Control of cellular redox potential as measured in a steady-state, cell-free system

A cell-free system consisting of rat liver mitochondria, liver cytosol, lactate, and the substrates intrinsic to the malate-aspartate shuttle was reconstituted for studies of steady-state substrate fluxes and, more specifically, to evaluate further the mechanism of control of the intra- and extramitochondrial steady states of the free NAD+/NADH ratios. Soluble (F1) ATPase or 2,4-dinitrophenol (DNP) were added in varying amounts to alter substrate fluxes and the constant energy state of this 'open' metabolizing system. The steady-state redox segregation (1.36 log NAD+/NADH ratio out vs NAD+/NADH in the mitochondrial matrix) was maximally about 3 kcal, and declined together with the membrane potential (delta psi) and log ATP/ADP, which obtain on imposing an increasing energy load on the system. It is concluded that transmembrane movement of reducing equivalents is coupled to electron transfer through delta psi, mediated by the electrogenic exchange of glutamate and aspartate. When delta psi was high (near State 4), delta G redox was approximately the same as that generated without flux of reducing equivalents [E. J. Davis, J. Bremer, and K. E. Akerman (1980) J. Biol. Chem. 255, 2277-2283], suggesting that delta Gredox is in near thermodynamic equilibrium with delta psi. If the steady-state ATP/ADP ratio was altered with an energy load (F1-ATPase), delta Gredox decreased more steeply than delta psi (tetraphenyl phosphonium-sensitive electrode used to measure delta psi). At comparable ranges of ATP/ADP, both delta Gredox and delta psi decreased more steeply with uncoupler than with an external ADP-regenerating system.     

Carbon source transport, nucleotide levels, and stable RNA synthesis in a mutant strain of Escherichia coli

We have previously described a temperature-sensitive mutant of Escherichia coli, 2S142 (rel-, met-, rns-, ilv-, ts-) which shows specific inhibition of stable RNA synthesis at 42 degrees C. This mutation mimics a carbon source downshift in that the decay of guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) is inhibited at the restrictive temperature. In this paper we show that the temperature-sensitive lesion in 2S142 does affect the uptake of glucose or alpha-D-methylglucopyranoside (alpha DMG) at 42 degrees C. However, restoration of glucose or alpha DMG uptake by the insertion of a constitutive galactose permease gene or further restriction of glucose uptake by insertion of a ptsG mutation into 2S142 have no effect on rRNA synthesis at 42 degrees C (although ppGpp levels are lowered in both cases). Furthermore, while restriction of uptake at 42 degrees C varies widely from carbon source to carbon source, severe restriction of rRNA synthesis is observed on all carbon sources tested at 42 degrees C. Levels of glycolytic intermediates, adenylate energy charge, ATP levels, and cAMP levels are all unaffected at the restrictive temperature. GTP levels decrease at 42 degrees C in glucose grown cells but that also does not appear to be related to the decrease in rRNA synthesis. These data were interpreted to suggest that the restriction of stable RNA synthesis in 2S142 at 42 degrees C can not be explained on the basis of decreased uptake and/or metabolism of carbon source. "Phantom spot" levels do decrease in 2S142 at 42 degrees C. In fact, "phantom spot" is the only putative regulatory molecule which correlates with restriction of rRNA synthesis on all carbon sources tested.     

Toad egg-jelly as a source of divalent cations essential for fertilization

Dejellied uterine eggs of the toad Bufo bufo japonicus are not fertilizable in 1/20 De Boer's solution (1/20 DB), but are fertilized when inseminated in a uv-solubilized jelly (UVJ) or the dialyzate of UVJ (UVJD). The present study was carried out to define this fertilization-supporting activity of egg-jelly. Dejellied eggs were fertilized in a high frequency when inseminated in a medium containing the ashes obtained by heating UVJD at 600 degrees C for 16 hr. Similarly, a reconstituted salt solution (RSS), which mimics the ionic composition of UVJD, supported a high rate of fertilization. To be effective in fertilization, however, RSS had to be present at the time of insemination. Analyses of individual salts revealed that dejellied eggs are successfully fertilized in CaCl2 and/or MgCl2 at 1-5 mM, only slightly in KCl at 10 mM, but not at all in NaCl at any of the concentrations tested. The activity of UVJD was lost reversibly when divalent cations were chelated by EDTA. The fertilization of dejellied eggs is therefore possible in a medium without any organic components of egg-jelly, provided that 2-5 mM Ca2+ or Mg2+ is present. Sperm were motile in media containing cations below 20-25 mM, regardless of the ionic composition. The egg-jelly possessed cations in a concentration of about 130 mM, but most ions were lost from intact jelly on immersion of eggs in water for 2-3 min, accompanied by the acquisition of fertilizability by sperm. Examination of the behavior of salts on dialysis or gel-filtration of jelly molecules revealed that the jelly retains Ca2+ and Mg2+, and possibly K+ as well, but not Na+ and Cl-. We propose that toad egg-jelly plays a function in fertilization by retaining Ca2+ and/or Mg2+ around each egg at the level necessary for successful sperm entrance into the egg.     

Free radicals from eumelanins: Quantum yields and wavelength dependence

Formation of light-induced free radicals from natural eumelanin (from bovine eyes) and synthetic melanin (from oxidation of 3,4-dihydroxyphenylalanine) has been studied by electron spin resonance spectroscopy. Action spectra measured for natural melanins are very similar to that found for synthetic melanin, and are unaffected by the removal of associated protein. A comparison of action spectra with optical absorbance spectra shows that the former has a more marked wavelength dependence, suggesting that the chromophore that is most active in free-radical production is not the major melanin chromophore that absorbs visible light. Measurements of quantum yields for freeradical production have been made over a wavelength range from 600 to 230 nm. The efficiency of radical production from natural eumelanin is about three times greater than from the synthetic material. Although production of the melanin radicals detected is independent of oxygen, some correlation with oxygen consumption is evident; quantum yields for radical production are approximately three times those for oxygen consumption obtained under similar conditions. Possible reasons for this are discussed.     

Effects of high K+ concentrations on the growth and development of ciliary ganglion neurons in cell culture

Extracts prepared from embryonic eye tissue permit all of the neurons present in embryonic ciliary ganglia to survive and develop in cell culture. High K⁺ concentrations stimulate growth of the neurons in culture above the maximal levels obtained with eye extract alone. Growth in 25 mM K⁺ produced parallel increases in the levels of choline acetyltransferase activity, lactate dehydrogenase activity (a common cytoplasmic enzyme), and total protein synthesis per neuron. The K⁺ effect appears to be mediated by membrane depolarization. Intracellular recording confirmed that the neurons were chronically depolarized in 25 mM K⁺. Veratridine produced the same stimulation of growth, while tetrodotoxin blocked the veratridine effect without preventing the K⁺ effect. Ca²⁺ may also play a role in the K⁺ effect. Two drugs thought to block Ca²⁺ channels (Mg²⁺ and D600) each blocked or reduced in the increase in growth caused by 25 mM K⁺. The drugs did not interfere with neuronal growth in control cultures, indicating that eye extract and membrane depolarization influence neuronal growth by different mechanisms.     

alpha-Fucose inhibits human mixed-lymphocyte culture reactions and subsequent suppressor cell generation

Carbohydrate moieties serve as important sites of interaction for many lymphocyte activities. The potential role of saccharides in the cellular interactions involved in mitogen-, antigen-, and alloantigen-induced proliferation was investigated. Eight different monosaccharides were tested for their inhibitory potential when added to uni- and bidirectional mixed-lymphocyte culture (MLC) reaction as well as to mitogen (Con A, PHA, PWM)-stimulated cultures. Only alpha-L-fucose blocked the MLC reaction in a dose-dependent fashion while having no effect on mitogen stimulation, although antigen-specific stimulation was also blocked by fucose. Similarly alpha-L-fucose specifically inhibited the MLC-induced generation of suppressor cells. Pretreatment of the MLC responder cells with fucose dehydrogenase abolished the MLC reaction while stimulator cell pretreatment had no effect, suggesting that the recognition site of the former contained alpha-L-fucose. The generation and the effector phase of Con A-induced suppressor cells was not affected by fucose, indicating that different receptors are involved in the latter. Apparent competitive inhibition by exogenous fucose of the cell-cell interaction required for the MLC reaction suggested that this monosaccharide is an essential constituent of allogeneic recognition sites.     

The interaction of calcium and procaine with hepatocyte and hepatoma tissue culture cell plasma membranes studied by fluorescence spectroscopy

The fluorescent probe l-anilinonaphthalene-8-sulfonate (ANS) has been used to investigate the properties of plasma membranes derived from normal hepatocytes and from hepatoma tissue culture (HTC) cells as well as used to study the effects of Ca²⁺ and procaine on these membrane systems. The interaction of ANS with hepatocyte plasma membranes (50 nmol/mg protein; K_D = 120,μM) resulted in a marked enhancement of fluorescence and a 20-nm blue shift. Both Ca²⁺ and procaine further increased the fluorescence intensity. Binding studies showed no alteration in the number of ANS binding sites but a significant decrease in K_D (40–50 μm). Procaine was also shown to completely displace Ca²⁺ from the membrane. The interaction of ANS with HTC cell plasma membranes again resulted in an enhancement in fluorescence intensity but with different binding properties (102 nmol/mg protein; K_D = 74 μM) from the hepatocyte system. The addition of Ca⁺² resulted in the formation of high and low affinity ANS binding sites as shown by Scatchard plot analysis with K_D values of 15 μm and 50 μm. The effect of procaine on ANS fluorescence in the normal and transformed cell membranes was indistinguishable; however, in the latter system procaine only displaced 60% of the bound Ca²⁺. These studies suggest several structural and binding alterations between plasma membranes derived from hepatocytes and HTC cells.     

Characterization of a cell culture model for the study of adenosine deaminase- and purine nucleoside phosphorylase-deficient immunologic disease.

The absence of erythrocytic adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP) has been associated with severe immunodeficiency disease in children. We have developed a cell culture model to study the possible relationships between purine salvage enzymes and immunologic function using an established T cell lymphosarcoma (S49) and a potent inhibitor of ADA, erythro-9(2-hydroxy-3-nonyl) adenine (EHNA). Wild-type S49 cells are killed by dexamethasone or dbc AMP, and adenosine (5 muM) in the presence of an ADA inhibitor (6 muM EHNA) also prevents the growth of and kills these S49 cells. It has been proposed that adenosine is toxic to lymphoid cells by virtue of its ability to increase the intracellular concentrations of cyclic AMP. We examined the sensitivity of three mutants of S49 cells, with distinctive defects in some component of cyclic AMP metabolism or action, to killing by adenosine and EHNA. All three mutants are resistant to killing by isoproterenol or cholera toxin and two are resistant to dbc AMP itself, but all are sensitive to killing by adenosine and EHNA. Similarly, two dexamethasone-resistant S49 mutants are as sensitive to adenosine and EHNA as are the wildtype cells. We have also simulated the purine nucleoside phosphorylase deficiency in S49 cells by adding inosine and adenosine to the growth medium. In the presence of EHNA or inosine, the toxic effects of adenosine can be partially reversed by addition of (10-20 muM) uridine, an observation suggesting that adenosine is toxic as the result of its inducing pyrimidine starvation.     

Paramagnetism in melanins: pH dependence

Changes in the paramagnetic properties of aqueous suspensions of melanin polymers have been monitored over a pH range from 1 to 12. Distinct changes in spin concentration and electron spin resonance spectral parameters (effective g value and line shape) are shown to occur. These data are interpreted in terms of pH- and temperaturedependent equilibria between diamagnetic and paramagnetic units on the melanin polymer, which can be partly or completely quenched if the melanin is precipitated by lowering the pH or by increasing the salt concentration. The heterogeneity of these units and possible chemical structures are discussed.     

Calorimetric study of carbohydrate binding to Concanavalin A

Flow microcalorimetry has been used to examine the ΔH of binding of two types of saccharides, a series of simple monosaccharides and a series of α-(1 → 4)-linked glucosides, to the lectin Concanavalin A. It has been found that the ΔH decreases with any change in the stereochemistry of a hydroxyl group relative to methyl α-d-mannopyranoside. The data have allowed the calculation of the relative contribution of two of the hydroxyl groups. The ΔH's of binding for the α-(1 → 4)-linked glucosides are approximately 31 kJ/mol, and the apparent association constants vary insignificantly with increasing length. This result indicates that only one glucose residue binds to concanavalin A by hydrogen bonds, and that the additional glucose residues have no interaction either by hydrogen bonds or by nonspecific hydrophobic interactions. This result confirms the absence of an extended binding site for α-(1 → 4)-linked glucopyranosides, in contrast to that proposed for α-(1 → 2)-linked mannopyranosides which show an increase in apparent association constants with increasing length.     

Tracing the development of oligodendrocytes from precursor cells using monoclonal antibodies, fluorescence-activated cell sorting, and cell culture

We have used antibody and complement-mediated cell killing, fluorescence-activated cell sorting and tissue culture to study the development of rat oligodendrocytes. We show that (1) three ligands that bind to the majority of CNS neurons (the monoclonal antibodies A4 and A2B5 and tetanus toxin) also bind to immature oligodendrocytes and to precursor cells in 14-day embryonic rat brain that develop into oligodendrocytes in vitro; and (2) precursor cells in 17- to 18-day embryonic rat optic nerve can develop into oligodendrocytes in vitro in the absence of living neurons.     

Differential gas-liquid chromatography method for determination of uronic acids in carbohydrate mixtures

An integrated gas-liquid chromatography method is described for the quantitation of mixtures containing simple monosaccharides in addition to mannuronic, glucuronic, and/or galacturonic acids. A hydrolyzed sample is divided into two portions. One portion is analyzed by the standard aldononitrile method. Glucuronic, galacturonic, and mannuronic acids are converted into compounds that do not chromatograph in the region of the standard aldononitrile acetates. Thus, this analysis gives an accurate estimation of the neutral monosaccharide content. The second portion is analyzed by a modified alditol acetate procedure. The reduction step is repeated three times to convert mannuronic, galacturonic, and glucuronic acids to their corresponding alditols via their intermediate lactones. The results of this gas-liquid chromatography analysis reflect the sum of the monosaccharides present plus their corresponding uronic acids. The difference between the values obtained by the aldononitrile acetate method and the modified alditol acetate method, therefore, is a measure of the uronic acid(s) present.     

Role of the carbohydrate part of yeast acid phosphatase

Acid phosphatase, purified from the yeast Saccharomyces cerevisiae, was completely deglycosylated by endo-beta-N-acetylglucosaminidase H or by HF treatment. Three protein bands were obtained on sodium dodecyl sulfate (SDS)-electrophoresis, with molecular weights of 73,000, 71,000 and 61,500. The released carbohydrate chains varied in size from 12 to 142 mannose units. To study the role of carbohydrate chains in the structure and function of acid phosphatase, a comparison of the properties of the partially deglycosylated enzyme with the native one was performed. The 60% deglycosylated enzyme retained the original activity, and CD and fluorescence spectra showed that the native conformation of the enzyme was preserved. The 90% deglycosylated enzyme showed a pronounced loss of enzyme activity, accompanied by the disruption of the three-dimensional structure. The partially deglycosylated enzyme was less soluble and more susceptible to denaturing effects of heat, pH, urea, and guanidine hydrochloride. Under conditions of electrophoresis, the partially deglycosylated enzyme dissociated, indicating a possible role of carbohydrate chains in maintaining the dimeric structure of the enzyme. Susceptibility of acid phosphatase toward proteolysis was drastically increased by deglycosylation.     

Purification and characterization of a vanadate-sensitive nucleotide tri- and diphosphatase with acid pH optimum from rat bone

Rat bone was extracted with KCl and Triton X-100, and a tartrate-resistant acid phosphatase activity was purified by protamine sulfate precipitation, ion-exchange chromatography (CM-cellulose), and gel filtration on Sephadex G-200 according to previously described procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining demonstrated a major band with an apparent monomer molecular size of approximately 14,000 Da. The enzyme is active with p-nitrophenylphosphate (p-NPP) but exhibits a 5- to 10-fold higher affinity towards several nucleotides of which ATP and ADP are the most readily hydrolyzed substrates based on kinetic studies. Based on sensitivity towards proteolytic treatment and detergent removal, as well as pH-optimum studies, a single enzyme was found to be responsible for activity towards nucleotide phosphates as well as p-NPP. This nucleotide tri- and diphosphatase constitutes around 15% of the total acid phosphatase activity in rat bone. The activity with ATP as substrate in contrast to that with p-NPP was inhibited in a noncompetitive fashion by MgCl2, sodium metavanadate, and p-chloromercuribenzoate. Enzyme activity with p-NPP and ATP is dependent on the presence of KCl and detergent and is activated by Fe3+ and ascorbate. The reported characteristics of the enzyme suggest that it functions as a unique membrane acid ATPase.     

Channel open time of acetylcholine receptors on Xenopus muscle cells in dissociated cell culture

The kinetics of acetylcholine (ACh) receptor channels on cultured myotomal muscle cells from Xenopus embryos were studied by analyzing focally recorded membrane currents. The mean open time for receptor channels on embryonic muscle cells grown in dissociated cell cultures showed a time-dependent decrease similar to that seen in vivo. The changes in power density spectra are consistent with the hypothesis that the decrease results from the appearance of a class of ACh receptor with a short mean channel open time (0.7 msec) and a decrease in the proportion of receptors with a long mean channel open time (3 msec). The addition of dissociated neural tube cells to muscle cell cultures resulted in an unexpected increase in mean channel open time for ACh receptors in both synaptic and nonsynaptic regions. These studies demonstrate that ACh receptor function may be altered in cultured muscle cells.     

Structural studies on a carbohydrate chain isolated from the lipopolysaccharide of Shigella boydii type 8

The lipopolysaccharide isolated from the cells of Shigella boydii type 8 bacteria gave precipitin bands against homologous antisera on Ouchterlony plates, whereas the carbohydrate-containing fractions obtained from it did not. One of the fractions was obtained in major proportion and contained 23.5% of sugars. A structure was assigned to the carbohydrate chain in this material by using the results of methylation, periodate oxidation, and deamination studies.     

Fructose 1,6-bisphosphate aldolase of Drosophila melanogaster: comparative sequence analyses around the substrate-binding lysyl residue

The amino acid sequence of a 103 residue segment encompassing the substrate-binding active site lysyl residue of fructose 1,6-bisphosphate aldolase from Drosophila melanogaster is determined. The sequence is identical to more than 70% with the structure of rabbit muscle aldolase and with the known partial sequences of the sturgeon muscle, trout muscle, and ox liver enzymes. The homology of the insect enzyme with the vertebrate aldolases strongly implies a similar tertiary structure folding.     

Influence of the redox potential on the activity of Clostridium pasteurianum and Chromatium hydrogenases

The oxidation of reduced methyl viologen by Clostridium pasteurianum or Chromatium hydrogenases as a function of the redox potential of the reaction mixture has been studied spectrophotometrically. The same results were obtained effecting the reduction of methyl viologen either with dithionite or with metallic zinc. With C. pasteurianum hydrogenase a gaussian pattern was obtained. This is indicative of a process involving two one-electron steps, which suggests that [4Fe-4S]²⁺ is the catalytically active species. On the contrary, in the case of Chromatium hydrogenase the data follow a sigmoidal pattern corresponding to a two-electron reduction process, which demonstrates that the redox site must be totally reduced to be active. This finding is at variance with the previously reported electron paramagnetic resonance spectra, which suggest that the single [4Fe-4S] cluster of this enzyme transfers or accepts only one electron.