Selective Irreversible Inhibition of Neuronal and Inducible Nitric-oxide Synthase in the Combined Presence of Hydrogen Sulfide and Nitric Oxide

Citrulline formation by both human neuronal nitric-oxide synthase (nNOS) and mouse macrophage inducible NOS was inhibited by the hydrogen sulfide (H₂S) donor Na₂S with IC₅₀ values of ∼2.4·10⁻⁵ and ∼7.9·10⁻⁵ m, respectively, whereas human endothelial NOS was hardly affected at all. Inhibition of nNOS was not affected by the concentrations of l-arginine (Arg), NADPH, FAD, FMN, tetrahydrobiopterin (BH4), and calmodulin, indicating that H₂S does not interfere with substrate or cofactor binding. The IC₅₀ decreased to ∼1.5·10⁻⁵ m at pH 6.0 and increased to ∼8.3·10⁻⁵ m at pH 8.0. Preincubation of concentrated nNOS with H₂S under turnover conditions decreased activity after dilution by ∼70%, suggesting irreversible inhibition. However, when calmodulin was omitted during preincubation, activity was not affected, suggesting that irreversible inhibition requires both H₂S and NO. Likewise, NADPH oxidation was inhibited with an IC₅₀ of ∼1.9·10⁻⁵ m in the presence of Arg and BH4 but exhibited much higher IC₅₀ values (∼1.0–6.1·10⁻⁴ m) when Arg and/or BH4 was omitted. Moreover, the relatively weak inhibition of nNOS by Na₂S in the absence of Arg and/or BH4 was markedly potentiated by the NO donor 1-(hydroxy-NNO-azoxy)-l-proline, disodium salt (IC₅₀ ∼ 1.3–2.0·10⁻⁵ m). These results suggest that nNOS and inducible NOS but not endothelial NOS are irreversibly inhibited by H₂S/NO at modest concentrations of H₂S in a reaction that may allow feedback inhibition of NO production under conditions of excessive NO/H₂S formation.     

Fast Inactivation of Delayed Rectifier K Conductance in Squid Giant Axon and Its Cell Bodies

Inactivation of delayed rectifier K conductance (g_K) was studied in squid giant axons and in the somata of giant fiber lobe (GFL) neurons. Axon measurements were made with an axial wire voltage clamp by pulsing to V_K (∼−10 mV in 50–70 mM external K) for a variable time and then assaying available g_K with a strong, brief test pulse. GFL cells were studied with whole-cell patch clamp using the same prepulse procedure as well as with long depolarizations. Under our experimental conditions (12–18°C, 4 mM internal MgATP) a large fraction of g_K inactivates within 250 ms at −10 mV in both cell bodies and axons, although inactivation tends to be more complete in cell bodies. Inactivation in both preparations shows two kinetic components. The faster component is more temperature-sensitive and becomes very prominent above 12°C. Contribution of the fast component to inactivation shows a similar voltage dependence to that of g_K, suggesting a strong coupling of this inactivation path to the open state. Omission of internal MgATP or application of internal protease reduces the amount of fast inactivation. High external K decreases the amount of rapidly inactivating I_K but does not greatly alter inactivation kinetics. Neither external nor internal tetraethylammonium has a marked effect on inactivation kinetics. Squid delayed rectifier K channels in GFL cell bodies and giant axons thus share complex fast inactivation properties that do not closely resemble those associated with either C-type or N-type inactivation of cloned Kv1 channels studied in heterologous expression systems.     

Voltage Gating of Shaker K+ Channels : The Effect of Temperature on Ionic and Gating Currents

Ionic (I_i) and gating currents (I_g) from noninactivating Shaker H4 K⁺ channels were recorded with the cut-open oocyte voltage clamp and macropatch techniques. Steady state and kinetic properties were studied in the temperature range 2–22°C. The time course of I_i elicited by large depolarizations consists of an initial delay followed by an exponential rise with two kinetic components. The main I_i component is highly temperature dependent (Q₁₀ > 4) and mildly voltage dependent, having a valence times the fraction of electric field (z) of 0.2–0.3 e_o. The I_g On response obtained between −60 and 20 mV consists of a rising phase followed by a decay with fast and slow kinetic components. The main I_g component of decay is highly temperature dependent (Q₁₀ > 4) and has a z between 1.6 and 2.8 e_o in the voltage range from −60 to −10 mV, and ∼0.45 e_o at more depolarized potentials. After a pulse to 0 mV, a variable recovery period at −50 mV reactivates the gating charge with a high temperature dependence (Q₁₀ > 4). In contrast, the reactivation occurring between −90 and −50 mV has a Q₁₀ = 1.2. Fluctuation analysis of ionic currents reveals that the open probability decreases 20% between 18 and 8°C and the unitary conductance has a low temperature dependence with a Q₁₀ of 1.44. Plots of conductance and gating charge displacement are displaced to the left along the voltage axis when the temperature is decreased. The temperature data suggests that activation consists of a series of early steps with low enthalpic and negative entropic changes, followed by at least one step with high enthalpic and positive entropic changes, leading to final transition to the open state, which has a negative entropic change.     

Calcium-Voltage Coupling in the Genesis of Early and Delayed Afterdepolarizations in Cardiac Myocytes

Early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs) are voltage oscillations known to cause cardiac arrhythmias. EADs are mainly driven by voltage oscillations in the repolarizing phase of the action potential (AP), while DADs are driven by spontaneous calcium (Ca) release during diastole. Because voltage and Ca are bidirectionally coupled, they modulate each other’s behaviors, and new AP and Ca cycling dynamics can emerge from this coupling. In this study, we performed computer simulations using an AP model with detailed spatiotemporal Ca cycling incorporating stochastic openings of Ca channels and ryanodine receptors to investigate the effects of Ca-voltage coupling on EAD and DAD dynamics. Simulations were complemented by experiments in mouse ventricular myocytes. We show that: 1) alteration of the Ca transient due to increased ryanodine receptor leakiness and/or sarco/endoplasmic reticulum Ca ATPase activity can either promote or suppress EADs due to the complex effects of Ca on ionic current properties; 2) spontaneous Ca waves also exhibit complex effects on EADs, but cannot induce EADs of significant amplitude without the participation of I_Ca,L; 3) lengthening AP duration and the occurrence of EADs promote DADs by increasing intracellular Ca loading, and two mechanisms of DADs are identified, i.e., Ca-wave-dependent and Ca-wave-independent; and 4) Ca-voltage coupling promotes complex EAD patterns such as EAD alternans that are not observed for solely voltage-driven EADs. In conclusion, Ca-voltage coupling combined with the nonlinear dynamical behaviors of voltage and Ca cycling play a key role in generating complex EAD and DAD dynamics observed experimentally in cardiac myocytes, whose mechanisms are complex but analyzable.     

Ion permeation and block of the gating pore in the voltage sensor of NaV1.4 channels with hypokalemic periodic paralysis mutations

Hypokalemic periodic paralysis and normokalemic periodic paralysis are caused by mutations of the gating charge–carrying arginine residues in skeletal muscle Na_V1.4 channels, which induce gating pore current through the mutant voltage sensor domains. Inward sodium currents through the gating pore of mutant R666G are only ∼1% of central pore current, but substitution of guanidine for sodium in the extracellular solution increases their size by 13- ± 2-fold. Ethylguanidine is permeant through the R666G gating pore at physiological membrane potentials but blocks the gating pore at hyperpolarized potentials. Guanidine is also highly permeant through the proton-selective gating pore formed by the mutant R666H. Gating pore current conducted by the R666G mutant is blocked by divalent cations such as Ba²⁺ and Zn²⁺ in a voltage-dependent manner. The affinity for voltage-dependent block of gating pore current by Ba²⁺ and Zn²⁺ is increased at more negative holding potentials. The apparent dissociation constant (K_d) values for Zn²⁺ block for test pulses to −160 mV are 650 ± 150 µM, 360 ± 70 µM, and 95.6 ± 11 µM at holding potentials of 0 mV, −80 mV, and −120 mV, respectively. Gating pore current is blocked by trivalent cations, but in a nearly voltage-independent manner, with an apparent K_d for Gd³⁺ of 238 ± 14 µM at −80 mV. To test whether these periodic paralyses might be treated by blocking gating pore current, we screened several aromatic and aliphatic guanidine derivatives and found that 1-(2,4-xylyl)guanidinium can block gating pore current in the millimolar concentration range without affecting normal Na_V1.4 channel function. Together, our results demonstrate unique permeability of guanidine through Na_V1.4 gating pores, define voltage-dependent and voltage-independent block by divalent and trivalent cations, respectively, and provide initial support for the concept that guanidine-based gating pore blockers could be therapeutically useful.     

Estimation of Sediment Denitrification Rates at In Situ Nitrate Concentrations

The denitrification rates in a marine sediment, estimated by using ¹⁵N-nitrate, V_max, K_m, and sediment nitrate concentrations, were 12.5 and 2.0 nmol of N₂-N cm⁻³ day⁻¹ at 0 to 1 and 1 to 3 cm, respectively, at 12°C. The total rate was 165 nmol of N₂-N m⁻² day⁻¹.     

Effect of Sarcoplasmic Reticulum (SR) Calcium Content on SR Calcium Release Elicited by Small Voltage-Clamp Depolarizations in Frog Cut Skeletal Muscle Fibers Equilibrated with 20 mM EGTA

Cut muscle fibers from Rana temporaria (sarcomere length, 3.5–3.9 μm; 14–16°C) were mounted in a double Vaseline-gap chamber and equilibrated with an external solution that contained tetraethyl ammonium– gluconate and an internal solution that contained Cs as the principal cation, 20 mM EGTA, and 0 Ca. Fibers were stimulated with a voltage-clamp pulse protocol that consisted of pulses to −70, −65, −60, −45, and −20 mV, each separated by 400-ms periods at −90 mV. The change in total Ca that entered into the myoplasm (Δ[Ca_T]) and the Ca content of the SR ([Ca_SR]) were estimated with the EGTA/phenol red method (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259–336). Fibers were stimulated with the pulse protocol, usually every 5 min, so that the resting value of [Ca_SR] decreased from its initial value of 1,700–2,300 μM to values near or below 100 μM after 18–30 stimulations. Three main findings for the voltage pulses to −70, −65, and −60 mV are: (a) the depletion-corrected rate of Ca release (release permeability) showed little change when [Ca_SR] decreased from its highest level (>1,700 μM) to ∼1,000 μM; (b) as [Ca_SR] decreased below 1,000 μM, the release permeability increased to a maximum level when [Ca_SR] was near 300 μM that was on average about sevenfold larger than the values observed for [Ca_SR] > 1,000 μM; and (c) as [Ca_SR] decreased from ∼300 μM to <100 μM, the release permeability decreased, reaching half its maximum value when [Ca_SR] was ∼110 μM on average. It was concluded that finding b was likely due to a decrease in Ca inactivation, while finding c was likely due to a decrease in Ca-induced Ca release.     

Arresting Amyloid with Coulomb’s Law: Acetylation of ALS-Linked SOD1 by Aspirin Impedes Aggregation

Although the magnitude of a protein’s net charge (Z) can control its rate of self-assembly into amyloid, and its interactions with cellular membranes, the net charge of a protein is not viewed as a druggable parameter. This article demonstrates that aspirin (the quintessential acylating pharmacon) can inhibit the amyloidogenesis of superoxide dismutase (SOD1) by increasing the intrinsic net negative charge of the polypeptide, i.e., by acetylation (neutralization) of multiple lysines. The protective effects of acetylation were diminished (but not abolished) in 100 mM NaCl and were statistically significant: a total of 432 thioflavin-T amyloid assays were performed for all studied proteins. The acetylation of as few as three lysines by aspirin in A4V apo-SOD1—a variant that causes familial amyotrophic lateral sclerosis (ALS)—delayed amyloid nucleation by 38% and slowed amyloid propagation by twofold. Lysines in wild-type- and ALS-variant apo-SOD1 could also be peracetylated with aspirin after fibrillization, resulting in supercharged fibrils, with increases in formal net charge of ∼2 million units. Peracetylated SOD1 amyloid defibrillized at temperatures below unacetylated fibrils, and below the melting temperature of native Cu₂,Zn₂-SOD1 (e.g., fibril T_m = 84.49°C for acetylated D90A apo-SOD1 fibrils). Targeting the net charge of native or misfolded proteins with small molecules—analogous to how an enzyme’s K_m or V_max are medicinally targeted—holds promise as a strategy in the design of therapies for diseases linked to protein self-assembly.     

Evolution of cardiac calcium waves from stochastic calcium sparks

We present a model that provides a unified framework for studying Ca2+ sparks and Ca2+ waves in cardiac cells. The model is novel in combining 1) use of large currents (approximately 20 pA) through the Ca2+ release units (CRUs) of the sarcoplasmic reticulum (SR); 2) stochastic Ca2+ release (or firing) of CRUs; 3) discrete, asymmetric distribution of CRUs along the longitudinal (separation distance of 2 microm) and transverse (separated by 0.4-0.8 microm) directions of the cell; and 4) anisotropic diffusion of Ca2+ and fluorescent indicator to study the evolution of Ca2+ waves from Ca2+ sparks. The model mimics the important features of Ca2+ sparks and Ca2+ waves in terms of the spontaneous spark rate, the Ca2+ wave velocity, and the pattern of wave propagation. Importantly, these features are reproduced when using experimentally measured values for the CRU Ca2+ sensitivity (approximately 15 microM). Stochastic control of CRU firing is important because it imposes constraints on the Ca2+ sensitivity of the CRU. Even with moderate (approximately 5 microM) Ca2+ sensitivity the very high spontaneous spark rate triggers numerous Ca2+ waves. In contrast, a single Ca2+ wave with arbitrarily large velocity can exist in a deterministic model when the CRU Ca2+ sensitivity is sufficiently high. The combination of low CRU Ca2+ sensitivity (approximately 15 microM), high cytosolic Ca2+ buffering capacity, and the spatial separation of CRUs help control the inherent instability of SR Ca2+ release. This allows Ca2+ waves to form and propagate given a sufficiently large initiation region, but prevents a single spark or a small group of sparks from triggering a wave.     

Methanol Promotes Atmospheric Methane Oxidation by Methanotrophic Cultures and Soils

Two methanotrophic bacteria, Methylobacter albus BG8 and Methylosinus trichosporium OB3b, oxidized atmospheric methane during batch growth on methanol. Methane consumption was rapidly and substantially diminished (95% over 9 days) when washed cell suspensions were incubated without methanol in the presence of atmospheric methane (1.7 ppm). Methanotrophic activity was stimulated after methanol (10 mM) but not methane (1,000 ppm) addition. M. albus BG8 grown in continuous culture for 80 days with methanol retained the ability to oxidize atmospheric methane and oxidized methane in a chemostat air supply. Methane oxidation during growth on methanol was not affected by methane deprivation. Differences in the kinetics of methane uptake (apparent K_m and V_max) were observed between batch- and chemostat-grown cultures. The V_max and apparent K_m values (means ± standard errors) for methanol-limited chemostat cultures were 133 ± 46 nmol of methane 10⁸ cells⁻¹ h⁻¹ and 916 ± 235 ppm of methane (1.2 μM), respectively. These values were significantly lower than those determined with batch-grown cultures (V_max of 648 ± 195 nmol of methane 10⁸ cells⁻¹ h⁻¹ and apparent K_m of 5,025 ± 1,234 ppm of methane [6.3 μM]). Methane consumption by soils was stimulated by the addition of methanol. These results suggest that methanol or other nonmethane substrates may promote atmospheric methane oxidation in situ.     

The First Identification of Carbohydrate Binding Modules Specific to Chitosan

Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (T_m) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)₆. The T_m elevation (ΔT_m) in DD1 was the highest (10.3 °C) upon the addition of (GlcN)₆ and was markedly higher than that in DD2 (1.0 °C). A synergistic effect was observed (ΔT_m = 13.6 °C), when (GlcN)₆ was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)_n; ΔG_r° values were −7.8 (n = 6), −7.6 (n = 5), −7.6 (n = 4), −7.6 (n = 3), and −7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)_n with the lower affinities (ΔG_r° = −5.0 (n = 3) ∼ −5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)₆ for individual DD1 and DD2 sites (ΔG_r° = −8.6 and −6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)_n (n = 2∼6) were found to bind to loops extruded from the core β-sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis.     

Mitochondrial Networks in Cardiac Myocytes Reveal Dynamic Coupling Behavior

Oscillatory behavior of mitochondrial inner membrane potential (ΔΨ_m) is commonly observed in cells subjected to oxidative or metabolic stress. In cardiac myocytes, the activation of inner membrane pores by reactive oxygen species (ROS) is a major factor mediating intermitochondrial coupling, and ROS-induced ROS release has been shown to underlie propagated waves of ΔΨ_m depolarization as well as synchronized limit cycle oscillations of ΔΨ_m in the network. The functional impact of ΔΨ_m instability on cardiac electrophysiology, Ca²⁺ handling, and even cell survival, is strongly affected by the extent of such intermitochondrial coupling. Here, we employ a recently developed wavelet-based analytical approach to examine how different substrates affect mitochondrial coupling in cardiac cells, and we also determine the oscillatory coupling properties of mitochondria in ventricular cells in intact perfused hearts. The results show that the frequency of ΔΨ_m oscillations varies inversely with the size of the oscillating mitochondrial cluster, and depends on the strength of local intermitochondrial coupling. Time-varying coupling constants could be quantitatively determined by applying a stochastic phase model based on extension of the well-known Kuramoto model for networks of coupled oscillators. Cluster size-frequency relationships varied with different substrates, as did mitochondrial coupling constants, which were significantly larger for glucose (7.78 × 10⁻² ± 0.98 × 10⁻² s⁻¹) and pyruvate (7.49 × 10⁻² ± 1.65 × 10⁻² s⁻¹) than lactate (4.83 × 10⁻² ± 1.25 × 10⁻² s⁻¹) or β-hydroxybutyrate (4.11 × 10⁻² ± 0.62 × 10⁻² s⁻¹). The findings indicate that mitochondrial spatiotemporal coupling and oscillatory behavior is influenced by substrate selection, perhaps through differing effects on ROS/redox balance. In particular, glucose-perfusion generates strong intermitochondrial coupling and temporal oscillatory stability. Pathological changes in specific catabolic pathways, which are known to occur during the progression of cardiovascular disease, could therefore contribute to altered sensitivity of the mitochondrial network to oxidative stress and emergent ΔΨ_m instability, ultimately scaling to produce organ level dysfunction.     

Population Shift Underlies Ca2+-induced Regulatory Transitions in the Sodium-Calcium Exchanger (NCX)

In eukaryotic Na⁺/Ca²⁺ exchangers (NCX) the Ca²⁺ binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca²⁺ sensor (Ca3–Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca²⁺-driven interdomain switch has been described, albeit how it couples Ca²⁺ binding with signal propagation remains unclear. To resolve the dynamic features of Ca²⁺-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium ⁴⁵Ca²⁺ binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg²⁺-bound forms of CBD12 are highly flexible, whereas Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca²⁺ is observed on the globally derived D_max (maximal interatomic distance), although under comparable conditions a normal [Ca²⁺]-dependent allosteric regulation occurs. Low affinity sites (Ca1–Ca2) of CBD1 do not contribute to Ca²⁺-induced population shift, but the occupancy of these sites by 1 mm Mg²⁺ shifts the Ca²⁺ affinity (K_d) at the neighboring Ca3–Ca4 sites from ∼ 50 nm to ∼ 200 nm and thus, keeps the primary Ca²⁺ sensor (Ca3–Ca4 sites) within a physiological range. Thus, Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment.     

Arachidonic acid inhibition of L-type calcium (CaV1.3b) channels varies with accessory CaVβ subunits

Arachidonic acid (AA) inhibits the activity of several different voltage-gated Ca²⁺ channels by an unknown mechanism at an unknown site. The Ca²⁺ channel pore-forming subunit (Ca_Vα₁) is a candidate for the site of AA inhibition because T-type Ca²⁺ channels, which do not require accessory subunits for expression, are inhibited by AA. Here, we report the unanticipated role of accessory Ca_Vβ subunits on the inhibition of Ca_V1.3b L-type (L-) current by AA. Whole cell Ba²⁺ currents were measured from recombinant channels expressed in human embryonic kidney 293 cells at a test potential of −10 mV from a holding potential of −90 mV. A one-minute exposure to 10 µM AA inhibited currents with β_1b, β₃, or β₄ 58, 51, or 44%, respectively, but with β_2a only 31%. At a more depolarized holding potential of −60 mV, currents were inhibited to a lesser degree. These data are best explained by a simple model where AA stabilizes Ca_V1.3b in a deep closed-channel conformation, resulting in current inhibition. Consistent with this hypothesis, inhibition by AA occurred in the absence of test pulses, indicating that channels do not need to open to become inhibited. AA had no effect on the voltage dependence of holding potential–dependent inactivation or on recovery from inactivation regardless of Ca_Vβ subunit. Unexpectedly, kinetic analysis revealed evidence for two populations of L-channels that exhibit willing and reluctant gating previously described for Ca_V2 channels. AA preferentially inhibited reluctant gating channels, revealing the accelerated kinetics of willing channels. Additionally, we discovered that the palmitoyl groups of β_2a interfere with inhibition by AA. Our novel findings that the Ca_Vβ subunit alters kinetic changes and magnitude of inhibition by AA suggest that Ca_Vβ expression may regulate how AA modulates Ca²⁺-dependent processes that rely on L-channels, such as gene expression, enzyme activation, secretion, and membrane excitability.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

Determination of Bacterial Cell Dry Mass by Transmission Electron Microscopy and Densitometric Image Analysis

We applied transmission electron microscopy and densitometric image analysis to measure the cell volume (V) and dry weight (DW) of single bacterial cells. The system was applied to measure the DW of Escherichia coli DSM 613 at different growth phases and of natural bacterial assemblages of two lakes, Piburger See and Gossenköllesee. We found a functional allometric relationship between DW (in femtograms) and V (in cubic micrometers) of bacteria (DW = 435 · V^0.86); i.e., smaller bacteria had a higher ratio of DW to V than larger cells. The measured DW of E. coli cells ranged from 83 to 1,172 fg, and V ranged from 0.1 to 3.5 μm³ (n = 678). Bacterial cells from Piburger See and Gossenköllesee (n = 465) had DWs from 3 fg (V = 0.003 μm³) to 1,177 fg (V = 3.5 μm³). Between 40 and 50% of the cells had a DW of less than 20 fg. By assuming that carbon comprises 50% of the DW, the ratio of carbon content to V of individual cells varied from 466 fg of C μm⁻³ for Vs of 0.001 to 0.01 μm³ to 397 fg of C μm⁻³ (0.01 to 0.1 μm³) and 288 fg of C μm⁻³ (0.1 to 1 μm³). Exponentially growing and stationary cells of E. coli DSM 613 showed conversion factors of 254 fg of C μm⁻³ (0.1 to 1 μm³) and 211 fg of C μm⁻³ (1 to 4 μm³), respectively. Our data suggest that bacterial biomass in aquatic environments is higher and more variable than previously assumed from volume-based measurements.     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.     

Single-channel analysis of the anion channel-forming protein from the plant pathogenic bacterium Clavibacter michiganense ssp. nebraskense

The anion channel protein from Clavibacter michiganense ssp. nebraskense (Schürholz, Th. et al. 1991, J. Membrane Biol. 123: 1-8) was analyzed at different concentrations of KCl and KF. At 0.8 M KCl the conductance G(V_m) increases exponentially from 21 pS at 50 mV up to 53 pS at V_m = 200 mV, 20°C. The concentration dependence of G(V_m) corresponds to a Michaelis-Menten type saturation function at all membrane voltage values applied (0-200 mV). The anion concentration K_0.5, where G(V_m) has its half-maximum value, increases from 0.12 M at 50 mV to 0.24 M at 175 mV for channels in a soybean phospholipid bilayer. The voltage dependence of the single channel conductance, which is different for charged and neutral lipid bilayers, can be described either by a two-state flicker (2SF) model and the Nernst-Planck continuum theory, or by a two barrier, one-site (2B1S) model with asymmetric barriers. The increase in the number of open channels after a voltage jump from 50 mV to 150 mV has a time constant of 0.8 s. The changes of the single-channel conductance are much faster (<1 ms). The electric part of the gating process is characterized by the (reversible) molar electrical work ΔG^θ_el = ρZ_gFV_m ≈ -1.3 RT, which corresponds to the movement of one charge of the gating charge number |Z_g| = 1 across the fraction ρ = ΔV_m/V_m = 0.15 of the membrane voltage V_m = 200 mV. Unlike with chloride, the single channel conductance of fluoride has a maximum at about 150 mV in the presence of the buffer PIPES (≥5 mM, pH 6.8) with K_0.5 ≈ 1 M. It is shown that the decrease in conductance is due to a blocking of the channel by the PIPES anion. In summary, the results indicate that the anion transport by the Clavibacter anion channel (CAC) does not require a voltage dependent conformation change of the CAC.     

Effect of cyanide in dark and light on the membrane potential and the ATP level of young and mature green tissues of higher plants

The effect of CN⁻ and N₂ on the electrical membrane potential (E_m) was compared with that of CN⁻ on the ATP levels in cotyledons of Gossypium hirsutum and in Lemna gibba L. In mature cotton tissue, CN⁻ depolarized E_m to the energy-independent diffusion potential (E_D) in the dark. In the light E_m recovered transiently. The same was observed in leaves of Nicotiana, Avena, Impatiens, Kalanchoë, and in Lemna. In contrast, in young cotton cotyledons and tobacco leaves and, to a large extent, in +sucrose-grown Lemna, E_m was depolarized to E_D also in the light in a similar way as in the dark.

In Lemna grown without sucrose, the energy-dependent component of E_m was only partially depolarized by CN⁻ in dark or light. Cyanide plus salicylhydroxamic acid completely reduced E_m to E_D, abolished respiration and photosynthesis, and severely diminished the ATP level. This suggests the operation of a CN⁻-insensitive respiration in uninjured Lemna. The initial CN⁻-induced decay of the ATP level in cotton and Lemna was more rapid than the decay of E_m. CN⁻-induced oscillations of the ATP level were followed by similar but slower oscillations of E_m. This supports the view of a general dependence of E_m on ATP. Discrepancies between inhibitor-induced changes of E_m and ATP levels are suggested to result from additional regulation of E_m by the cytoplasmatic pH value.

A comparison of E_D in young and mature cotton cotyledons in the dark and in the light suggests that in growing young cotyledons the different effect of CN⁻ in the light is due to a less effective photosynthesis together with high mitochondrial respiration. In Lemna and in mature cotton tissue, E_m in the light is maintained by noncyclic photophosphorylation and photosystem II, which is only partly inhibited by CN⁻, thus resulting in an incomplete depolarization and recovery of E_m. Complete inhibition of photosynthetic O₂ evolution and membrane depolarization by CN⁻ plus salicylhydroxamic acid are suggested to result from photooxidation.