Factors modulating alkaline phosphatase activity in the diazotrophic rice-field cyanobacterium, Anabaena oryzae

Summary Alkaline phosphatases (APase), both phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) were studied in the cyanobacterium Anabaena oryzae for their specific requirements of temperature, pH, micro- and macronutrients and their activities in the presence of salinity and heavy metal stress. The alkaline phosphatases (PMEase and PDEase) are quite stable enzymes and require a narrow range of pH (pH 10–10.2) and temperature (35–40 °C) for their optimal activity.A pH of 10, 10.2 and 10.2 supported optimal activity of cellular PMEase, cellular PDEase and extracellular PMEase, respectively, whereas temperatures of 35, 38 and 40 °C were required for their optimal activity. The requirement for Ca²⁺ and Mg²⁺ as macronutrients and the significance of the micronutrients Zn²⁺, Co²⁺, Fe²⁺, Mn²⁺ and Cu²⁺ in APase activity in the cyanobacterium suggests nutritional regulation of enzyme activity in A. oryzae. The metals Pb²⁺, Cr⁶⁺ and Ni²⁺ severely inhibited APase activity, whereas the NaCl stress had a dual role, which was concentration dependent. NaCl stress at lower concentrations (≤20 mM) caused an increase in cellular PMEase activity while its higher concentration (>20 mM) favoured release of the extracellular PMEase. The decrease in cellular activity and an increase in extracellular activity suggest that the higher concentrations of salt stimulate the release of the enzyme.The data suggest that the cyanobacterium A. oryzae possess a potential application as biofertilizer in high salinity and alkaline (Ca²⁺-rich) soils because of its ability to release PO₄³⁻ enzymatically under these conditions.     

Colonization of biofilms by bacteria capable of biodegrading sodium dodecyl sulphate (SDS) at clean and polluted riverine sites

Fifty cyanobacterial strains (10 genera) were tested in batch culture for their ability to use organic phosphorus compounds (1 mg liter⁻¹ P) as their sole P source. Two monoesters, Na₂-β-glycerophosphate and π-nitrophenyl phosphate (πNPP), supported growth of all strains, and the diester bis-π-nitrophenyl phosphate (bis-π-NPP) and herring sperm DNA supported almost all strains. ATP was either a very favorable or poor P source and failed to support growth of nine strains, seven of which were Rivulariaceae with trichomes ending in a hair or long tapered region. Phytic acid was in general the least favorable P source. P-limited cultures grown initially with inorganic phosphate to conditions of P limitation were also tested for cell-bound and extracellular phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities at two pH values (7.6, 10.3) using πNPP and bis-πNPP as substrates. Cell-bound PMEase was inducible in all strains and cell-bound PDEase in most strains. Most showed extracellular PMEase, but not extracellular PDEase. The highest values (μM πNPP or bis-πNPP hydrolyzed mg dry weight⁻¹ hour⁻¹) all occurred in strains ofGloeotrichia as follows: cell-bound PMEase at pH 7.6, 2.7 μM in strain D602; cell-bound PMEase at pH 10.3, 5.2 μM in D602; extracellular PMEase at pH 7.6, 0.73 μM in D281; extracellular PMEase at pH 10.3, 6.6 μM in D281; cell-bound PDEase at 7.6, 0.40 μM in D613; cell-bound PDEase at pH 10.3, 1.0 μM in D613. The results were compared to see if they indicated possible relationships between phosphatase activity and taxonomic or ecological grouping. The following differences were significant (P<0.05). Rivulariaceae produced higher yields than filamentous non-Rivulariaceae with β-glycerophosphate, πNPP, and DNA. Rivulariaceae with the ability to form hairs in culture showed poorer growth in ATP than non-hair-forming Rivulariaceae, but were more effective at utilizing phytic acid. Strains from calcareous environments had higher PMEase activity at pH 10.3 than strains from noncalcareous environments (P<0.01).     

Production and characterization of polygalacturonase fromGeotrichum candidum

An extracellular polygalacturonase (EC 3.2.1.15) fromGeotrichum candidum ATCC 34614 grown onsauerkraut brine was produced and characterized. Polygalacturonic acid markedly increased the enzyme yield in the brine. The fungus produced the highest activity (290 U/l) in brine with 0.3% (w/v) polygalacturonic acid. The pH and temperature optima of the enzymes were 4.5 to 5.0 and 30°C, respectively. It was stable from pH 4.0 to 5.8 and at 30°C but lost its activity at higher temperatures. The K_m and V_max values for polygalacturonic acid were 4.2 mg/ml and 0.19mm galacturonic acid/min, respectively. The enzyme was not substrate inhibited.     

Phosphatase activities of the aquatic moss <Emphasis Type="Italic">Warnstorfia fluitans</Emphasis> (Hedw.) Loeske from an acidic stream in North-East England

A study was made of the aquatic environment, tissue nutrient composition and surface phosphatase activities of the aquatic moss Warnstorfia fluitans in Brandon Pithouse Stream, a small acidic stream in N-E England. The water, which originates from an underground spring, had been pH 2.6 for at least 30 years, but about 3.9 during the present study. The moss was by far the most abundant phototroph during all this period. Seasonal changes in aqueous nitrogen and phosphorus fractions were measured over a 2-year period near the source. Most of the filtrable N and P were at times organic, but the very high N:P ratio (even if organic N is excluded) suggests that only organic phosphate is likely to be important for the moss. There was a high peak in organic phosphate in late spring in both study years. Surface phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities were highly correlated in the field and in axenic culture, though there were some differences in response to environmental factors. Axenic material showed higher PMEase and PDEase activities when grown with organic P than with inorganic P. Although the data suggest that internal P content is an important factor influencing phosphatase activities, PDEase activity was especially marked when the moss was grown with the diester, DNA, as P source, indicating that at least one of its surface phosphatases can also respond directly to the environment. Handling editor: S. M. Thomaz     

Diphenolases from <Emphasis Type="Italic">Anoxybacillus kestanbolensis</Emphasis> strains K1 and K4 <Superscript>T</Superscript>

Summary Diphenolases from Anoxybacillus kestanbolensis strains K1 and K4^T, highly active against 4-methylcatechol were characterized in terms of pH- and temperature-optima, pH- and temperature-stability, kinetic parameters, and inhibition/activation behaviour towards some general polyphenol oxidase (PPO) inhibitors and metal ions. The temperature-activity optima, for Anoxybacillus kestanbolensis K1 and K4^T catecholases in the presence of 4-methylcatechol, were 80 and 70 °C, respectively. Although catecholase from A. kestanbolensis K4^T lost no activity after a period of 1 h incubation at its optimum temperature, the enzyme pH from K1 was stimulated by keeping at 80 °C. Both of the enzymes possessed pH optima at 9.5, and the pH-stability profiles showed that cathecholases from both preparations retained their activities at alkaline pH values. Both A. kestanbolensis K1 and K4^T catecholase activities were totally inhibited by addition of 0.01 mM sodium metabisulphite, ascorbic acid and l-cysteine. 1 mM Mn²⁺ increased the activities of A. kestanbolensis K1 and K4^T catecholases by 6.4- and 5.3-fold, respectively. These results indicate that both A. kestanbolensis K1 and K4^T strains possess thermo- and alkalostable catecholases.     

Enzymatic splitting of penicillin V for the production of 6-APA using immobilized penicillin V acylase

Penicillin V acylase from Fusarium sp. SKF 235 was immobilized on several cation-exchange resins, of which Amberlite CG-50 was preferred. Maximum activity of the immobilized penicillin V acylase was 250 to 280 IU/g dry beads. The pH and temperature optima of the enzyme shifted from 6.5 to 6.8 and 55°C to 60°C, respectively, as a result of immobilization. However, the K _m for penicillin V remained at 10mm. Parameters for producing 6-aminopenicillanic acid were investigated and the immobilized penicillin V acylase was used for 68 cycles in a stirred tank reactor.     

Purification and some properties of a starch debranching enzyme ofHendersonula toruloidea

An extracellular, debranching isoamylase fromHendersonula toruloidea ATCC 64930, grown on starch, was purified 12-fold to an electrophoretically homogeneous state. The purified enzyme (estimated mol wt 83000) was optimally active at pH 6.0 and 50°C and remained active when held at 70°C (30 min) and at pH 6 to 8 for 24 h. Na⁺, Fe²⁺ and Ba²⁺ (at 5mm) enhanced enzyme activity while Hg²⁺, Zn²⁺ and Cu²⁺ (at 5mm) were inhibitory. The enzyme hydrolysed amylopectin (Km, 0.25 mg/ml), forming maltose, maltotriose and maltotetraose and hydrolyzed glycogen (Km, 0.29 mg/ml) and soluble starch (Km, 0.42 mg/ml) forming maltotriose and maltotetraose. Pullulan was not hydrolyzed.     

Partial purification and some properties of an alkaline cellulase from an alkalophilic Bacillus sp.

A newly isolated Bacillus species, which grew optimally at 30°C and pH 10, produced a carboxymethylcellulase in a medium containing 10 g CM-cellulose/l. The enzyme, when partially purified by gel filtration, had a mass of about 29 kDa as determined by both SDS-PAGE and gel filtration chromatography. It was optimally active at pH 9.5 and 40°C, and was stable from pH 7 to 11 at 4°C for 24 h. The enzyme was stimulated by Ca²⁺ (1mm) but was completely inhibited by Hg²⁺ (1mm). Neither EDTA nor EGTA (10mm) affected the activity.The author is with the Department of Biological Sciences, University of Jordan. PO Box 2686, Amman 11181, Jordan     

Purification and characterization of thermostable leucine dehydrogenase from Bacillus stearothermophilus

Leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) has been purified to homogeneity from a moderate thermophilic bacterium, Bacillus stearothermophilus. Am improved method of preparative slab gel electrophoresis was used effectively to purify it. The enzyme has a molecular mass of about 300,000 and consists of six subunits with identical molecular mass (Mr, 49,000). The enzyme does not lose its activity by heat treatment at 70° C for 20 min, and incubation in the pH range of 5.5–10.0 at 55° C for 5 min. It is stable in 10 mM phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol at over 1 month, and is resistant to detergent and ethanol treatment. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their keto analogs in the presence of NAD⁺ and NADH, respectively, as the coenzymes. The pH optima are 11 for the deamination of l-leucine, and 9.7 and 8.8 for the amination of -ketoisocaproate and -ketoisovalerate, respectively. The Michaelis constants were determined: 4.4 mM for l-leucine, 3.3 mM for l-valine, 1.4 mM for l-isoleucine and 0.49 mM for NAD⁺ in the oxidative deamination. The B. stearothermophilus enzyme shows similar catalytic properties, but higher activities than that from Bacillus sphaericus.Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

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.     

Purification and characterisation of two extremely halotolerant xylanases from a novel halophilic bacterium

The present work reports for the first time the purification and characterisation of two extremely halotolerant endo-xylanases from a novel halophilic bacterium, strain CL8. Purification of the two xylanases, Xyl 1 and 2, was achieved by anion exchange and hydrophobic interaction chromatography. The enzymes had relative molecular masses of 43 kDa and 62 kDa and pI of 5.0 and 3.4 respectively. Stimulation of activity by Ca²⁺, Mn²⁺, Mg²⁺, Ba²⁺, Li²⁺, NaN₃ and isopropanol was observed. The K_m and V_max values determined for Xyl 1 with 4-O-methyl-d-glucuronoxylan are 5 mg/ml and 125,000 nkat/mg respectively. The corresponding values for Xyl 2 were 1 mg/ml and 143,000 nkat/mg protein. Xylobiose and xylotriose were the major end products for both endoxylanases. The xylanases were stable at pH 4–11 showing pH optima around pH 6. Xyl 1 shows maximal activity at 60°C, Xyl 2 at 65°C (at 4 M NaCl). The xylanases showed high temperature stability with half-lives at 60°C of 97 min and 192 min respectively. Both xylanases showed optimal activity at 1 M NaCl, but substantial activity remained for both enzymes at 5 M NaCl.Communicated by W.D. Grant     

Response of chloride efflux from skeletal muscle ofRana pipiens to changes of temperature and membrane potential and diethylpyrocarbonate treatment

Summary Efflux of³⁶Cl^– from frog sartorius muscles equilibrated in two depolarizing solutions was measured. Cl^– efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases.For temperatures between 0 and 20°C, the measured activation energy is 7.5 kcal/mol for Cl^– efflux at pH 5 and 12.6 kcal/mol for the pH-dependent Cl^– efflux. The pH-dependent Cl^– efflux can be described by the relationu=1/(1+10n(pK _a -pH)), whereu is the Cl^– efflux increment obtained on stepping from pH 5 to the test pH, normalized with respect to the increment obtained on stepping from pH 5 to 8.5 or 9.0. For muscles equilibrated in solutions containing 150mm KCl plus 120mm NaCl (internal potential about –15 mV), the apparent pK _a is 6.5 at both 0 and 20°C, andn=2.5 for 0°C and 1.5 for 20°C. For muscles equilibrated in solutions containing 7.5mm KCl plus 120mm NaCl (internal potential about –65 mV), the apparent pK _a at 0°C is 6.9 andn is 1.5. The voltage dependence of the apparent pK _a suggests that the critical pH-sensitive moiety producing the pH-dependent Cl^– efflux is sensitive to the membrane electric field, while the insensitivity to temperature suggests that the apparent heat of ionization of this moiety is zero. The fact thatn is greater than 1 suggests that cooperativity between pH-sensitive moieties is involved in determining the Cl^– efflux increment on raising external pH.The histidine-modifying reagent diethylpyrocarbonate (DEPC) applied at pH 6 reduces the pH-dependent Cl^– efflux according to the relation, efflux=exp(–k·[DEPC]·t), wheret is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mm). At 17°C,k ^–1=188mm·min. For temperatures between 10 and 23°C,k has an apparent Q₁₀ of 2.5. The Cl^– efflux inhibitor SCN^– at a concentration of 20mm substantially retards the reduction of the pH-dependent Cl^– efflux by DEPC. The findings that the apparent pK _a is 6.5 in depolarized muscles, that DEPC eliminates the pH-dependent Cl^– efflux, and that this action is retarded by SCN^– supports the notion that protonation of histidine groups associated with Cl^– channels is the controlling reaction for the pH-dependent Cl^– efflux.     

Chloride transport in apical membrane vesicles from bovine tracheal epithelium: Characterization using a fluorescent indicator

Summary Cl transport in apical membrane vesicles derived from bovine tracheal epithelial cells was studied using the Cl-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl) quinolinium. With an inwardly directed 50 mM Cl gradient at 23°C, the initial rate of Cl entry (J _Cl) was increased significantly from 0.32±0.12 nmol · sec^–1 · mg protein^–1 (mean±sem) to 0.50±0.07 nmol · sec^–1 · mg protein^–1 when membrane potential was changed from 0 to +60 mV with K/valinomycin. At 37°C, with membrane potential clamped at 0 mV, there was a 34±7% (n=5) decrease inJ _Cl from a control value of 0.37±0.03 nmol · sec^–1 · mg protein^–1 upon addition of 0.2mm diphenylamine-2-carboxylate. The following did not alterJ _Cl significantly (J _Cl values gives as percent change from control): 50mm cis Na (–1±5%), 0.1mm furosemide (–3±4%), 0.1mm furosemide in the presence of 50mm cis Na (–5±2%), 0.1mm H₂DIDS (–18±9%), a 1.5 pH unit inwardly directed H gradient (–7±7%), and 0.1mm H₂DIDS in the presence of a 1.5 unit pH gradient (4±18%). With inward 50mm anion gradients, the initial rates of Br and I entry (J _Br andJ ₁, respectively) were not significantly different fromJ _Cl.J _Cl was a saturable function of Cl concentration with apparentK _d of 24mm and apparentV _max of 0.54 nmol · sec^–1 · mg protein^–1. Measurement of the temperature dependence ofJ _Cl yielded an activation energy of 5.0 kcal/mol (16–37°C). These results demonstrate that Cl transport in tracheal apical membrane vesicles is voltage-dependent and inhibited by diphenylamine-2-carboxylate. There is no significant contribution from the Na/K/2Cl, Na/Cl, or Cl/OH(H) transporters. The conductive pathway does not discriminate between Cl, Br, and I and is saturable. The low activation energy supports a pore-type mechanism for the conductance.     

Phosphate transport in human red blood cells: Concentration dependence and pH dependence of the unidirectional phosphate flux at equilibrium conditions

Summary The concentration dependence and the pH dependence of the phosphate transport across the red cell membrane were investigated. The unidirectional phosphate fluxes were determined by measuring the³²P-phosphate self-exchange in amphotericin B (5 mol/liter) treated erythrocytes at 25°C.The flux/concentration curves display anS-shaped increase at low phosphate concentrations, a concentration optimum in the range of 150 to 200mm phosphate and a self-inhibition at high phosphate concentrations. The apparent half-saturation concentrations,P _(0.5), range from 50 to 70mm and are little affected by pH. The self-inhibition constants, as far as they can be estimated, range from 400 to 600mm. The observed maximal phosphate fluxes exhibit a strong pH dependence. At pH 7.2, the actual maximal flux is 2.1×10^–6 moles·min^–1·g cells^–1. The ascending branches of the flux/concentration curves were fitted to the Hill equation. The apparent Hill coefficients were always in the range of 1.5–2.0. The descending branches of the flux/concentration curves appear to follow the same pattern of concentration response.The flux/pH curves were bell-shaped and symmetric with regard to their pH dependence. The pH optimum is at approximately pH 6.5–6.7. The apparent pK of the activator site is in the range of 7.0 to 7.2, while the apparent pK for the inactivating site is in the range of 6.2 to 6.5. The pK-values were not appreciably affected by the phosphate concentration.According to our studies, the transport system possesses two transport sites and probably two modifier sites as indicated by the apparent Hill coefficients. In addition, the transport system has two proton binding sites, one with a higher pK that activates and one with a lower pK that inactivates the transport system. Since our experiments were executed under self-exchange conditions, they do not provide any information concerning the location of these sites at the membrane surfaces.     

Separation and characterization of endoglucanases from culture filtrates of Cellulomonas sp.

Summary Six major components exhibiting endo-1,4-\-d-glucanase activity were partially purified from culture filtrates of a newly isolated Cellulomonas sp. using ion-exchange chromatography. Molecular weights (44,000 to 140,000), pH optima (6.0 to 7.0), temperature optima (40 to 50°C), half-life, energy of activation, K _mand other kinetic parameter investigations indicate the existence of 6 different endoglucanases.Further support for this assumption comes from inhibition studies, whereby glucose inhibited the enzyme activities between 15 and 50% at a concentration of 0.034% (1.65 mM) and cellobiose between 0 and 50% at a concentration of 0.1% (2.92 mM). Of all the metals (Hg²⁺, Co²⁺, Cu²⁺, Ca²⁺, Mg²⁺, Zn²⁺, Fe³⁺) tested, only Hg²⁺ exhibited a 55% inhibition at 5.0 mM.     

Structural characteristics of the saxitoxin receptor on nerve

Summary The effects of uranyl ion (UO ₂ ²⁺ ; at low concentrations binds specifically to phosphate groups) and the cationic dye methylene blue (MB⁺; binds strongly to carboxyl groups) on saxitoxin (STX) potency in crayfish axon has been studied by means of intracellular microelectrodes. At pH 6.00±0.05 and 13.5mm Ca²⁺, addition of 10.0 m UO ₂ ²⁺ +5.0nm STX had only slightly, if any, less effect on the spike's maximum rate of rise [0.79±0.04 (viz., mean±sem) of control value] than did addition of 5.0nm STX alone (0.72±0.05). Under the same conditions of pH and Ca²⁺ concentration, 1.0mm MB⁺ had approximately the same effect: 1.0mm MB⁺+5.0nm STX, 0.76±0.03; 5.0nm STX alone, 0.70±0.04. However, at pH 7.00±0.05 and lower Ca²⁺ concentrations, 1.0mm MB⁺ significantly reduced STX potency. Using 6.0mm Ca²⁺: 1.0mm MB⁺+5.0nm STX, 0.92±0.01; 5.0nm STX alone, 0.68±0.08. Using 3.0mm Ca²⁺, the corresponding values were 0.94±0.03 and 0.67±0.04. It is concluded that: (1) In accord with previous suggestions, the ionized acidic group known to exist in the Na channel (and to which a guanidinium group of STX appears to bind) is very likely a carboxyl group and not a phosphate group. (2) The accessible part of the Na channel mouth serving as the saxitoxin receptor probably does not include phospholipid in its structure proper.     

Stimulant-evoked depolarization and increase in [Ca2+] i in insulin-secreting cells is dependent on external Na+

Summary The patch-clamp technique and measurements of single cell [Ca²⁺] _i have been used to investigate the importance of extracellular Na⁺ for carbohydrate-induced stimulation of RINm5F insulin-secreting cells. Using patch-clamp whole-cell (current-clamp) recordings the average cellular transmembrane potential was estimated to be –60±1 mV (n=83) and the average basal [Ca²⁺] _i 102±6nm (n=37). When challenged with either glucose (2.5–10mm) ord-glyceraldehyde (10mm) the cells depolarized, which led to the initiation of Ca²⁺ spike potentials and a sharp rise in [Ca²⁺] _i . Similar effects were also observed with the sulphonylurea compound tolbutamide (0.01–0.1mm). Both the generation of the spike potentials and the increase in [Ca²⁺] _i were abolished when Ca²⁺ was removed from the bathing media. When all external Na⁺ was replaced with N-methyl-d-glucamine, in the continued presence of either glucose,d-glyceraldehyde or tolbutamide, a membrane repolarization resulted, which terminated Ca²⁺ spike potentials and attenuated the rise in [Ca²⁺] _i . Tetrodotoxin (TTX) (1–2 m) was also found to both repolarize the membrane and abolish secretagogue-induced rises in [Ca²⁺] _i .     

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.     

Purification and some properties of a thermostable protease ofThermoactinomyces thalpophilus

Summary A thermostable protease fromThermoactinomyces thalpophilus was purified to give a single protein band on disc PAGE with a molecular size of 55000 Da. Optimal proteolytic activity of the purified protease was at pH 6.0 and 70°C. The enzyme was maximally stable between pH 5.0 and 8.0 and retained 62% of its original activity at 70°C after 30 min. Temperature stability was not improved in the presence of Ca²⁺ (1mm). Enzyme activity was inhibited by AG⁺, Hg²⁺, Ba²⁺ and Co²⁺, partially inhibited byo-phenanthroline but not by diisopropylfluorophosphate (5mm).

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Resumen Se purificó una proteína termoestable deThermoactinomyces thalpophilus que dió una sola banda proteica al someterla a una electroforesis en columna de poliacrilamida (PAGE) y un tamaño molecular de 55.000 Da. La actividad proteolítica de la proteína purificada era óptima a pH 6.0 y 70°C. El enzima tenía máxima estabilidad entre pH 5.0 y 8.0 y retuvo un 62% de su actividad original despues de 30 min at 70°C. La estabilidad térmica no mejoró en presencia de Ca²⁺ (1mm). La actividad enzimática fue Inhibida por Ag⁺

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Résumé Une protéase thermostable deThermoactinomyces thalpophilus a été purifiée jusqu'à donner une bande protéique unique sur un disque PAGE avec un poids moléculaire de 55000 daltons. L'activé protéolytique optimum de la protéase purifiée se situe à pH 6.0 at à 70°C. L'enzyme présente son maximum de stabilité entre pH 5.0 et 8.0 et conserve 62% de son activité originelle après 30 min à 70°C. La stabilité à la température n'est pas améliorée en présence de Ca²⁺ 1mm. L'activité enzymatique est inhibée par Ag⁺, Hg²⁺, Ba²⁺ et Co²⁺. Elle est partiellement inhibée par l'o-phénanthroline mais elle n'est pas inhibée par le di-iso-propylfluorophosphate 5mm.

     

Single-channel K+ currents inDrosophila muscle and their pharmacological block

Summary Four types of nonvoltage-activated potassium channels in the body-wall muscles ofDrosophila third instar larvae have been identified by the patch-clamp technique. Using the inside-out configuration, tetraethylammonium (TEA). Ba²⁺, and quinidine were applied to the cytoplasmic face of muscle membranes during steady-state channel activation. The four channels could be readily distinguished on the basis of their pharmacological sensitivities and physiological properties. The K_ST channel was the only type that was activated by stretch. It had a high unitary conductance (100 pS in symmetrical 130/130mm KCl solution), was blocked by TEA (K _d 35mm), and was the most sensitive to Ba²⁺ (complete block at 10^–4 m). A Ca²⁺-activated potassium channel. K_CF 72pS (130/130mm KCl), was gated open at>10^–8 m Ca²⁺, was the least sensitive to Ba²⁺ (K _d of 3mm) and TEA (K _d of 100mm), and was not affected by quinidine. K₂ was a small conductance channel of 11 pS (130/2 KCl, pipette/bath), and was very sensitive to quinidine, being substantially blocked at 0.1mm. It also exhibited a half block at 0.3mm Ba²⁺ and 25mm TEA. A fourth channel type, K₃, was the most sensitive to TEA (half block<1mm). It displayed a partial block to Ba²⁺ at 10mm, but no block by 0.1mm quinidine. The blocking effects of TEA, Ba²⁺ and quinidine were reversible in all channels studied. The actions of TEA and Ba²⁺ appeared qualitatively different: in all four channels. TEA reduced the apparent unitary conductance, whereas Ba²⁺ decreased channel open probability.