Characteristics of protease synthesis in Bacteroides splanchnicus NCTC 10825

Studies to determine the physiological and nutritional characteristics of protease synthesis by Bacteroides splanchnicus NCTC 10825 showed that the proteases were constitutive and cell-associated during exponential growth in batch culture. As growth slowed and the bacteria entered the stationary phase, proteases that had accumulated intracellularly were released into the culture media. In continuous cultures, [dilution rate (D)=0.03 h^–1 to D=0.29 h^–1], protease activity was completely cell-bound and maximal during nitrogen-limited growth at high dilution rates. The proteases hydrolysed a relatively restricted range of protein substrates including casein, azocasein and gelatine (comparative maximum rates of hydrolysis were 1.0, 4.1 and 2.7 units mg^–1 protein respectively). B. splanchnicus proteases exhibited arylamidase activities against leucine p-nitroanilide, valylalanine p-nitroanilide and glycylproline p-nitroanilide. Inhibition experiments indicated that the bacterium produced a mixture of serine, thiol and, possibly, metalloproteases. Protease activities were affected by reducing agents and divalent metal ions. Mercaptoethanol at 1 mm was slightly stimulatory; however, dithiortheitol and dithioerythritol (each 10 mm) respectively inhibited protease activities by 91% and 100%. Calcium ions (5 mm) stimulated protease activity by 30%, whereas Mn²⁺ and Mg²⁺ had little or no effect. Protease and arylamidase activities had neutral to alkaline pH optima. Together, these results show that with respect to the types of protease formed and the physiology of the process, B. splanchnicus proteolysis is similar in many respects to that occurring in species belonging to the B. fragilis group. Correspondence to: G. T. Macfarlane     

EDTA activation of H2 photoproduction by Rhodospirillum rubrum

Summary The effect of trace elements (Fe, Ni) and chelating compounds on the activity of hydrogen (H₂) uptake (Hup) hydrogenase, nitrogenase and rate and yield of H₂ photoproduction from l-lactate in photosynthetic cultures of Rhodospirillum rubrum was investigated. Hup activity depended on the availability of Ni²⁺ and was inhibited by EDTA (0.3–0.5 mm ethylenedinitrilotetraacetic acid). Addition of EDTA (0.5 mm) to the culture medium caused a nearly complete inactivation of Hup activity and activation of nitrogenase, which was paralleled by a threefold increase in total H₂ photoproduced from lactate. Hup^– mutants, isolated by transposon Tn5 mutagenesis, produced maximally twofold more H₂ than the wild-type. Experiments with different chelating agents [EDTA, NTA (nitrilotriacetic acid), citrate, isocitrate] and varying concentrations of Fe²⁺ and Fe³⁺ showed that photosynthetic growth and nitrogenase activity of R. rubrum were strongly influenced by the iron supply. It is concluded that EDTA enhanced H₂ photoproduction by (I) inhibition of biosynthesis of Hup hydrogenase and (II) mobilization of iron, thereby activating the biosynthesis of the nitrogenase complex. Correspondence to: M. Kern     

Comparative studies of fructose 1,6-diphosphate aldolase fromEscherichia coli 518 andLactobacillus casei var.rhamnosus ATCC 7469

A comparative study has been carried out with FDP aldolases fromEscherichia coli 518 andLactobacillus casei ATCC 7469, which had been purified 17.6- and 65-fold, respectively. The aldolase ofL.casei was stable only in the presence of mercaptoethanol, whereas that ofE.coli was strongly inhibited at low (1.0×10^–4 m) and activated at high concentrations (2.0×10^–1 m) of the same compound.p-Chloromercuric benzoic acid inhibited both aldolases, with 40% inhibition at 2×10^–5 m withE.coli aldolase against at 2×10^–4 m withL.casei aldolase. Significant differences were also observed in pH optima and Km values.E.coli aldolase exhibited a maximal activity at pH 9.0 and gave a Km value of 1.76×10^–3 m FDP with strong substrate inhibition above 7×10^–3 m, against pH 6.8–7.0 and a Km of 7.04×10^–3 m FDP forL.casei aldolase. Strong resistance ofL.casei aldolase against inhibition by EDTA, Ca²⁺ and Mn²⁺ was observed compared with complete inhibition at concentrations of 20mm, 40mm and 20mm, respectively, withE. coli aldolase. Polyacrylamide gel electrophoresis did not reveal any differences between the two enzyme preparations.The differences of the properties of FDP aldolases from different bacterial genera are discussed in relation to other Class II aldolases.     

Effects of Thiol-Modifying Agents on a K(Ca2+) Channel of Intermediate Conductance in Bovine Aortic Endothelial Cells

Ca²⁺-activated K⁺ channels (K(Ca²⁺)) constitute key regulators of the endothelial cell electrophysiological response to InsP₃-mobilizing agonists. Inside-out and outside-out patch clamp experiments were thus undertaken to determine if the gating properties of a voltage-insensitive K(Ca²⁺) channel of intermediate conductance present in bovine aortic endothelial (BAE) cells could be modified by specific sulfhydryl (SH) oxidative and/or reducing reagents. The results obtained first indicate that cytosolic application of hydrophilic oxidative reagents such as 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) (0.2 to 5 mm) or [(O-carboxyphenyl)thio]ethyl mercury sodium salt (thimerosal) (0.5 to 5 mm) reduces gradually the K(Ca²⁺) channel activity with no modification of the channel unitary conductance. The inhibitory action of DTNB (1 to 5 mm) or thimerosal (1 to 5 mm) was not reserved following withdrawal of the oxidative agents, but channel activity could partly be restored by the addition of the SH group reducing agents dithiothreitol (DTT) (5 mm) or reduced glutathione (GSH) (5 mm) in 53% and 50% of the inside-out experiments performed with DTNB and thimerosal respectively. Similar results were obtained using H₂O₂ at concentrations ranging from 500 μm to 10 mm as oxidative reagent. In contrast, the lipid soluble oxidative agent 4,4′-dithiodipyridine (4-PDS) (1 mm) appeared in inside-out experiments less potent than DTNB and thimerosal at inhibiting the K(Ca²⁺) channel activity, suggesting that the critical SH groups involved in channel gating are localized at the inner face of the cell membrane. This conclusion was further substantiated by a series of outside-out patch clamp experiments which showed that DTNB (5 mm) and thimerosal (5 mm) were unable to inhibit the K(Ca²⁺) channel activity when applied to the external surface of the excised membrane. Finally, no significant changes of the gating properties of the K(Ca²⁺) channel were observed in inside-out experiments where the SH group reducing agents DTT and GSH were applied immediately following membrane excision. However, the application of either GSH or DTT was found to partly restore channel activity in experiments where the K(Ca²⁺) channels showed significant rundown. Received: 7 August 1996/Revised: 20 February 1997     

Purification and Characterization of a Thermostable Neutral Metalloprotease I from Chloroflexus aurantiacus J-10-fl

Chloroflexus aurantiacus J-10-fl was found to contain two types (protease I and protease II) of thermostable proteases which were separated by Butyl-Toyopearl 650M chromatography. Protease I was purified to electrophoretic homogeneity from the culture broth of C. aurantiacus J-10-fl. The molecular mass of protease I was estimated to be approximately 66 kDa by SDS-PAGE, and the value of approximately 66kDa was also obtained by the Hedrick-Smith method, indicating that protease I was a monomer. The isoelectric point was 6.2. Protease I activity was inhibited by metalloprotease inhibitors such as EDTA, EGTA, and o-phenanthroline. The optimum pH for the activity of protease I was around 8.0. Addition of Ca²⁺ increased the pH and heat stabilities of protease I. The activity was stable between pH 4.0–11.0 and up to 75°C, and the maximum activity was observed at 70°C in the presence of 2mM CaCl₂. Protease I was resistant to the treatment by denaturing reagents (8 M urea or 1% SDS) at pH 8.0 and 20°C for 24 h. The sites of cleavage. in oxidized insulin B chain by protease I were similar to those by other microbial neutral metalloproteases. Elastase activity of protease I was not detected.     

Purification and characterization of an alkaline cellulase from a newly isolated alkalophilic Bacillus sp. HSH-810

An alkaline cellulase from Bacillus sp. HSH-810 was purified 8.7-fold with a 30% yield and a specific activity of 71 U mg^–1 protein. It was optimally active at pH 10 and 50 °C and was stable from pH 6 to 10 with more than 60% activity remaining after heating at 60 °C for 60 min. The molecular mass of cellulase was 80 kDa. It was inhibited by 50% by Fe³⁺ (1 mM) and Mn²⁺ (0.1 mM) but was relatively insensitive to Hg²⁺ and Pb²⁺ at 1 mM.Revisions requested: 8 October 2004/1 December 2004; Revisions received 29 November 2004/5 January 2005     

Volume-dependent Glutamate Permeation Depends on Transmembrane Ionic Strength and Extracellular Cl<Superscript>−</Superscript>

Many mammalian cells regulate their volume by the osmotic movement of water directed by anion and cation flux. Ubiquitous volume-dependent anion currents permit cells to recover volume after swelling in response to a hypotonic environment. This study addressed competition between glutamate (Glu) and Cl^– permeation in volume-activated anion currents in order to provide insight into the ionic requirements for volume regulation, volume-dependent anion channel activity and to the architecture of the channel pore. The effect of changing the intracellular molar fraction (MF) of Glu and Cl^– on conductance and relative anion permeability was evaluated as a function of the extracellular permeant anion and/or the ionic strength. Relative permeability of Glu to Cl^– was determined by measuring reversal potentials under defined ionic conditions. Under conditions with high (150 mM) or low (50 mM) ionic strength solutions on both sides of the membrane, Cl^– was always more permeable than Glu. When a transmembrane ionic strength gradient (150 mM extracellular: 50 mM intracellular) was set to drive water into the cell, and in the presence of extracellular Cl^–, Glu became up to 16-fold more permeable than Cl^–. Replacement of extracellular Cl^– with Glu abolished this effect. These results indicate that it is possible for Glu to move into the extracellular environment during volume-regulatory events and they support the emerging role of glutamate as a modulator of anion channel activity.     

Swelling-activated Chloride and Potassium Conductance in Primary Cultures of Mouse Proximal Tubules. Implication of KCNE1 Protein

Volume-sensitive chloride and potassium currents were studied, using the whole-cell clamp technique, in cultured wild-type mouse proximal convoluted tubule (PCT) epithelial cells and compared with those measured in PCT cells from null mutant kcne1 –/– mice. In wild-type PCT cells in primary culture, a Cl^– conductance activated by cell swelling was identified. The initial current exhibited an outwardly rectifying current-voltage (I-V) relationship, whereas steady-state current showed decay at depolarized membrane potentials. The ion selectivity was I^– > Br^– > Cl^– >> gluconate. This conductance was sensitive to 1 mM 4,4-Diisothiocyanostilbene-2,2-disulfonic acid (DIDS), 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 1 mM diphenylamine-2-carboxylate (DPC). Osmotic stress also activated K⁺ currents. These currents are time-independent, activated at depolarized potentials, and inhibited by 0.5 mM quinidine, 5 mM barium, and 10 µM clofilium but are insensitive to 1 mM tetraethylammonium (TEA), 10 nM charybdotoxin (CTX), and 10 µM 293B. In contrast, the null mutation of kcne1 completely impaired volume-sensitive chloride and potassium currents in PCT. The transitory transfection of kcne1 restores both Cl^– and K⁺ swelling-activated currents, confirming the implication of KCNE1 protein in the cell-volume regulation in PCT cells in primary cultures.     

Influence of lead ions on cation permeability in human red cell ghosts

Summary Intracellular Pb²⁺ ions can replace Ca²⁺ ions in stimulating the Ca-dependent K permeability of human red blood cells. In metabolically depleted resealed ghosts, the threshold for stimulation of⁸⁶Rb efflux by internal Pb²⁺ is around 5×10^–10 m, and stimulation is half-maximal at about 2×10^–9 m, and maximal at 10^–8 m Pb²⁺. There is no effect on²²Na efflux in this concentration range.⁸⁶Rb efflux is antagonized by internal Mg²⁺ ions, and by the channel-blocking drugs quinidine and diS-C₂(5), as observed for the Ca-dependent K permeability in red cells. In ghosts containing EDTA, which prevents any internal effects of Pb²⁺ ions, external Pb²⁺ increases both²²Na and⁸⁶Rb permeability when its concentration exceeds 6×10^–7 m. This effect is seemingly unrelated to the Ca-dependent K permeability. This work makes extensive use of Pb²⁺ ion buffers, and gives information about their preparation and properties.     

Effects of Cd++ on short-circuit current across epithelial membranes

Summary Cadmium ion (Cd⁺⁺) significantly increased potential difference (PD) and short-circuit current (SCC) across isolated frog skin when added to the outside Ringer's solution at 10^–4, 10^–3 and 5×10^–3 m concentration. Resistance was reduced by 10^–4 m Cd⁺⁺ but not significantly changed by the higher concentrations. When SCC was first stimulated by vasopressin, 10^–4 and 10^–3 m Cd⁺⁺ produced additive stimulation which was reversible by washing with Cd⁺⁺-free Ringer's. If SCC was first stimulated by Cd⁺⁺, further stimulation by vasopressin was additive with 10^–4 m Cd⁺⁺ but completely inhibited by 10^–3 m Cd⁺⁺. Elevating the calcium ion (Ca⁺⁺) concentration of the outer Ringer's from 10^–3 m to 5×10^–3 m or 10^–2 m prior to Cd⁺⁺ treatment did not reduce the magnitude of SCC stimulation by Cd⁺⁺. Removal of Ca⁺⁺ from the outside Ringer's with 2×10^–3 m EDTA increased SCC as predicted. Subsequent addition of 5×10^–3 m Cd⁺⁺ drastically reduced SCC below control levels while equimolar concentrations of Cd⁺⁺ and EDTA reduced SCC only to control levels. These results suggest that Cd⁺⁺ interacts with the components of the apical plasma membranes of epithelial cells which are associated with the stimulation of SCC by vasopressin and Ca⁺⁺ removal and may be a useful probe for elucidating these components.     

Pyrimidine biosynthesis in Serratia marcescens: Polypeptide interactions of three nonsequential enzymes

Orotidine-5-monophosphate pyrophosphorylase (OMPppase, E.C. 2.4.2.10) and orotidylate decarboxylase (OMPdecase, E.C. 4.1.1.23) were purified from Serratia marcescens HY. These enzymes required physical association for maximal catalytic activities and formed a fragile complex with dihydroorotase (DHOase, E.C. 3.5.2.3.). OMPppase reversibly lost 50% of its activity upon separation from DHOase. The kinetic characteristics of OMPppase were modified by this separation. In the presence of DHOase, the K _ms for PRPP and orotate were stoichiometric: 2.3×10^–6 m and 2.6×10^–6 m, respectively. Following separation, the K _ms were significantly different: 1.3 × 10^–6 m for PRPP and 4.1×10^–6 m for orotate. OMPppase and OMPdecase could be reversibly separated by acrylamide gel electrophoresis, but the separation was accompanied by a loss of catalytic efficiency for both enzymes. DHOase readily associated into multiple molecular forms and could not be purified. The DHOase-OMPppase-OMPdecase interactions demonstrate that a weakly aggregated, multifunctional enzyme complex participates in the biosynthesis of pyrimidine nucleotides in S. marcescens. This unique association of nonsequential biosynthetic enzymes may represent a larger complex which provides a channeling or regulatory unit.This work was supported by grants from the National Science Foundation (NSF GB 5811) and the Office of Naval Research (Nonr 4413). One of us (J.W.) was a National Science Foundation Graduate Fellow.     

The effect of cations on reassociation of the components of the cellulosome cellulase complex synthesized by the bacterium Clostridium thermocellum

The cellulosome multienzyme complex was dissociated into 12–14 components when incubated at 30° C in a reaction mixture that was buffered at pH 5.0 and was 50 mm with respect to sodium dodecyl sulphate and 10 mm with respect to both ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT). The dissociated components reassociated into a complex when dialysed against 20 mm TRIS/HCl buffer, pH 7.7, containing 2.5 mm DTT. When incubated in the presence of Ca²⁺ and DTT the reassociated complex had the same activity to hydrogen-bond-ordered cellulose as the undissociated cellulosome. However, when Ca²⁺ ions were incorporated into the TRIS/HCl-DTT dialysis medium the reconstituted complex had very little activity towards cellulose. Other divalent cations such as Mg²⁺ and Ba²⁺ had the same effect, but the monovalent cation Na⁺ resulted in a complex that was very active on crystalline cellulose. The results are interpreted as indicating that the divalent cations bind to one or more of the dissociated polypeptide components and induce changes in conformation that prevent their reassociation into a complex with activity towards crystalline cellulose. Correspondence to: T. M. Wood     

Neuropharmacological analysis of synaptic transmission in the Lorenzinian ampulla of the skate Raja clavata

Summary Dissected ampullae of Lorenzini of the skate (Raja clavata) were studied with the aim of determining the synaptic transmitter between electroreceptor cell and afferent fibre. Resting activity and stimulus-evoked activity in response to electrical pulses were recorded in single afferent units at constant perfusion with normal and test solutions containing different putative neurotransmitters. Presynaptic transmitter release was blocked by Mg²⁺ (up to 50 mM) to investigate the effects of the test substances upon the postsynaptic membrane. l-Glutamate (l-GLU) and l-aspartate (l-ASP), both at concentrations between 10^-7 and 10^-3 M, enlarged strongly resting and stimulus-evoked discharge frequency in the afferent fibre. If transmission was blocked by high Mg²⁺, resting discharge frequency could be restored by l-GLU or l-ASP. The glutamate agonists quisqualate (10^-8–10⁵ M) and N-methyl-D-aspartate (10^-5–10^-3 M) enlarged spontaneous activity in the afferent fiber. The same was found for kainic acid (10^-9–10^-5 M). Taurine at concentrations between 10^-5 and 10^-3 M caused a concentration-dependent decrease in afferent activity. The same was found for gammaaminobutyric acid (GABA; 10^-5–10^-4 M), and for the catecholamines adrenaline and noradrenaline, both in concentrations between 10^-5 and 10^-3 M. Serotonine (10^-5–10^-3 M) and dopamine (10^-5-10^-3 M) had no effect on resting or evoked activity in the Lorenzinian ampulla afferents. Acetylcholine (ACh; 10^-4 M) enlarged discharge frequency in those units with initial rates lower than 22–25 Hz, but diminished discharge frequency in fibres with initial activity higher than 25 Hz. When synaptic transmission was blocked by high Mg²⁺ solution, perfusion with additional ACh did not restore resting activity in the afferent fibre. The results suggest that the most probable transmitter in the afferent synapse of the ampullae of Lorenzini is l-GLU or l-ASP, or a substance of similar nature.Abbreviations ACh acetylcholine - GABA gamma aminobutyric acid - KA kainic acid - l-ASP l-aspartate - l-GLU l-glutamate - NMDA N-methyl-D-aspartate - Q quisqualate - n.s. normal solution     

Paramecium Na+ channels activated by Ca2+-calmodulin: Calmodulin is the Ca2+ sensor in the channel gating mechanism

Paramecium Na⁺ channels, which were Ca²⁺-calmodulin activated, were studied in the inside-out mode of patch clamp. After excision of the membrane patch, they were active in the presence of 10^–5 to 10^–3 m Ca²⁺ in the bath. They became much less active in the presence of 10^–6 m Ca²⁺, and their activity subsided completely at 10^–8 m Ca²⁺. A Hill plot showed a dissociation constant of 6 m for Ca²⁺ binding. This dissociation constant shifted to a submicromolar range in the presence of 1 mm Mg²⁺. The channels also exhibited a mild voltage dependence. When exposed to 10^–8 m Ca²⁺ for an extended period of 2–4 min, channels were further inactivated even after bath Ca²⁺ was restored to 10^–4 m. Whereas neither high voltage (+100 mV) nor high Ca²⁺ (10^–3 m) was effective in reactivation of the inactive channels, addition of Paramecium wild-type calmodulin together with high Ca²⁺ to the bath restored channel activity without a requirement of additional Mg²⁺ and metabolites such as ATP. The channels reactivated by calmodulin had the same ion conductance, ion selectivity and Ca²⁺ sensitivity as those prior to inactivation. These inactivation and reactivation of the channels could be repeated, indicating that the direct calmodulin effect on the Na⁺ channel was reversible. Thus, calmodulin is a physiological factor critically required for Na⁺ channel activation, and is the Ca²⁺ sensor of the Na⁺-channel gating machinery.We thank C. Kung for his kind support, and A. Boileau for critical reading. Supported by grants from National Institutes of Health GM 22714-20 and 36386-09.     

Some effects of trinitrocresolate and valinomycin on Na and K transport across thin lipid bilayer membranes: A steady-state analysis with simultaneous tracer and electrical measurements

Summary This paper describes the effect of trinitrocresolate anions (TNC^–) on the electrical conductance (G _m ), and tracer-measured unidirectional Na and K fluxes (M _Na andM _K) across bilayers formed from sheep red cell lipids dissolved in decane. In the absence of TNC^–, typical low conductances were observed, while the cation fluxes were too low to measure by our techniques (<10^–12 moles cm^–2 sec^–1). In the presence of TNC^– (10^–2 m),G _m increased and TNC^– was the main charge carrier in the system. The cationic fluxes were also much increased, but the membranes showed no significant selectivity between K and Na. Furthermore, the Na and K fluxes were at least two orders of magnitude larger than the ionic fluxes calculated fromG _m . Thus, almost all of the K and Na transport across the membrane in the presence of TNC^– is electrically silent and is probably carried out as KTNC and NaTNC ion pairs.In the presence of valinomycin (10^–6 m) and no TNC^–, both the ion fluxes andG _m were 10³ times larger in KCl than in NaCl, thus exhibiting the characteristic high selectivity of valinomycin for K over Na. In the presence of both valinomycin (10^–6 m) and TNC^– (10^–2 m), this selectivity disappeared in that bothG _m andM _Na in the NaCl system were similar to the respective values in the KCl system. Even under these conditions, most of the Na is still transported by a process which does not carry charge.BothG _m andM _x increased alike and monotonically with increasing temperature over the range 7 to 30°C. In the absence of TNC^– the enthalpies of activation were invariably higher in KCl than in NaCl. Addition of TNC^– produced equal enthalpies of activation for both Na and K containing systems suggesting a common, temperature-dependent, ratedetermining step in charge transfer and the electrically silent cation fluxes.     

Purification and properties ofl-serine dehydratase fromLactobacillus fermentum ATCC 14931

l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM l-cysteine in 50 mM phosphate buffer. M_r=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (M_r=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent K_m forl-serine was 65 mM. Fe⁺⁺ was required for the enzymatic activity, and the apparent K_m value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol^–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit.     

Red cell hydrolases

Summary A 0.1% Triton X-100 extract of human erythrocyte plasma membranes contained high proteolytic activity as determined by a very sensitive assay utilizing³H-acetylated hemoglobin (162 cpm/pmole) as a substrate. Two proteolytic enzymes having optimum activity at pH 3.4 and pH 7.4 were isolated from Sephadex G-100. The protease active at pH 3.4 was 75 times as active as the pH 7.4 enzyme and it was purified 182-fold over the original homogenate and characterized. A linear relationship for activity versus time and activity versus concentration of enzyme was found. The optimum temperature was 37°C and theK _m was 1×10^–5 m hemoglobin. No enzyme activation was observed with any cation studied and EDTA had no inhibitory effect; (10mm Fe⁺³ and Hg⁺² were inhibitory). The pH 3.4 protease was stable indefinitely at –20°C in 0.1% Triton X-100. Gel electrophoresis was performed on a sodium dodecylsulfate-mercaptoethanol enzyme preparation and two protein bands (mol. wt. 33,000 and 54,000) were evident for the Sephadex G-200 eluate containing the pH 3.4 protease.     

The effect of p-hydroxymercuribenzoate and congeners on microsomal glucose-6-phosphatase

Summary Iodoacetamide, N-ethylmaleimide, p-hydroxy-mercuribenzoate (p-MB) and HgCl₂ were tested as inhibitors of microsomal glucose-6-phosphatase. Iodoacetamide had no effect at 2mm. N-ethylmaleimide inhibited only crude, but not purified microsomal preparations (M₂) or crude microsomes exposed to deoxycholate.¹⁴C-labelled N-ethylmaleimide was not bound by the M₂ protein fraction. p-MB inhibited all types of preparations and the inhibition was not counteracted by detergent. A more detailed study was carried out with the purified M₂ fraction (specific activity: 2–4µmoles P_i/min/mg protein). Glucose-6-phosphate hydrolysis was inhibited 50% by 5 × 10^–5 m p-MB. The inhibition was completely reversible by dithiothreitol except when the enzyme was pre-incubated with p-MB in the absence of substrate. Then p-MB accelerated the temperature-dependent inactivation of glucose-6-phosphatase. Binding studies showed that around 3µmoles¹⁴C-p-MB were incorporated into 100 mg M₂ protein regardless of the concentration of mercurial in the incubation mixture. That is, over a 25 fold range of p-MB concentration, causing up to 80% inhibition of enzyme activity, no difference was seen in the amount of labelled p-MB which was irreversibly bound to M₂ protein. Kinetically p-MB behaved like a reversible inhibitor and this was confirmed by dilution experiments. Several compounds, including some amino acids, antagonized the inhibition by p-MB. The order of effectiveness was EDTA > barbital > tryptophan > histidine > lysine > other amino acids. Glycine, Tris and urea were ineffective competitors of p-MB inhibition. Double reciprocal plots showed that the K_m for glucose-6-phosphate was increased and the V_max reduced in the presence of p-MB. HgCl₂ was a more effective inhibitor than p-MB with a K_i of 6 × 10^–6 m. We conclude that a reaction of p-MB with M₂ sulfhydryls does not play a part in the inhibition of enzyme activity. It is suggested that p-MB may interact with one or more amino acid side chains in such a way that enzyme conformation is altered.Supported by U.S. Public Health Service Grant No. AM11448-08 and General Research Support Grant No. RR05486-12.     

Sodium-selective channels in membranes of rat macrophages

With the use of the patch-clamp technique, highly selective nonvoltage-gated sodium channels were found in the membrane of rat peritoneal macrophages. The inward single channel currents were measured in cell-attached and outside-out mode experiments at different holding membrane potentials within the range of-60 to +40 mV. The channels had a unitary conductance of 10.2 ± 0.2 pS with 145 mm Na⁺ in the external solution at 23–24°C. The results of ion-substitution experiments confirmed that this novel type of cation channel in macrophages is characterized by high selectivity for Na⁺ over K⁺ (as for Cs⁺, NH₄ ⁺, Ca²⁺, Ba²⁺) ions, whose conduction through these sodium-permeable channels was not measurable. Lithium is the only other ion that is transported by this pathway; the unitary conductance was equal to 3.9 ± 0.2 pS in the Li⁺-containing external solution. Single channel currents and conductance were found to be linearly dependent on the external sodium concentration. Sodium channels in macrophage membrane patches were not blocked by tetrodotoxin (0.01–1 m). Single sodium currents were reversibly inhibited by the external application of amiloride (0.1–2 mm) and its derivative ethylisopropilamiloride (0.01–0.1 Mm). The mechanism of channel block by amiloride and its analogue seems to be different.We thank Dr. G.N. Mozhayeva and Dr. A.P. Naumov for useful discussions. This work has been supported by a grant from the Russian Basic Research Foundation, 93-04-21722.     

Proteolytic activation of a hyperpolarization- and calcium-dependent potassium channel inParamecium

Summary The effects of proteolysis on a hyperpolarization- and Ca²⁺-dependent K channel from the surface membrane ofParamecium tetraurelia were examined in the inside-out excised patch mode. Treatment with trypsin, pronase or thermolysin removed the Ca²⁺-dependence of the channel activation, yielding an increase in channel activity greater than 2.5-fold at all Ca²⁺ concentrations between 10^–4 and 10^–8 m. Thermolysin addition-ally removed the voltage dependence of channel opening and gave the most activation among the three proteases tested. Proteolysis did not affect the single-channel conductance. In an analogy to the mechanism of activation of many Ca²⁺-dependent enzymes it is suggested that thisParamecium channel has a cytoplasmic inhibitory domain which can be removed by proteolysis, and that the physiological activation by Ca²⁺ is due to a temporary removal of this inhibition. Moreover, these findings indicate structural differences between depolarization-, Ca²⁺-dependent K channels (BK channels) and the hyperpolarization-, Ca²⁺-dependent K channels inParamecium.