Regulation of glutamine synthetase. VI. Interactions of inhibitors for Bacillus licheniformis glutamine synthetase

The relationships of five feedback inhibitors for the Bacillus licheniformis glutamine synthetase were investigated. The inhibitors were distinguishable by differences in their competitive relationship for the substrates of the enzyme. Mixtures of l-glutamine and adenosine-5'-monophosphate (AMP) or histidine and AMP caused synergistic inhibition of glutamine synthesis. Histidine, alanine, and glycine acted antagonistically toward the l-glutamine inhibition. Alanine acted antagonistically toward the glycine and histidine inhibitions. Independence of inhibitory action was observed with the other pairs of effectors. Possible mechanisms by which the inhibitors may interact to control glutamine synthesis are discussed. The low rate of catalysis of the glutamyl transfer reaction by the B. licheniformis glutamine synthetase can be attributed to the fact that l-glutamine serves both as a substrate and an inhibitor for the enzyme. Effectors which act antagonistically toward the l-glutamine inhibition stimulated glutamotransferase activity. The stimulation was not observed when d-glutamine was used as substrate for the glutamyl transfer reaction.     

Disposable sensor for measuring the biochemical oxygen demand for nitrification and inhibition of nitrification in wastewater

A disposable-type microbial sensor was developed for the determination of both the biochemical oxygen demand for nitrification (N-BOD) and inhibiting effects on nitrifying bacteria. The sensor was based on the respiratory activity of nitrifying bacteria immobilized on a miniature oxygen electrode. Typical response times for measuring N-BOD of ammonium standard solutions as well as of wastewater samples were in the range of 6–12 min. A dynamic evaluation of the signals after a measuring time of 120 s also resulted in good reproducibility and sensitivity. A daily profile of a municipal sewage plant was recorded, comparing the biosensor data with two standard methods. For the measurement of nitrification-inhibiting effects a 120-s dynamic signal evaluation was preferred to a steady-state method because of the long recovery times resulting from extended exposure to inhibitors. However, steady-state measurement techniques allowed allylthiourea detection with a ten times higher sensitivity. Because of the advantages of this miniaturized electrode, e.g. short response time, simple measuring procedure and low costs of production, this sensor system is considered to be suitable for commercial application in environmental analysis. Received: 30 April 1998 / Received revision: 4 September 1998 / Accepted: 13 September 1998     

Inhibited enzyme electrodes. Part 3. A sensor for low levels of H2S and HCN

It is shown that an inhibited enzyme electrode, using cytochrome oxidase, will respond to H2S, HCN and azide ion. For all three inhibitors the kinetics of the inhibiton and recovery processes have been analysed using the theoretical model presented previously (Albery et al., 1990a). Rearrangement of the differential equation describing inhibition and the development of the necessary software has enabled us to obtain values of the concentration of inhibitor in a matter of seconds after exposure of the sensor. The sensor will measure concentrations of H2S down to 1 ppm in the gas phase and concentrations of HCN and azide ion down to 0.4 mumol dm-3 in the solution phase.     

Comparison of methods for measuring oxygen consumption in tumor cells in vitro

The oxygen consumption rate of tumor cells affects tumor oxygenation and response to therapies. Highly sensitive methods for determining cellular oxygen consumption are, therefore, needed to identify treatments that can modulate this parameter. We compared the performances of three different methods for measuring cellular oxygen consumption: electron paramagnetic resonance (EPR) oximetry, the Clark electrode, and the MitoXpress fluorescent assay. To compare the assays, we used K562 cells in the presence of rotenone and hydrocortisone, compounds that are known to inhibit the mitochondrial electron transport chain to different extents. The EPR method was the only one that could identify both rotenone and hydrocortisone as inhibitors of tumor cell oxygen consumption. The Clark electrode and the fluorescence assay demonstrated a significant decrease in cellular oxygen consumption after administration of the most potent inhibitor (rotenone) but failed to show any significant effect of hydrocortisone. EPR oximetry is, therefore, the most sensitive method for identifying inhibitors of oxygen consumption on cell assays, whereas the Clark electrode offers the unique opportunity to add external compounds during experiments and still shows great sensitivity in studying enzyme and chemical reactions that consume oxygen (non-cell assays). Finally, the MitoXpress fluorescent assay has the advantage of a high-sample throughput and low bulk requirements but at the cost of a lower sensitivity.     

Inhibited enzyme electrodes. Part 3: A sensor for low levels of H2S and HCN

It is shown that an inhibited enzyme electrode, using cytochrome oxidase, will respond to H₂S, HCN and azide ion. For all three inhibitors the kinetics of the inhibition and recovery processes have been analysed using the theoretical model presented previously (Albery et al., 1990a). Rearrangement of the differential equation describing inhibition and the development of the necessary software has enabled us to obtain values of the concentration of inhibitor in a matter of seconds after exposure of the sensor. The sensor will measure concentrations of H₂S down to 1 ppm in the gas phase and concentrations of HCN and azide ion down to 0·4 μmol dm⁻³ in the solution     

Heat induction of heat shock protein 25 requires cellular glutamine in intestinal epithelial cells

Glutamine is considered a nonessential amino acid; however, it becomes conditionally essential during critical illness when consumption exceeds production. Glutamine may modulate the heat shock/stress response, an important adaptive cellular response for survival. Glutamine increases heat induction of heat shock protein (Hsp) 25 in both intestinal epithelial cells (IEC-18) and mesenchymal NIH/3T3 cells, an effect that is neither glucose nor serum dependent. Neither arginine, histidine, proline, leucine, asparagine, nor tyrosine acts as physiological substitutes for glutamine for heat induction of Hsp25. The lack of effect of these amino acids was not caused by deficient transport, although some amino acids, including glutamate (a major direct metabolite of glutamine), were transported poorly by IEC-18 cells. Glutamate uptake could be augmented in a concentration- and time-dependent manner by increasing either media concentration and/or duration of exposure. Under these conditions, glutamate promoted heat induction of Hsp25, albeit not as efficiently as glutamine. Further evidence for the role of glutamine conversion to glutamate was obtained with the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON), which inhibited the effect of glutamine on heat-induced Hsp25. DON inhibited phosphate-dependent glutaminase by 75% after 3 h, decreasing cell glutamate. Increased glutamine/glutamate conversion to glutathione was not involved, since the glutathione synthesis inhibitor, buthionine sulfoximine, did not block glutamine’s effect on heat induction of Hsp25. A large drop in ATP levels did not appear to account for the diminished Hsp25 induction during glutamine deficiency. In summary, glutamine is an important amino acid, and its requirement for heat-induced Hsp25 supports a role for glutamine supplementation to optimize cellular responses to pathophysiological stress. IEC-18; NIH/3T3; glutaminase; 6-diazo-5-oxo-L-norleucine; glutathione     

Glycosyltransferases of the human cervical epithelium. I. Characterization of a beta-galactoside alpha-2-L-fucosyltransferase and the identification of a beta-N-acetylglucosaminide alpha-3-L-fucosyltransferase

Using phenyl beta-D-galactoside as an acceptor, alpha-2-L-fucosyltransferase activity was identified in human cervical epithelium with pH optima at 6.0 and 7.2. The different response to p-chloromercuribenzoate, and ability to utilise asialofetuin as an acceptor, suggests the presence of two fucosyltransferases. The acid form is probably involved in glycoprotein synthesis in vivo. At pH 6.0, fucosyltransferase has a temperature optimum of 25 degrees C, requires the presence of Triton X-100 and either manganese or magnesium for maximal activity, and has Km values for GDP-L-[14-C]fucose and phenyl beta-D-galactoside of 32.1 . 10(-6) M and 8.2 . 10(-3) M, respectively. Guanosine nucleotides are potent inhibitors of the fucosyltransferase reaction; GDP is a competitive inhibitor while, depending on its concentration, GTP can either inhibit or activate the reaction. The alpha-L-fucosidase present in cervical tissue has negligible activity towards the enzyme product, phenyl-alpha-2-L-[14C]fucosyl-beta-D-galactoside. The use of high and low molecular weight acceptors indicates the presence of a beta-N-acetylglucosaminide alpha-3-L-fucosyltransferase and an N-acetylgalactosaminide fucosyltransferase.     

Possible mechanisms of the efflux of glutamate from kidney mitochondria generated by the activity of mitochondrial glutaminase

The transport of glutamate across the inner membrane of kidney mitochondria and the influx of glutamine into the mitochondria was studied using an oxygen electrode, the swelling technique and by continous recording of the activity of the mitochondrial glutaminase by an NH₄⁺-sensitive electrode. It is well known that the enzyme is activated by inorganic phosphate and strongly inhibited by glutamate.

1. 1. Avenaciolide, Bromocresal purple and Bromothymol blue inhibited the respiration of the mitochondria almost completely in the presence of glutamate as substrate but not in the presence of glutamine. Production of aspartate during the oxidation of glutamine was not significantly inhibited by avenaciolide but it was markedly suppressed by Bomocresol purple and Bromothymol blue.

2. 2. Swelling of kidney mitochondria in an isosmotic solution of glutamine and ammonium phosphate was not inhibited by avenaciolide or Bromocresol purple indicating that these substances do not inhibit the penetration of the mitochondrial membrane by glutamine or phosphate.

3. 3. The activity of the mitochondrial glutaminase was strongly inhibited by avenaciolide or Bromocresol purple in the presence of inhibitors of respiration or an uncoupler but not in their absence. Experimental data suggest that this was caused by the inhibition of glutamate efflux. The addition of a detergent removed this inhibition.

On the basis of these observations it was concluded that two mechanisms exist which enable glutamate to leave the inner space of kidney mitochondria: (a) an electrogenic efflux coupled to the respiration-driven proton translocation and the presence of a membrane potential (positive outside) and (b) an electroneutral glutamate-hydroxyl antiporter which is inhibited by avenaciolide and which operates in both directions. Our observations do not support the existence of the electrogenic glutamine-glutamate antiporter or glutamate-aspartate exchange in the mitochondria studied.     

Microbial electrode sensor for alcohols

A microbial electrode consisting of immobilized microorganisms, a gas permeable Teflon membrane, and an oxygen electrode was prepared for the continuous determination of methyl and ethyl alcohols. Immobilized Trichosporon brassicae was employed for a microbial electrode sensor for ethyl alcohol. When a sample solution containing ethyl alcohol was injected into a microbial electrode system, the current of the electrode decreased markedly with time until a steady state was reached. The response time was within 10 min by the steady state method and within 6 min by the pulse method. A linear relationship was observed between the current decrease and the concentration of ethyl alcohol below 22.5 mg/liter. The current was reproducible within ± 6% of the relative error when a sample solution containing 16.5 mg/liter ethyl alcohol. The standard deviation was 0.5 mg/liter in 40 experiments. The selectivity of the microbial electrode sensor for ethyl alcohol was satisfactory. The microbial electrode sensor was applied to a fermentation broth of yeasts and satisfactory comparative results were obtained (correlation coefficient 0.98). The current output of the microbial electrode sensor was almost constant for more than three weeks and 2100 assays. A microbial electrode sensor using immobilized bacteria for methyl alcohol was also described.     

Demonstration of an intramitochondrial invertase activity and the corresponding sugar transporters of the inner mitochondrial membrane in Jerusalem artichoke (<Emphasis Type="Italic">Helianthus tuberosus</Emphasis> L.) tubers

Genetic evidences indicate that alkaline/neutral invertases are present in plant cell organelles, and they might have a novel physiological function in mitochondria. The present study demonstrates an invertase activity in the mitochondrial matrix of Helianthus tuberosus tubers. The pH optimum, the kinetic parameters and the inhibitor profile of the invertase activity indicated that it belongs to the neutral invertases. In accordance with this topology, transport activities responsible for the mediation of influx/efflux of substrate/products were studied in the inner mitochondrial membrane. The transport of sucrose, glucose and fructose was shown to be bidirectional, saturable and independent of the mitochondrial respiration and membrane potential. Sucrose transport was insensitive to the inhibitors of the proton-sucrose symporters. The different kinetic parameters and inhibitors as well as the absence of cross-inhibition suggest that sucrose, glucose and fructose transport are mediated by separate transporters in the inner mitochondrial membrane. The mitochondrial invertase system composed by an enzyme activity in the matrix and the corresponding sugar transporters might have a role in both osmoregulation and intermediary metabolism.     

Extended-gate FET-based enzyme sensor with ferrocenyl-alkanethiol modified gold sensing electrode

We developed a field-effect transistor (FET)-based enzyme sensor that detects an enzyme-catalyzed redox-reaction event as an interfacial potential change on an 11-ferrocenyl-1-undecanethiol (11-FUT) modified gold electrode. While the sensitivity of ion-sensitive FET (ISFET)-based enzyme sensors that detect an enzyme-catalyzed reaction as a local pH change are strongly affected by the buffer conditions such as pH and buffer capacity, the sensitivity of the proposed FET-based enzyme sensor is not affected by them in principle. The FET-based enzyme sensor consists of a detection part, which is an extended-gate FET sensor with an 11-FUT immobilized gold electrode, and an enzyme reaction part. The FET sensor detected the redox reaction of hexacyanoferrate ions, which are standard redox reagents of an enzymatic assay in blood tests, as a change in the interfacial potential of the 11-FUT modified gold electrode in accordance with the Nernstian response at a slope of 59 mV/decade at 25 degrees C. Also, the FET sensor had a dynamic range of more than five orders and showed no sensitivity to pH. A FET-based enzyme sensor for measuring cholesterol level was constructed by adding an enzyme reaction part, which contained cholesterol dehydrogenase and hexacyanoferrate (II)/(III) ions, on the 11-FUT modified gold electrode. Since the sensitivity of the FET sensor based on potentiometric detection was independent of the sample volume, the sample volume was easily reduced to 2.5 microL while maintaining the sensitivity. The FET-based enzyme sensor successfully detected a serum cholesterol level from 33 to 233 mg/dL at the Nernstian slope of 57 mV/decade.     

Effects of thiol protease inhibitors on intracellular degradation of exogenous beta-galactosidase in cultured human skin fibroblasts

The effects of low molecular weight (LMW) protease inhibitors of microbial origin were evaluated on the intracellular degradation of beta-galactosidase purified from Aspergillus oryzae and taken up by cultured human skin fibroblasts with beta-galactosidase deficiency. Only thiol protease inhibitors showed an effect to increase the enzyme activity. E-64, a specific inhibitor of thiol proteases, prolonged 3-fold a half life of the exogenous beta-galactosidase and when the enzyme was supplied as liposomes, the half life was prolonged 9-fold in these cells. The role of thiol proteases in the degradation of enzyme molecules was discussed.     

Asparagine uptake in rat hepatocytes: Resolution of a paradox and insights into substrate-dependent transporter regulation

Summary. Extracellular asparagine has previously been shown to markedly stimulate both ornithine decarboxylase and System N-mediated glutamine transport activities in hepatocytes by a transport-dependent mechanism. However, as a weak substrate of its inferred transporter System N, the specific route of asparagine uptake has remained enigmatic. In this study, asparagine transport was studied in detail and shown to be Na⁺-dependent, Li⁺-tolerant, stereospecific, and inhibited profoundly by glutamine and histidine. Coupled with competitive inhibition by glutamine (K_i = 2.63 ± 1.11 mM), the data indicated that asparagine was indeed slowly transported by System N in rat hepatocytes, albeit at rates an order of magnitude less than for glutamine. The differential substrate transport velocities were shown to be attributable to a low transporter asparagine affinity (K_m = 9.3 − 17.5 mM) compared to glutamine (K_m∼ 1 mM). Consistent with its slow uptake, asparagine accumulated to a fivefold lesser degree than glutamine after 60 min, yet stimulated System N activity to the same extent as glutamine. The transaminase inhibitor aminooxyacetate and starvation of the donor animal each enhanced asparagine uptake twofold and augmented subsequent transporter activation. Conversely, asparagine-dependent System N stimulation was abrogated by hyperosmotic media and blunted 30%–40% by phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. Collectively, the data suggest that System N-mediated asparagine uptake serves an autostimulatory role, mediated by cellular swelling and in part by a PI3K-dependent signal transduction pathway. Received January 3, 2000 Accepted May 18, 2000     

Possible reasons for the failure of glutamine to influence GABA release in rat hippocampal slices; Effect of nipecotic acid and methionine sulfoximine

Although labelled glutamine is readily incorporated into labelled releasable GABA, it has been shown recently that high concentrations (0.1–0.5 mM) glutamine do not increase the release of GABA from brain slices, while greatly enhancing that of glutamate. Two possible reasons for this discrepancy were investigated: (a) That released GABA, in contrast to glutamate is not freshly synthesized but derives from GABA taken up by terminals. The possibility was made unlikely by the present finding which showed that even in the presence of the uptake inhibitor nipecotic acid, glutamine failed to enhance GABA release. (b) That glutamine is transported into GABA-ergic terminals by a high-affinity transport system which is saturated even at low glutamine concentrations obtained without adding glutamine to the superfusion fluid. However, when glutamine efflux was further reduced by prolonging depolarization with 50 mM K⁺ and by pretreatment with the glutamine synthetase inhibitor methionine sulfoximine, GABA release was depressed only very little and this decrease was related to the duration of depolarization and not to extracellular glutamine levels. These results can be reconciled with the ready incorporation of labelled glutamine into releasable GABA by assuming that GABA originates from a glutamate pool to which both glutamine and glucose contribute. The formation of releasable GABA however, is not governed by the supply of glutamate in this pool but by the activity of the rate-limiting enzyme glutamate decarboxylase.     

The mechanism of human NK cell-mediated cytotoxicity. Mode of action of surface-associated proteases in the early stages of the lytic reaction

The possible involvement of cell surface-associated proteolytic enzymes in human NK cell-mediated cytotoxicity and the mechanism by which such enzymes exert their activity have been studied. The treatment of intact cells with 3H-DFP under restricted conditions that predominantly bind surface-associated enzymes resulted in the labeling of five to six enzyme bands. Among these were a 35,000-dalton enzyme, which may be a previously identified trypsin-like proteinase engaged in cytotoxicity, and a 58,000-dalton elastase. The latter seems not to be involved in the reaction, as potent inhibitors of this enzyme have negligible effect on cytotoxicity. Of the membrane-associated enzymes, those engaged in cytotoxicity seem to be concealed from the external environment, as pretreatment of the effector cells with protease inhibitors such as trasylol and PMSF have no effect on the reaction. Immediately upon addition of the target cells and the initiation of cytotoxicity, the reaction becomes highly sensitive to inhibitors for a limited time interval of 2 to 5 min when trasylol is employed and 5 to 10 min when TPCK is the inhibitor, suggesting that target cell binding triggers the exposure of the enzymes to the external environment, rendering them accessible to the inhibitors. This short sensitivity period parallels the interval in which the reaction is sensitive to the microfilament inhibitor cytochalasin B. As the reaction proceeds, it becomes increasingly resistant to inhibitors of both proteolysis and cytoskeleton, at the same time suggesting that microfilament action and the unraveling of proteases may be processes that bear a close linkage with one another. The surface-associated elastase on the other hand maintains a constitutive mode of activity distinctive and unrelated to that of enzymes engaged in cytotoxicity. These findings suggest the existence on the surface of the NK lymphocyte of a mechanism that associates the receptor for target cells with an array of enclaved proteolytic enzymes via microfilaments. The resting cytotoxic structures become activated as the receptor attaches to the target cell, triggers the exposure of the proteolytic moiety, and initiates the lytic phase of the reaction.     

Inactivation of glucosamine-6-phosphate synthetase from Salmonella typhimurium LT2 by fumaroyl diaminopropanoic acid derivatives, a novel group of glutamine analogs

A novel group of glutamine analogs, N3-fumaroyl-L-2,3-diaminopropanoic acid (FDP) and its derivatives and analogs including amide (FCDP), methyl ester (FMDP) and its homologue, N4-(4-methoxyfumaroyl)-L-2,4-diaminobutanoic acid, inactivate glucosamine-6-phosphate synthetase (L-glutamine: D-fructose-6-phosphate aminotransferase (hexose-isomerizing), EC 2.6.1.16), isolated from Salmonella typhimurium, by covalent modification. For comparative purposes, selected known glutamine analogs were also examined. Anticapsin, 6-diazo-5-oxo-L-norleucine and, at high concentration, azaserine inactivate the enzyme. The pseudo-first-order rate constants show a hyperbolic dependence on inhibitor concentration for all the above-mentioned inhibitors, suggesting the formation of a reversible complex prior to covalent modification. Dissociation constants for inhibitors were determined and ranged from 10(-4) M for FCDP to 10(-6) M for FMDP. Albizziin, gamma-glutamylhydroxamate and, at low concentration, azaserine inhibit glucosamine synthetase only reversibly. All inhibitors tested are competitive in relation to glutamine. and competitive inhibitors, albizziin and gamma-glutamylhydroxamate protect the enzyme against inactivation. Fructose 6-phosphate accelerates the rate of inactivation. Some analogs of FDP, such as SMDP, CRDP, O-FMSer, MMDP and AADP, are not active against glucosamine-6-phosphate synthetase. The structure-activity relationship of the novel group of glutamine analogs is discussed and structural requirements for the activity of these compounds is established. It is postulated that the compounds examined can be classified as mechanism-based enzyme inactivators.     

Importance of the flux of phosphate across the inner membrane of kidney mitochondria for the activation of glutaminase and the transport of glutamine

The effect of mersalyl, an inhibitor of phosphate transport across the inner mitochondrial membrane, was investigated on the uncoupled respiration of pig kidney mitochondria in the presence of glutamine as substrate and on the activity of the phosphate-dependent glutaminase in the intact organelles. In addition, the submitochondrial location of the enzyme was reinvestigated.

1. (1) It was found that mersalyl completely inhibits uncoupled respiration of the mitochondria in the presence of glutamine as substrate, whereas respiration with glutamate was not affected. The same amount of mersalyl which inhibits coupled oxidation of glutamine also inhibits coupled oxidation of glutamate and some other substrates.

2. (2) Mersalyl strongly inhibited the activation of glutaminase in intact mitochondria only in the presence of inhibitors of electron transport or of an uncoupler. The addition of a detergent prevented or fully released the inhibition. The effect of mersalyl was observed even when the mitochondria were pre-incubated with phosphate or incubated in the phosphate-free medium. If mersalyl and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were added 3 min after pre-incubation with phosphate the same intramitochondrial concentration of the anion as in control experiments was found, whereas the activity of glutaminase was severely inhibited. These findings suggest that the activation of the enzyme by phosphate in intact nonenergized mitochondria occurs only if the activator moves across the inner mitochondrial membrane.

3. (3) Mersalyl (plus CCCP) markedly decreased [¹⁴C]glutamine- and [³²P]-phosphate-permeable mitochondrial spaces. A close correlation between the decrease of phosphate and glutamine permeable spaces and the inhibition of glutaminase activity was found.

4. (4) If the activation energy of the enzyme was determined with frozen mitochondrial preparations, a discontinuity or break in the Arrhenius plot was observed, whereas the presence of a detergent completely abolished the break. Digitonin or ultrasonic treatment of the mitochondria followed by separation of the membrane and the soluble fraction revealed that glutaminase is a membrane-bound enzyme.

On the basis of these findings it is concluded that there is an association between the transport of phosphate on one side and the transport of glutamine and glutaminase activity on the other. It is possible that the movement of phosphate across the membrane activates the enzyme which facilitates diffusion of glutamine down a concentration gradient. However, the existence of a specific glutamine-phosphate carrier is not ruled out.     

Characterisation and optimisation of AC conductimetric biosensors

Urease, immobilised on interdigitated gold electrodes, is employed as a model enzyme for characterisation and optimisation of a.c. conductimetric sensors. The sensors' response is measured over a frequency range of 20 Hz to 300 kHz and an optimum operating frequency established. The activity of the urease, both in solution and immobilised states, is investigated and Km values obtained. The effect of method of immobilisation and enzyme loading on the sensors' performance are studied and urease electrodes are characterised as a function of temperature, pH and electrolyte concentration. An important finding, particularly for conductimetric sensors designed for clinical use, is that proper consideration of the effects of electrode polarisation must be taken into account in order to maintain high sensor sensitivity at physiological electrolyte concentrations. Measurements of urea concentration in untreated serum are described.     

Purification and characterization of a newly screened microbial peptide amidase

A microbial peptide amidase was found in a limited screening and purified about 500-fold from Stenotrophomonas maltophilia. The native enzyme has a molecular mass of 38 kDa (gel filtration). The sequence of the first 16 amino acids was determined by Edman degradation. The isoelectric point was found to be around 5.8. The peptide amidase exhibited a pH optimum of 6.0 and a temperature optimum of about 39–45°C. The enzyme is stable in 50 mM TRIS/HCl, pH 7.5, at 30°C, and the residual activity was found to be above 90% after 1 week of incubation. The biocatalyst is not inhibited by potential inhibitors like Hg²⁺, EDTA, d-cycloserine or dithiothreitol and only weakly influenced by inhibitors of serine proteases. The peptide amidase deamidates selectively C-terminal amide groups in peptide amides without hydrolysing internal peptide bonds or amide functions in the side-chain of glutamine or asparagine. Unprotected amino acid amides are not hydrolysed. The enzyme is stereoselective with regard to l-enantiomers in the C-terminal position.     

Phase fluorometric sterilizable optical oxygen sensor

We report here on a low-cost, optical oxygen sensor as an attractive alternative to the widely used amperometric Clark-type oxygen electrode for measuring dissolved oxygen tensions in cell cultures and bioreactor. Our sensor is based on the defferential quenching of the fluorescence lifetime of chromophore in response to the partial pressure of oxygen. This is measured as a phase shift in fluorescence emission from the chromophore due to oxygen quenching when excited by an intensity modulated beam of light. In this article we demonstrate the advantages of lifetime-based optical methods over both intensity based optical methods and amperometric electrodes. Our sensor is particularly suitable for measuring dissolved oxygen in bioreactors. It is autoclavable, is free of maintenance requirements, and solvents the problems of long-term stability, calibration drifts, and reliable measurement of low oxygen tensions in dense microbial cultures that limit the utility of Clark-type elcectordes. (c) 1994 John Wiley & Sons, Inc.