Purification and characterization of a new type of serine carboxypeptidase from<Emphasis Type="Italic"> Monascus purpureus</Emphasis>

Carboxypeptidase produced by Monascus purpureus IFO 4478 was purified to homogeneity. The purified enzyme is a heterodimer with a molecular mass of 132 kDa and consists of two subunits of 64 and 67 kDa. It is an acidic glycoprotein with an isoelectric point of 3.67 and 17.0% carbohydrate content. The optimum pH and temperature were 4.0 and 40 °C, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 °C for 1 h, and up to 50 °C at pH 5.0 for 15 min. The enzyme was strongly inhibited by piperastatin A, diisopropylfluoride phosphate (DFP), phenylmethylsulfonylfluoride (PMSF), and chymostatin, suggesting that it is a chymotrypsin-like serine carboxypeptidase. Monascus purpureus carboxypeptidase was also strongly inhibited by p-chloromercuribenzoic acid (PCMB) but not by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, indicating that it requires cysteine residue but not metal ions for activity. Benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrate of the enzyme. The K_m, V_max, K_cat, and K_cat/K_m values of the enzyme for Z-Tyr-Glu at pH 4.0 and 37 °C were 0.86 mM, 0.917 mM min^–1, 291 s^–1, and 339 mM^–1 s^–1, respectively.     

Genetic mapping,distribution, and properties of an aconitase isozyme in Anopheles albimanus (Diptera:Culicidae)

Electrophoretic analysis of the developmental stages and tissues of Anopheles albimanus showed that qualitatively similar allozymes of aconitase (Acon-2) occur at all stages, and the enzyme is widespread in every larval and adult tissues. Relative heat stabilities of the allozymes were investigated by electrophoresis of heated aqueous extracts and by heating the enzyme in situ in acrylamide gels after electrophoretic separation in Tris-citrate and Tris-maleate buffer systems. The pupal aconitase in the crude extract is more stable to heat than the larval and adult enzyme. The presence of citrate ions in the gel increased the stability of aconitase to heat. Studies of substrate specificities indicated that cis-aconitic acid is the best substrate but citric acid can also serve as a substrate. Zymograms developed with isocitric acid as a substrate showed no aconitase electromorphs and produced only isocitrate dehydrogenase bands. Aconitase has a pH optimum of 8.0 and this enzyme is completely inhibited if treated in situ with ethylenediaminetetra-acetic acid (EDTA), p-chloromercuribenzoate (PCMB), and urea at concentrations higher than 5mm, 5×10^–5 m, and 2 m, respectively. Acon-2¹⁰⁰ and Acon-2¹⁰⁵ do not respond differently to the above treatments. Genetic crosses involving a holandric translocation, pericentric inversions, visible mutants, and allozyme markers were analyzed to map the aconitase (Acon-2) locus on the left arm of chromosome 3. The gene sequence (and map distances) on 3L is centromere—esterase-8 (Est-8)—2—esterase-4 (Est-4)—25—esterase-2 (Est-2)—9—Acon-2—5—phosphoglucomutase (Pgm)—7—esterase-6 (Est-6).     

Allelic expression in intergeneric fox hybrids (Alopex lagopus × Vulpes vulpes). II. Erythrocyte glucose-6-phosphate dehydrogenase in arctic and silver foxes: Purification and properties

The functional properties of purified glucose-6-phosphate dehydrogenase (G6PD) from the erythrocytes of Arctic foxes (Alopex lagopus) and silver foxes (Vulpes vulpes) were investigated. It was found that pH optima for G6PD range from 8.15 to 8.25 in Arctic foxes and from 10.2 to 10.4 in silver foxes. For G6P, the estimated K _m values were 74×10^–6 m (at pH 8.2) and 166×10^–6 m (at pH 10.2) in Arctic foxes and 58×10^–6 m (at pH 10.2) and 40×10^–6 m (at pH 8.2) in silver foxes. The K _m values for NADP were estimated as 62×10^–6 m (at pH 8.2) and 86×10^–6 m (at pH 10.2) in the Arctic foxes and 15×10^–6 m (at pH 10.2) and 12×10^–6 m (at pH 8.2) in the silver foxes. It was found that Mg²⁺ ions exert a significant activating effect on G6PD in the Arctic fox and do not affect appreciably its activity in the silver fox. The experimental data indicate that slight differences in the electrophoretic mobility of G6PD are associated with considerable functional differences in this enzyme between the two fox species.     

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.     

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.     

Purification and characterization of two ascorbate peroxidases of rice (<Emphasis Type="Italic">Oryza sativa </Emphasis>L.) expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To clarify the diversity and function of isozymes of ascorbate peroxidase (APX) in plants, a method of producing large quantities of these proteins is needed. Here, we describe an Escherichia coli expression system for the rapid and economic expression of two rice APX genes, APXa and APXb (GeneBank accession Nos. D45423 and AB053297, respectively). The two genes were cloned into the pGEX-6p-3 vector to allow expression of APX as a glutathione-S-transferase (GST) fusion protein. The GST-APXa and GST-APXb fusion proteins were purified by affinity chromatography using a glutathione-Sepharose 4B column, with final yields of 40 and 73 mg g^–1 dry cells, respectively. Specific activities were 15 and 20 mM ascorbate min^–1 mg^–1 protein, respectively. The K_m values for ascorbate were 4 and 1 mM, respectively, and those for H₂O₂ were 0.3 and 0.7 mM, respectively indicating that the two rice isoenzymes have different properties.Revisions requested 27 September 2004; Revisions received 12 November 2004     

Rat osseous plate alkaline phosphatase: mechanism of action of manganese ions

Polidocanol-solubilized osseous plate alkaline phosphatase was modulated by manganese ions in a similar way as by zinc ions. For concentrations up to 1.0 nm, the enzyme was stimulated by manganese ions, showing site-site interactions (n = 2.2). However, larger concentrations (> 0.1 m) were inhibitory. Manganese ions could play the role of zinc ions stimulating the enzyme synergistically in the presence of magnesium ions (K _d = 7.2 m; V = 1005.5 U mg^–1). Manganese ions could also play the role of magnesium ions, stimulating the enzyme synergistically in the presence of zinc ions (K _d = 2.2 m; V = 1036.7 U mg^–1). However, manganese ions could not substitute for zinc and magnesium at the same time since ion assymetry is necessary for full activity of the enzyme. A steady-state kinetic model for the modulation of enzyme activity by manganese ions is proposed.     

Electrical characteristics of stomatal guard cells: The contribution of ATP-dependent, “Electrogenic” transport revealed by current-voltage and difference-current-voltage analysis

Summary The steady-state, current-voltage (I–V) characteristics of stomatal guard cells fromVicia faba L. were explored by voltage clamp using conventional electrophysiological techniques, but with double-barrelled microelectrodes containing 50mm K⁺-acetate. Attention was focused, primarily, on guard cell response to metabolic blockade. Exposures to 0.3–1.0mm NaCN and 0.4mm salicylhydroxamic acid (SHAM) lead consistently to depolarizing (positive-going) shifts in guard cell potentials (V _m ), as large as +103 mV, which were generally complete within 60–90 sec (mean response half-time, 10.3±1.7 sec); values forV _m in NaCN plus SHAM were close or positive to –100 mV and well removed from the K⁺ equilibrium potential. Guard cell ATP content, which was followed in parallel experiments, showed a mean half-time for decay of 10.8±1.9 ([ATP] _t=0, 1.32±0.28mm; [ATP] _{t=60–180sec}, 0.29±0.40mm). In respiring cells, theI–V relations were commonly sigmoid aboutV _m or gently concave to the voltage axis positive toV _m . Inward- and outward-rectifying currents were also observed, especially near the voltage extremes (nominally –350 and +50 mV). Short-circuit currents (atV=0 mV) were typically about 200–500 mA m^–2. The principal effect of cyanide early on was to linearize theI–V characteristic while shifting it to the right along the voltage axis, to decrease the membrane conductance, and to reduce the short-circuit current by approx. 50–75%. The resulting difference-current-voltage (dI–V) curves (±cyanide) showed a marked sensitivity to voltages negative from –100 mV and, when clamp scans had been extended sufficiently, they revealed a distinct minimum near –300 mV before rising at still more negative potentials. The difference currents, along with changes in guard cell potential, conductance and ATP content are interpreted in context of a primary, ATP-consuming ion pump. FittingdI–V curves to reaction kinetic model for the pump [Hansen, U.-P., et al. (1981)J. Membrane Biol. 63:165; Blatt, M.R. (1986)J. Membrane Biol. 92:91] implicates a stoichiometry of one (+) charge transported outward for each ATP hydrolyzed, with pump currents as high as 200 mA m^–2 at the free-running potential. The analysis indicates that the pump can comprise more than half of the total membrane conductance and argues against modulations of pump activity alone, as an effective means to controlling K⁺ transport for stomatal movements.     

Michaelis constant (K m ) of acid phospatase as affected by montmorillonite,illite, and kaolinite clay minerals

The influence of Ca homoionic clay minerals (montmorillonite, illite, and kaolinite) on the activity,K _m , andV _m values of acid phosphatase was examined in model experiments. At each substrate (p-nitrophenyl phosphate) level tested, the addition of increasing amounts of clays (50, 100, and 150 mg, respectively) decreased the activity and increased theK _m value from 1.43×10^–3 m PNP (in the soluble state) to 82.3×10^–3M (montmorillonite), 8.02×10^–3 m (kaolinite), and 7.65×10^–3 m (illite) at the 150 mg level. The maximum enzyme reaction velocity (V _m ) remained nearly constant at different amounts of kaolinite and illite, but increased remarkably with rising quantities of montmorillonite. Apparently, the substrate affinity of sorbed acid phosphatase is significantly lower with montmorillonite than with kaolinite or illite. This may be ascribed to an intensive sorption of both substrate and enzyme to the surface as well as to interlattice sites of montmorillonite.     

Communicating junctions and calmodulin: Inhibition of electrical uncoupling inXenopus embryo by calmidazolium

Summary This paper reports the inhibitory effects of calmidazolium (CDZ), a calmodulin inhibitor, on electrical uncoupling by CO₂. Membrane potential and coupling ratio (V ₂/V₁) are measured in two neighboring cells ofXenopus embryos (16 to 64 cell stage) for periods as long as 5.5 hr. Upon exposure to 100% CO₂, control cells consistently uncouple even if the CO₂ treatments are repeated every 15 min for 2.5 hr. CDZ (5×10^–8–1×10^–7 m) strongly inhibits uncoupling. The inhibition starts after 30, 50 and 60 min of treatment with 1×10^–7, 7×10^–8 and 5×10^–8 m CDZ, respectively, is concentration-dependent and partially reversible. In the absence of CO₂, CDZ also improves electrical coupling. CDZ has no significant effect on membrane potential and nonjunctional membrane resistance. These data suggest that calmodulin or a calmodulin-like protein participates in the uncoupling mechanism.     

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.     

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.     

Enzyme kinetics of the prime K+ channel in the tonoplast of Chara: Selectivity and inhibition

Summary The prime potassium channel from the tonoplast of Chara corallina has been analyzed in terms of an enzyme kinetic model (Gradmann, Klieber & Hansen 1987, Biophys. J. 53:287) with respect to its selectivity for K⁺ over Rb⁺ and to its blockage by Cs⁺ and by Ca²⁺. The channel was investigated by patchclamp techniques over a range of membrane voltages (V _m , referred to an extracytoplasmic electrical potential of zero) from –200 mV to + 200 mV under various ionic conditions (0 to 300 mM K⁺, Rb⁺, Cs⁺, Ca²⁺, and Cl^–) on the two sides of isolated patches. The experimental data are apparent steady-state currentvoltage relationships under all experimental conditions used and amplitude histograms of the seemingly noisy open-channel currents in the presence of Cs⁺. The used model for K⁺ uniport comprises a reaction cycle of one binding site through four states, i.e., (1) K⁺-loaded and charged, facing the cytoplasm, (2) K⁺-loaded and charged facing the vacuole, (3) empty, facing the vacuole, and (4) empty, facing the cytoplasm. V _m enters the system in the form of a symmetric Eyring barrier between state 1 and 2. The numerical results for the individual rate constants are (in 10⁶s^–1 for zero voltage and 1 m substrate concentration): k ₁₂: 1,410, k ₂₁: 3,370, k ₂₃: 105,000, k ₃₂: 10,600, k ₃₄: 194, k ₄₃: 270, k ₄₁: 5,290, k ₁₄: 15,800. For the additional presence of an alternate transportee (here Rb⁺), the model can be extended in an analog way by another two states ((5) Rb⁺-loaded and charged, facing cytoplasm, and (6) Rb⁺-loaded and charged, facing vacuole) and six more rate constants (k ₄₅: 300, k ₅₄: 240, k ₅₆: 498, k ₆₅: 4,510, k ₆₃: 4,070, k ₃₆: 403). This six-state model with its unique set of fourteen parameters satisfies the complete set of experimental data. If the competing substrate can be bound but not translocated (here Cs⁺ and Ca²⁺), k ₅₆ and k ₆₅ of the model are zero, and the stability constants K _cyt (= k ₃₆/k ₆₃) and K _vac (= k ₄₅/k ₅₄) turn out to be K _cyt(Ca²⁺): 250 m ^–1 · exp(V _m /(64 mV)), k _vac(Ca²⁺): 10 m ^–1 · exp(–V _m /(66 mV)), K _cyt(Cs⁺): 0, and K _vac(Cs⁺): 46 m ^–2 · exp(–V _m /(12.25 mV)). With the assumption that the current fluctuations in the presence of Cs⁺ consist of incompletely resolved, short periods of complete openings and complete closures, the amplitude histograms of the noisy open channel currents can be described by a beta distribution, yielding the rate constants for binding (92 · 10⁶ sec^–1 · m ^–2 · exp(–V _m /(22.5 mV)) and debinding (2, 10⁶ sec^–1 · m ^–2 · exp(V _m /(22.5 mV)) of Cs⁺ to the vacuolar side of the channel as functions of the [Cs⁺] and of V _m . Considering these data and those from the literature, an asymmetry of the channel can be assessed, with a high charge density at the cytoplasmic side (Eisenman-series Nr. XI) and a low charge density at the vacuolar side (Eisenman-series Nr. I). Furthermore, the results provide an example for intimate linkage between conduction and switching of a channel.This work has been supported by the Deutsche Forschungsgemeinschaft.     

Electrogenic Cl− absorption byAmphiuma small intestine: Dependence on serosal Na+ from tracer and Cl− microelectrode studies

Summary The Na⁺ requirement for active, electrogenic Cl^– absorption byAmphiuma small intestine was studied by tracer techniques and double-barreled Cl^–-sensitive microelectrodes. Addition of Cl^– to a Cl^–-free medium bathingin vitro intestinal segments produced a saturable (K _m =5.4mm) increase in shortcircuit current (I _sc) which was inhibitable by 1mm SITS. The selectivity sequence for the anion-evoked current was Cl^–=Br^–>SCN^–>NO ₃ ^– >F^–=I^–. Current evoked by Cl^– reached a maximum with increasing medium Na concentration (K _m =12.4mm). Addition of Na⁺, as Na gluconate (10mm), to mucosal and serosal Na⁺-free media stimulated the Cl^– current and simultaneously increased the absorptive Cl^– flux (J _ms ^Cl ) and net flux (J _net ^Cl ) without changing the secretory Cl^– flux (J _sm ^Cl ). Addition of Na⁺ only to the serosal fluid stimulatedJ _ms ^Cl much more than Na⁺ addition only to the mucosal fluid in paired tissues. Serosal DIDS (1mm) blocked the stimulation. Serosal 10mm Tris gluconate or choline gluconate failed to stimulateJ _ms ^Cl . Intracellular Cl^– activity (a _Cl ⁱ ) in villus epithelial cells was above electrochemical equilibrium indicating active Cl^– uptake. Ouabain (1mm) eliminated Cl^– accumulation and reduced the mucosal membrane potential _m over 2 to 3 hr. In contrast, SITS had no effect on Cl^– accumulation and hyperpolarized the mucosal membrane. Replacement of serosal Na⁺ with choline eliminated Cl^– accumulation while replacement of mucosal Na⁺ had no effect. In conclusion by two independent methods active electrogenic Cl^– absorption depends on serosal rather than mucosal Na⁺. It is concluded that Cl^– enters the cell via a primary (rheogenic) transport mechanism. At the serosal membrane the Na⁺ gradient most likely energizes H⁺ export and regulates mucosal Cl^– accumulation perhaps by influencing cell pH or HCO ₃ ^– concentration.     

Na−K−2Cl cotransport in winter flounder intestine and bovine kidney outer medulla: [3H] bumetanide binding and effects of furosemide analogues

Summary The effects of several sulfamoyl benzoic acid derivatives on Na–K–Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC₅₀ values) and order of potency of these derivatives were benzmetanide, 5×10^–8 m> bumetanide 3×10^–7 m>piretanide 3×10^–6 m>furosemide 7×10^–6 m> amino piretanide 1×10^–5 3-amino-4-penoxy-5-sulfamoyl benzoic acid. Binding of [³H] bumetanide was studied in microsomal membranes from winter flounder intestine and compared to that in bovine kidney outer medulla. Binding was also studied in brush-border membranes from winter flounder intestine. The estimated values forK _d and number of binding sites (n) were: bovine kidney,K _d =1.6×10^–7,n=10.5 pmol/mg protein; winter flounder intestine,K _d 1.2×10^–7,n=7.3 pmol/mg protein, and brush-border membranes from winter flounder,K _d =5.3×10^–7,n=20.4 pmol/mg protein. The estimatedK _d for bumetamide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8×10^–7 m. The similarity in magnitudes of IC₅₀ andK _d for bumetanide suggests that the brush-border cotransporter is ordinarily rate-limiting for transmural salt absorption and that bumetanide specifically binds to the cotransporter. Measurement of bumetanide binding at various concentrations of Na, K and Cl showed that optimal binding required all three ions to be present at about 5mm concentrations. Higher Na and K concentrations did not diminish binding but higher Cl concentrations (up to 100mm Cl) inhibited bumetanide binding by as much as 50%. Still higher Cl concentrations (500 and 900mm) did not further inhibit bumetanide binding. Scatchard analysis of bumetanide binding at 5 and 100mm Cl concentrations showed that bothK _d andn were lower at the higher Cl concentration (5mm Cl:K _d =5.29×10^–7 m,n=20.4 pmol/mg protein; 100mm Cl:K _d =2.3×10^–7 m,n=8.8 pmol/mg protein). These data suggest two possibilities: that bumetanide and Cl binding are not mutually exclusive (in contrast to pure competitive inhibition) and that they each bind to separate sites or that two distinct bumetanide binding sites exist, only one of which exhibits Cl inhibition of binding. This inhibition would then be consistent with a competitive interaction with Cl.     

Ion fluxes inAcetabularia: Vesicular shuttle

Summary Ion flux relations in the unicellular marine algaAcetabularia have been investigated by uptake and washout kinetics of radioactive tracers (²²Na⁺,⁴²K⁺,³⁶Cl^– and⁸⁶Rb⁺) in normal cells and in cell segments with altered compartmentation (depleted of vacuole or of cytoplasm). Some flux experiments were supplemented by simultaneous electrophysiological recordings. The main results and conclusions about the steady-state relations are: the plasmalemma is the dominating barrier for translocation of K⁺ with influx and efflux of about 100 nmol·m^–2·sec^–1×K⁺ passes three- to sevenfold more easily than Rb⁺ does. Under normal conditions, Cl^– (the substrate of the electrogenic pump, which dominates the electrical properties of the plasmalemma in the resting state) shows two efflux components of about 17 and 2 mol·m^–2·sec^–1, and a cytoplasmic as well as vacuolar [Cl^–] of about 420mm ([Cl^–] _o =529mm). At 4°C, when the pump is inhibited, both influx and efflux, as well as the cellular [Cl^–], are significantly reduced. Na⁺ ([Na⁺] _i : about 70mm, [Na⁺] _o : 461mm), which is of minor electrophysiological relevance compared to K⁺, exhibits rapid and virtually temperature-insensitive (electroneutral) exchange (two components with about 2 and 0.2 mol·m^–2·sec^–1 for influx and efflux). Some results with Na⁺ and Cl^– are inconsistent with conventional (noncyclic) compartmentation models: (i) equilibration of the vacuole (with the external medium) can be faster than equilibration of the cytoplasm, (ii) absurd concentration values result when calculated by conventional compartmental analysis, and (iii) large amounts of ions can be released from the cell without changes in the electrical potential of the cytoplasm. These observations can be explained by the particular compartmentation of normalAcetabularia cells (as known by electron micrographs) with about 1 part cytoplasm, 5 parts central vacuole, and 5 parts vacuolar vesicles. These vesicles communicate directly with the central vacuole, with the cytoplasm and with the external medium.     

A Calcium-activated and nucleotide-sensitive nonselective cation channel in M-1 mouse cortical collecting duct cells

We recently reported that M-1 mouse cortical collecting duct cells show nonselective cation (NSC) channel activity (Proc. Natl. Acad. Sci. USA 89:10262–10266, 1992). In this study, we further characterize the M-1 NSC channel using single-channel current recordings in excised inside-out patches. The M-1 NSC channel does not discriminate between Na⁺, K⁺, Rb⁺, Cs⁺, and Li⁺. It has a linear I-V relation with a conductance of 22.7±0.5 pS (n=78) at room temperature. The P_cation/ P_anion ratio is about 60 and there is no measurable conductance for NMDG, Ca²⁺, Ba²⁺, and Mn²⁺. Cytoplasmic calcium activates the M-1 NSC channel at a threshold of 10^–6 m and depolarization increases channel activity (NP _o ). Cytoplasmic application of adenine nucleotides inhibits the M-1 NSC channel. At doses of 10^–4 m and 10^–3 m, ATP reduces NP _o by 23% and 69%, respectively.Furthermore, since ADP (10^–3 m) reduces NP _o by 93%, the inhibitory effect of adenine nucleotides is not dependent on the presence of a -phosphoryl group and therefore does not involve protein phosphorylation. The channel is not significantly affected by 8-Br-cGMP (10^–4 m) or by cGMP-dependent protein kinase (10^–7 m) in the presence of 8-Br-cGMP (10^–5 m) and ATP (10^–4 m). The NSC channel is not sensitive to amiloride (10^–4 m cytoplasmic and/or extracellular) but flufenamic acid (10^–4 m) produces a voltage-dependent block, reducing NP _o by 35% at depolarizing voltages and by 80% at hyperpolarizing voltages.We conclude that the NSC channel of M-1 mouse cortical collecting duct cells belongs to an emerging family of calcium-activated and nucleotide-sensitive nonselective cation channels. It does not contribute to amiloride-sensitive sodium absorption and is unlikely to be a major route for calcium entry. The channel is normally quiescent but may be activated under special physiological conditions, e.g., during volume regulation.The expert technical assistance of U. Fink and I. Doering-Hirsch is gratefully acknowledged. We thank A. Rabe and Dr. J. Disser for programming the computer software.This work was supported by a grant from the Deutsche Forschungsge-meinschaft (DFG grant Fr 233/9-1) and a grant from the National Institutes of Health (NIH grant DK-17433).     

Purification and characterization of a high molecular mass serine carboxypeptidase from <Emphasis Type="Italic">Monascus pilosus</Emphasis>

Two serine carboxypeptidases, MpiCP-1 and MpiCP-2, were purified to homogeneity from Monascus pilosus IFO 4480. MpiCP-1 is a homodimer with a native molecular mass of 125 kDa composed of two identical subunits of 61 kDa, while MpiCP-2 is a high mass homooligomer with a native molecular mass of 2,263 kDa composed of about 38 identical subunits of 59 kDa. This is unique among carboxypeptidases and distinguishes MpiCP-2 as the largest known carboxypeptidase. The two purified enzymes were both acidic glycoproteins. MpiCP-1 has an isoelectric point of 3.7 and a carbohydrate content of 11%, while for MpiCP-2 these values were 4.0 and 33%, respectively. The optimum pH and temperature were around 4.0 and 50°C for MpiCP-1, and 3.5 and 50°C for MpiCP-2. MpiCP-1 was stable over a broad range of pH between 2.0 and 8.0 at 37°C for 1 h, and up to 55°C for 15 min at pH 6.0, but MpiCP-2 was stable in a narrow range of pH between 5.5 and 6.5, and up to 50°C for 15 min at pH 6.0. Phenylmethylsulfonylfluoride strongly inhibited MpiCP-1 and completely inhibited MpiCP-2, suggesting that they are both serine carboxypeptidases. Of the substrates tested, benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu) was the best for both enzymes. The K_m, V_max, K_cat and K_cat/K_m values of MpiCP-1 for Z-Tyr-Glu at pH 4.0 and 37°C were 1.33 mM, 1.49 mM min^–1, 723 s^–1 and 545 mM^–1 s^–1, and those of MpiCP-2 at pH 3.5 and 37°C were 1.55 mM, 1.54 mM min^–1, 2,039 s^–1 and 1,318 mM^–1 s^–1, respectively.     

<Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> NAD-dependent, <Emphasis Type="SmallCaps">D</Emphasis>-fructose reducing,polyol dehydrogenases activity: screening,medium optimisation and application for enzymatic polyol production

Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehydrogenase production. The highest enzyme activities were obtained when this strain was cultivated on a medium consisting of 30 g glycerol l^–1, 7.2 g peptone l–1 and 1.8 g yeast extract l^–1. Two D-fructose reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6), while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5–10.5). The mannitol dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used to produce D-mannitol and D-sorbitol enzymatically from D-fructose. Efficient coenzyme regeneration was accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂.     

Rabit distal colon epithelium: II. Characterization of (Na+, K+, Cl−)-cotransport and [3H]-bumetanide binding

Summary Loop diuretic-sensitive (Na⁺,K⁺,Cl^–)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na⁺,K⁺) gradien-driven³⁶Cl fluxes weree half-maximally inhibited by 0.14 m bumetanide and 44 m furosimide. While⁸⁶Rb uptake rates showed hyperbolic dependencies on Na⁺ and K⁺ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl^– concentration, Hill coefficient 1.8, indicating a 1 Na⁺: 1 K⁺:2 Cl^– stoichiometry of ion transport.The interaction of putative (Na⁺, K⁺, Cl^–)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [³H]-bumetanide. The requirement for the simulataneous presence of Na⁺,K⁺, and Cl^–, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na⁺, K⁺, Cl^–)-cotransporter. [³H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K _D1=0.13 m;B _max1 =6.4 pmol/mg of protein) and low-affinity (K _D2=34 m;B _max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl^– (K ₁=60mm), whereas low-affinity sites seem to be unaffected by Cl^–. We have shown that only high-affinity [³H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.