. These data strongly suggest that in parallel with alteration of cytoplasmic membrane structures during carcinogen feeding glucose-6-phosphatase is detached from heavier components of the cytoplasm while total activity decreased. Possible mechanisms of these findings are discussed.     

Linear and cyclic ester Oligomers of succinic acid and 1,4-butanediol: Biocatalytic synthesis and characterization

Abstract

The lipase-catalyzed synthesis of cyclic ester oligomers from non-activated succinic acid (A) and 1,4-butanediol (B) in the presence of immobilized Candida antarctica lipase B was investigated. Batch and pulse fed-batch systems were implemented to increase the formation of cyclic ester products. The substrate conversions after 24 h were 86% and 95% under batch and fed-batch operation, respectively and the product of the reaction was, for both systems, a mixture of cyclic (CEOs) and linear (LEOs) ester oligomers. Fed-batch operation afforded a product containing 71% cyclic ester oligomers (CEOs) as compared with only 52% CEOs in batch operation. Cyclic ester oligomers accumulated as the reaction progressed, with the dimer CEO₁ the most predominant product (i.e. 50% of the total products formed in fed-batch operation). The pulse fed-batch operation system was superior to the batch operation not only because higher substrate conversion and more CEOs were obtained, but also because it resulted in products with a higher degree of polymerization (DP up to 7). Cyclic ester oligomers are produced from the early stage of the reaction simultaneously with the linear ester oligomers by a ring-closure reaction on the active site of the enzyme, and not as a result of ring-chain equilibria.     

'Steady/equilibrium approximation' in relaxation and fluctuation. II. Mathematical theory of approximations in first-order reaction

A mathematical theory of the steady/equilibrium approximation for first-order reactions is presented. This gives the theoretical basis for the methods of simplifying the complex first-order reactions described in the preceding work The steady/equilibrium relation holds on every fast component after a proper inducation period T degrees T degrees is either of O(1) or less, or nearly of O(1/epsilon) depending on the reaction scheme and on the initial condition but is always less than O(1/epsilon) (as in the preceding paper [1], we use the symbol O(1) to denote a positive number of the order of unity). In the open group, the determinant of the submatrix M(p), representing the interconversion between the fast components in the group and their dissipation, is of O(1). The concentration of the fast components in the open group can thus be expressed as a linear combination of those components neighboring the group after the establishment of a steady/equilibrium relation, and can be eliminated from the reaction scheme leaving the pathway through them. On the other hand, in the closed group the determinant of Mp is of O(epsilon) or less and the components in the group are in quasi equilibrium with each other after T degrees . They are eliminated from the reaction scheme leaving the sum of the components in the closed group as a slow component.     

Low-temperature spectral properties of the respiratory chain cytochromes of mitochondria from Crithidia fasciculata

Highly purified mitochondrial preparations from the trypanosomatid hemoflagellate, Crithidia fasciculata (A.T.C.C. No.11745), were examined by low-temperature difference spectroscopy. The cytochrome a+a₃ maximum of hypotonically-treated mitochondria reduced with succinate, was shifted from 605 nm at room temperature to 601 nm at 77 °K. The Soret maximum, found at 445 nm at 23 °C, was split at 77 °K into two approximately equally absorbing species with maxima at 438 and 444 nm. A prominent shoulder observed at 590 nm with hypotonically-treated mitochondria was not present in spectra of isotonic controls.

The cytochrome b maxima observed in the presence of succinate plus antimycin A were shifted from the 431 and 561 nm positions observed at 23 °C to 427 and 557 nm at 77 °K. Multiple b cytochromes were not apparent.

Unlike other soluble c-type cytochromes, the maximum of cytochrome c₅₅₅ was not shifted at 77 °K although it was split to give a 551 nm shoulder adjacent to the 555 nm maximum. This lack of a low-temperature blue shift was true for partially purified hemoprotein preparations as well as in situ in the mitochondrial membrane.

Using cytochrome c₅₅₅-depleted mitochondria, a cytochrome c₁ pigment was observed with a maximum at 420 nm and multiple maxima at 551, 556, and 560 nm. After extraction of non-covalently bound heme, the pyridine hemochromogen difference spectrum of cytochrome c₅₅₅-depleted preparations exhibited an maximum at 553 nm at room temperature.

The reduced rate of succinate oxidation by cytochrome c₅₅₅-depleted mitochondria and the ferricyanide requirement for the reoxidation of cytochrome c₁, even in the presence of antimycin, indicated that cytochrome c₅₅₅-mediated electron transfer between cytochromes c₁ and a+a₃ in a manner analagous to that of cytochrome c in mammalian mitochondria.     

Silica supported 12-tungstophosphoric acid catalysts for synthesis of 1,4-dihydropyridines under solvent-free conditions

12-Tungstophosphoric acid (PW) supported on different metal oxides (SiO₂, γ-Al₂O₃, KSF, K10) and activated carbon were prepared by impregnation method and their catalytic performances were evaluated in three component condensation of benzaldehyde, ethyl acetoacetate and ammonium acetate to afford corresponding 1,4-dihydropyridine. A high catalytic activity was found over silica supported PW. Effect of PW loading, catalyst loading and solvent was studied to introduce the best reaction condition. Based on the above experimental finding, catalytic performances was optimized with a loading of 40% PW onto SiO₂ (0.2 g) under solvent-free condition. The characterization data derived from FT-IR, XRD, and TGA-DSC techniques reveal that the PW on silica support exists in Keggin structure. In addition, acidity measurements were performed by potentiometric titration with n-butylamine. The activity of the catalysts is strongly dependent on their acidic characteristic which, in turn, depended on PW loading. Finally, a series of 4-aryl, N-alkyl, and N-aryl substituted 1,4-dihydropyridines have been synthesized in high to excellent yield in short reaction times. PW/SiO₂ was found to be reusable and a considerable catalytic activity still could be achieved after fourth run.