Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H₂. The enzyme showed a maximal activity of 120±40 mol H₂ oxidized · min^–1 · min^–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H₂ · min^–1 · mg^–1 with methyl viologen as electron donor, and an apparent K _m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S^2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F₄₂₀ or ferredoxin, nor with FAD, FMN, or NAD(P)⁺. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)     

The distribution and turnover of tryptamine in the brain and spinal cord

Tryptamine levels have been determined in mouse brain regions and spinal cord and in rat spinal cord. They were; caudate nucleus 2.5 ng·g^–1, hypothalamus <0.5 ng·g^–1, hippocampus <0.7 ng·g^–1, olfactory bulb <0.7 ng·g^–1, olfactory tubercles <0.6 ng·g^–1, brain stem <0.4 ng·g^–1, cerebellum <1.0 ng·g^–1, and the rest 0.9 ng·g^–1. The mouse whole brain was found to have 0.5 ng·g^–1, the mouse spinal cord 0.3 ng·g^–1, and the rat spinal cord 0.3 ng·g^–1. These concentrations increased rapidly to 22.8 ng·g^–1, 14.2 ng·g^–1, and 6.6 ng·g^–1 respectively at 1 hr after 200 mg·kg^–1 pargyline. The turnover rates and half lives of tryptamine in the mouse brain and spinal cord and rat spinal cord were estimated to be 0.14 nmol·g^–1·h^–1 and 0.9 min; 0.054 nmol·g^–1·h^–1 and 1.5 min and 0.04 nmol·g^–1·h^–1 and 1.6 min respectively. The aromaticl-aminoacid decarboxylase inhibitors NSD 1034 and NSD 1055 reduced synthesis of tryptamine in controls and pargyline pretreated animals. Tryptophan increased the concentrations of mouse striatal tryptamine and 5-hydroxytryptamine and brain stem 5-hydroxyindole acetic acid.p-Chlorophenylalanine reduced formation of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid but did not change that of tryptamine.     

Time course of vasopressin-induced formation of microvilli in granular cells of toad urinary bladder

Summary Vasopressin-induced transformation of ridges to microvilli on the surface of granular cells of toad urinary bladder occurs in conjunction with induced alterations in the water permeability of the luminal membrane. This study was designed to establish the relationship between the time course for induction of microvilli and the time course for induction of increased water permeability after vasopressin stimulation. Hemibladders were examined at 2.5, 5, 10, 20 and 30 min following exposure to 20 mU/ml of vasopressin and at 5, 10, 20, 30, 40, 50 and 60 min after washout of vasopressin. Within 2.5 min, vasopressin initiated complete transformation of ridges to microvilli on approximately 13% of the granular cells, while osmotic water flow (Jv) was 0.31±0.10 l·min^–1·cm^–2. Five minutes following vasopressin stimulation, microvilli were present on approximately 30% of granular cells andJv was 2.27±0.13 l·min^–1·cm^–2. At 10 minJv was maximum at 4.03±0.15 l·min^–1·cm^–2 and 50% of the granular cells were covered with microvilli. This percentage increased to 70% at 20 min and was maintained at 30 min, althoughJv decreased to 3.9±0.35 l·min^–1·cm^–2 at 30 min. Five minutes following vasopressin washout, ridges interspersed with microvilli reappeared asJv fell to 1.10±0.30 l·min^–1·cm^–2. At 10 min after vasopressin washout,Jv approached basal levels, but the reversal of microvilli to ridges remained incomplete. At 60 min after vasopressin washout, the granular cells had regained their original ridgelike surface structures. Thus, these studies establish a temporal relationship between the induction and reversibility of vasopressin-induced microvillous formation and alterations in the osmotic water permeability of the apical plasmalemma.     

Continuous monitoring of cellulase action on microcrystalline cellulose

Summary Cellobiose oxidase from Phanerochaete chrysosporium was used for continuous monitoring of cellulase action on microcrystalline cellulose (Avicel). Two protocols are described, the parameter monitored being either the decline in electrode potential as ferricyanide is reduced or consumption of dioxygen. Most experiments used a commercial cellulase preparation from Trichoderma reesei and ferricyanide as acceptor. Within 1 min of an addition of cellulase, ferricyanide reduction reached a steady rate. This was converted into a rate of production of substrate for celobiose oxidase, in mol·min^–1. Experiments were conducted either with a constant concentration of cellulase and increasing Avicel, or with constant Avicel and increasing cellulase. Kinetic analysis of the experiments with constant cellulase indicated a K _mof 4.8 ± 1.0 (g cellulose)·1^–1, which was close to the value predicted from binding studies. The specific activity of the cellulase was measured as 375±25 mol·(g cellulase)^–1·min^–1 in experiments with a high cellulose concentration, but was less than half this value when the cellulose was saturated with cellulase. The maximal rate of cellulose degradation was 9.6±1.3 mol·(g cellulose)^–1·min^–1.     

Caecal water and electrolyte absorption and the effects of acetate and glucose, in dehydrated, low-NaCl diet hens

Summary In vivo electrolyte transport and water absorption from the caeca of dehydrated, low-NaCl diet hens are reported. In the absence of luminal glucose or acetate, net electrolyte transport rates and water absorption are small. When physiological concentrations of acetate (40 mM) are included in the perfusate, Na⁺ transport and water absorption increase significantly (P<0.01): 38±7 eqNa⁺/caecum kg·h and 256±33 l H₂O/caecum · kg · h.A similar increase in water absorption occurs with the inclusion of 15 mM glucose in the perfusate (219±30 l H₂O/caecum · kg · h), however both net Na⁺ and Cl^– absorption increase: 28±6 eq Na⁺/caecum · kg · h and 21±5 eq Cl^–/caecum kg · h.These pronounced increases in electrolyte and water absorption are not accompanied by any significant increase in transmural potential difference.The data presented establish caeca as important sites in the recuperation of water and electrolytes in dehydrated, low-NaCl diet hens.Abbreviations ECPD electrochemical potential difference - PD (transmural) potential difference - PEG polyethylene glycol     

Compartmental nitrate concentrations in barley root cells measured with nitrate-selective microelectrodes and by single-cell sap sampling

Nitrate-selective microelectrodes were used to measure intracellular nitrate concentrations (as activities) in epidermal and cortical cells of roots of 5-d-old barley (Hordeum vulgare L.) seedlings grown in nutrient solution containing 10 mol · m^–3 nitrate. Measurements in each cell type grouped into two populations with mean (±SE) values of 5.4 ± 0.5 mol · m^–3 (n=19) and 41.8 ± 2.6 mol · m^–3 (n = 35) in epidermal cells, and 3.2 ± 1.2 mol · m^–3 (n = 4) and 72.8 ± 8.4 mol · m^–3 (n = 13) in cortical cells. These could represent the cytoplasmic and vacuolar nitrate concentrations, respectively, in each cell type. To test this hypothesis, a single-cell sampling procedure was used to withdraw a vacuolar sap sample from individual epidermal and cortical cells. Measurement of the nitrate concentration in these samples by a fluorometric nitrate-reductase assay confirmed a mean vacuolar nitrate concentration of 52.6 ± 5.3 mol · m^–3 (n = 10) in epidermal cells and 101.2 ± 4.8 mol · m^–3 (n = 44) in cortical cells. The nitrate-reductase assay gave only a single population of measurements in each cell type, supporting the hypothesis that the higher of the two populations of electrode measurements in each cell type are vacuolar in origin. Differences in the absolute values obtained by these methods are probably related to the fact that the nitrate electrodes were calibrated against nitrate activity but the enzymic assay against concentration. Furthermore, a 28-h time course for the accumulation of nitrate measured with electrodes in epidermal cells showed the apparent cytoplasmic measurements remained constant at 5.0 ± 0.7 mol · m^–3, while the vacuole accumulated nitrate to 30–50 mol · m^–3. The implications of the data for mechanisms of nitrate transport at the plasma membrane and tonoplast are discussed.Symbol _n ² Chi-squared with n degrees of freedom R.-G.Z. was awarded a Sino-British Friendship Scholarship sponsored by the British Council and H.-W.K. was supported by an AFRC Linked Research Grant to A.D.T for collaboration with R.A.L. We wish to thank Dr. K. Goulding for advice on ion chromatography, Dr. K. Moore for assistance with statistical analysis and Dr. J.H. Williams for advice on the microsample analysis.     

Adaptation of renal function to hypotonic medium in the winter flounder (Pseudopleuronectes americanus)

Summary The kidneys of winter flounders transferred to hypotonic medium were investigated for glomerular and tubular handling of fluid and electrolytes and for the urinary excretion of proteins. Media were sea water (925 mosm·kg^–1) and brackish water (70 mosm·kg^–1).In sea water, the urine was hypertonic to the plasma in 7 fish of this study. Urine flow rate was correlated with the GFR. After adaptation to brackish water a delay of 1 to 3 days was observed until the kidneys switched from fluid retention to the excretion of large amounts of dilute urine. GFR and urine flow rate were increased from 0.61±0.08 to 1.58±0.29 ml·h^–1·kg^–1 and from 0.14±0.02 to 0.68±0.08 ml·h^–1·kg^–1, respectively . With increased filtered load the tubular reabsorption of fluid decreased from 74±2.4% to 45±11.2%. The excretion rates of sodium and potassium were increased due to decreased fractional sodium and potassium reabsorption. The urinary excretion of divalent cations, however, was reduced because the net tubular reabsorption of calcium was increased and the net secretion of magnesium was diminished.Both the urinary total protein concentration and the protein pattern showed no significant change, but the rate of protein excretion was increased from 0.21±0.04 to 0.60±0.05 mg·h^–1·kg^–1. The comparison of protein patterns obtained from urine and serum samples revealed that high molecular weight (HMW) proteins prevail in the serum whereas low molecular weight (LMW) proteins dominate in the urine. The diminished quantity of the HMW-protein fraction in the urine thus may reflect size selectivity of the glomerular filtration barrier for serum proteins also in the winter flounder.Abbreviations BW brackish water - SW sea water - GFR glomerular filtration rate - HMW heigh molecular weight - LMW low molecular weight     

Functional Characterization of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchangers in Primary Cultures of Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. We previously reported Na⁺/H⁺ exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I_sc, (11.6 ± 0.5 µA · cm^–2), potential differences, V_t (2.1 ± 0.2 mV), and resistance, R_t (169 ± 12 · cm²). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable ²²Na⁺ uptake under a H⁺ gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of ²²Na⁺ uptake was 21.4 ± 1.3 nmol · mg prot^–1 · min^–1, of which 9.5 ± 0.7 (~45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for ~6%, ~66% and ~28% of GBECs total NHE activity, respectively. GBECs exhibited saturable NHE kinetics (V_max 9.2 ± 0.3 nmol · mg prot^–1 · min^–1; K_m 11.4 ± 1.4 mM Na⁺). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na⁺ transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.     

Kinetic studies on the stimulation of Na+−H+ exchange activity in renal brush border membranes isolated from thyroid hormone-treated rats

Summary Na⁺–H⁺ exchange activity in renal brush border membrane vesicles isolated from hyperthyroid rats was increased. When examined as a function of [Na⁺], treatment altered the initial rate of Na⁺ uptake by increasingV _m (hyperthyroid, 18.9±1.1 nmol Na⁺ · mg^–1 · 2 sec^–1; normal, 8.9±0.3 nmol Na⁺ · mg^–1 · 2 sec^–1), and not the apparent affinityK _Na ⁺ (hyperthyroid, 7.3±1.7mm; normal, 6.5±0.9mm). When examined as a function of [H⁺] and at a subsaturating [Na⁺] (1mm), hyperthyroidism resulted in the proportional increase in Na⁺ uptake at every intravesicular pH measured. A positive cooperative effect on Na⁺ uptake was found with increased intravesicular acidity in vesicles from both normal and hyperthyroid rats. When the data were analyzed by the Hill equation, it was found that hyperthyroidism did not change then (hyperthyroid, 1.2±0.06; normal, 1.2±0.07) or the [H⁺]_0.5 (hyperthyroid, 0.39±0.08 m; normal, 0.44±0.07 m) but increased the apparentV _m (hyperthyroid, 1.68±0.14 nmol Na⁺ · mg^–1 · 2 sec^–1; normal 0.96±0.10 nmol Na⁺ · mg^–1 · 2 sec^–1). The uptake of Na⁺ in exchange for H⁺ in membrane vesicles from normal and hyperthyroid animals was not influenced by membrane potential. H⁺ translocation or debinding was rate limiting for Na⁺–H⁺ exchange since Na⁺–Na⁺ exchange activity was greater than Na⁺–H⁺ exchange activity. Hyperthyroidism caused a proportional increase and hypothyroidism caused a proportional decrease in Na⁺–Na⁺ and Na⁺–H⁺ exchange. We conclude that hyperthyroidism leads to either an increase in the number of functional exchangers in the membrane or exactly proportional increases in the rate-limiting steps for Na⁺–Na⁺ and Na⁺–H⁺ exchange activity.     

Decrease of polypeptides in the PS I antenna complex with increasing growth irradiance in the red alga Porphyridium cruentum

Thylakoids isolated from cells of the red alga Porphyridium cruentum exhibit an increased PS I activity on a chlorophyll basis with increasing growth irradiance, even though the stoichiometry of Photosystems I and II in such cells shows little change (Cunningham et al. (1989) Plant Physiol 91: 1179–1187). PS I activity was 26% greater in thylakoids of cells acclimated at 280 mol photons · m^–2 · s^–1 (VHL) than in cells acclimated at 10 mol photons · m^–2 · s^–1 (LL), indicating a change in the light absorbance capacity of PS I. Upon isolating PS I holocomplexes from VHL cells it was found that they contained 132±9 Chl/P700 while those obtained from LL cells had 165±4 Chl/P700. Examination of the polypeptide composition of PS I holocomplexes on SDS-PAGE showed a notable decrease of three polypeptides (19.5, 21.0 and 22 kDa) in VHL-complexes relative to LL-complexes. These polypeptides belong to a novel LHC I complex, recently discovered in red algae (Wolfe et al. (1994a) Nature 367: 566–568), that lacks Chl b and includes at least six different polypeptides. We suggest that the decrease in PS I Chl antenna size observed with increasing irradiance is attributable to changes occurring in the LHC I-antenna complex. Evidence for a Chl-binding antenna complex associated with PS II core complexes is lacking at this point. LHC II-type polypeptides were not observed in functionally active PS II preparations (Wolfe et al. (1994b) Biochimica Biophysica Acta 1188: 357–366), nor did we detect polypeptides that showed immunocross-reactivity with LHC II specific antisera (made to Chlamydomonas and Euglena LHC II).Abbreviations Bis-Tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - DCPIP 2,6-dichlorophenol indophenol - -dm dodecyl--d-maltoside - HL high light of 150 mol photons · m^–2 · s^–1 - LGB lower green band - LHC I light-harvesting complex of PS I - LHC II light-harvesting complex of PS II - LL low light of 10 mol photons · m^–2 · s^–1 - ML medium light of 50 mol photons · m^–2 · s^–1 - MES 2-(N-morpholino) ethanesulfonic acid - P700 reaction center of PS I - PFD photon flux density - Trizma tris(hydroxymethyl)aminomethane - UGB upper green band - VHL very high light of 280 mol photons · m^–2 · s^–1     

Secretion of electrolytes,protein and urea by the mandibular gland of the common wombat (Vombatus ursinus)

Saliva was collected from the mandibular glands of anaesthetized common wombats (Vombatus ursinus) to ascertain maximal flow rates, salivary compostion and possible adaptations, particularly PO₄ ^3- secretion, to assist digestion. After temporary catheterization of the main duct through its oral opening, salivary secretion was evoked at flow rates ranging from 0.02±0.002 (±SEM) ml·min^-1 (0.7±0.07 l·min^-1·kg body weight^-1) to 0.4±0.05 ml·min^-1(14±1.9 l·min^-1·kg body weight^-1) by ipsilateral intracarotid infusion of acetylcholine. The [Na⁺] (15±5.1 to 58±8.6 mmol·l^-1) and [HCO₃ ^-] (35±1.9 to 60±1.9 mmol·l^-1) were positively correlated with salivary flow rate. The [K⁺] (58±5.2 to 30±2.4 mmol·l^-1), [Ca²⁺] (10.4±1.67 to 4.1±0.44 mmol·l^-1), [Mg²⁺] (0.94±0.137 to 0.17±0.032 mmol·l^-1), [Cl^-] (71±9.2 to 45±6.0 mmol·l^-1), [urea] (9.3±0.79 to 5.1±0.54 mmol·l^-1), H⁺ activity (29±1.6 to 17±1.6 nEq·l^-1) and amylase activity (251±57.4 to 92±23.3 kat·l^-1) were negatively correlated with flow. Both concentration and osmolality fell with increasing flow at the lower end of the flow range but osmolality always increased again by maximal flow whereas the relation between protein and flow was not consistent at the higher levels of flow and stimulation. Salivary [PO₄ ³⁺] was not correlated with flow and at 3–14% of the plasma concentration was extremely low. Thus, in contrast to its nearest relative, the koala (Phascolarctos cinereus), the wombat secretes little PO₄ ³⁺ presumably because it does not need high levels of PO₄ ³⁺ in its saliva to facilitate microbial digestion of plant fibre.Abbreviations bw body weight - ww wet weight     

Chloride cells and chloride exchange in the skin of a sea-water teleost,the shanny (Blennius pholis L.)

Summary The fine structure of the skin and its importance in chloride outfluxes were investigated in a sea-water teleost, the shanny (Blennius pholis L.).The epidermis is composed of three cells types: epithelial cells, mucous cells and chloride cells. These chloride cells typically contain a great number of mitochondria and an extensive agranular reticulum extending through the whole cell body. They open at the surface of the epidermis into an apical pit. An undifferentiated small cell is often observed near these chloride cells and probably corresponds to the adjacent chloride cell.The values of chloride outfluxes through the skin and the gills are respectively 5333±884 Eq·h^–1·kg^–1 and 4479±2521 Eq·h^–1·kg ^–1; n=6; t=13±0.5°C. Thus the ratio between skin chloride outflux and total chloride outflux is 64.7±9.3%.     

Beta-adrenergic control of trans-cutaneous evaporative cooling mechanisms in birds

Summary The decreasing effect of -adrenergic blockade on skin resistance to vapor diffusion and the onset of cutaneous water evaporation in the pigeon (Columba livia) was investigated. Oral administration of 1, 2.3 and 5 mg propranolol to pigeons (268±53 g) initiated intensive trans-cutaneous water evaporation (CWE) up to 29.1 mg H₂O·cm^–2·h^–1 in resting birds at 30°C air temperature (Ta), but had only a slight effect on CWE of birds exposed to 50 °C Ta.After 7 h of effective -adrenergic blockade (oral administration of 5 mg propranolol), skin and body temperature stabilized at 39.0±0.5 °C and 41.0±0.7 °C, compared to 40.2±0.8 °C and 41.9±0.6 °C in the control group, respectively. A slight hypothermia was accompanied by feather fluffing.Intradermal injection of 0.001, 0.01 and 0.12 mg propranolol also caused intensive CWE. Local -adrenergic blockade in relatively low blocker doses (0.001 and 0.01 mg propranolol) decreased skin resistance from a high value of 44.5 s·cm^–1 to about 6.0 s·cm^–1, and caused a sharp increase in CWE from a control value of about 4 to a high of 26.4 mg H₂O·cm^–2·h^–1 during the first two hours of exposure to 30°C Ta.The possible role of -adrenergic blockade in regulation of trans-cutaneous water evaporation of latent heat dissipation is discussed.     

Light response of D1 turnover and photosystem II efficiency in the seagrassZostera capricorni

Biochemical and biophysical parameters, including D1-protein turnover, chlorophyll fluorescence, oxygen evolution activity and zeaxanthin formation were measured in the marine seagrassZostera capricorni (Aschers) in response to limiting (100 mol·m^–2·^–1), saturating (350 mol·m^–2·s^–1) or photoinhibitory (1100 mol·m^–2·s^–1) irradiances. Synthesis of D1 was maximal at 350 mol·m^–2·s^–1 which was also the irradiance at which the rate of photosynthetic O₂ evolution was maximal. Degradation of D1 was saturated at 350 mol·m^–2·s^–1. The rate of D1 synthesis at 1100 mol·m^–2·s^–1 was very similar to that at 350 mol·m^–2·s^–1 for the first 90 min but then declined. At limiting or saturating irradiance little change was observed in the ratio of variable to maximal fluorescence (Fv/Fm) measured after dark adaptation of the leaves, while significant photoinhibition occurred at 1100 mol·m^–2·s^–1. The proportion of zeaxanthin in the total xanthophyll pool increased with increasing irradiance, indicative of the presence of a photoprotective xanthophyll cycle in this seagrass. These results are consistent with a high level of regulatory D1 turnover inZostera under non-photoinhibitory irradiance conditions, as has been found previously for terrestrial plants.We would like to thank Professor Peter Böger (Department of Plant Biochemistry, University of Konstanz, Germany) for the kind gift of D1 antibodies. This work was partly supported by a University of Queensland Enabling Grant to CC.     

Down-regulation of blood-brain glucose transport in the hyperglycemic nonobese diabetic mouse

The intracarotid injection method has been utilized to examine blood-brain barrier (BBB) glucose transport in hyperglycemic (4–6 days) mice. In anesthetized mice, Brain Uptake Indices were measured over a range of glucose concentrations from 0.010–50 mmol/l; glucose uptake was found to be saturable and kinetically characterized. The maximal velocity (V_max) for glucose transport was 989±214 nmol·min^–1·g^–1· and the half-saturation constant estimated to be 5.80±1.38 mmol/l. The unsaturated Permeability Surface are product (PS) is=171+8 l·min.^–1·g^–1. A rabbit polyclonal antiserum to a synthetic peptide encoding the 13 C-terminal amino acids of the human erythrocyte glucose transporter immunocytochemically confirmed the presence of the GLUT1 isoform in non-obese diabetic (NOD) mouse brain capillary endothelia. These studies indicate that a down-regulation of BBB glucose transport occurs in these spontaneously hyperglycemic mice; both BBB glucose permeability (as indicated by PS product) and transporter maximal velocity are reduced (in comparison to normoglycemic CD-1 mice), but the half-saturation constant remains unchanged.     

Fluorescence polarization studies of different forms of angiotensin-converting enzyme

The interaction of three forms of bovine angiotensin-converting enzyme (ACE) with the competitive peptide inhibitor lisinopril with a fluorescent label was studied by the fluorescence polarization technique. The dissociation constants K _d of the enzyme-inhibitor complexes in 50 mM Hepes-buffer, pH 7.5, containing 150 mM NaCl and 1 M ZnCl₂ at 37°C were (2.3 ± 0.4)·10^–8, (2.1 ± 0.3)·10^–8, and (2.1 ± 0.2)·10^–8 M for two-domain somatic ACE, single-domain testicular ACE, and for the N-domain of the enzyme, respectively. The interaction of the enzyme with the inhibitor strongly depended on the presence of chloride in the medium, and the apparent dissociation constant of the ACE-chloride complex was (1.3 ± 0.2)·10^–3 M for the somatic enzyme. The dissociation kinetics of the complex of the inhibitor with somatic ACE did not fit the kinetics of a first-order reaction, but it was approximated by a model of simultaneous dissociation of two complexes with the dissociation rate constants (0.13 ± 0.01) sec^–1 and (0.026 ± 0.001) sec^–1 that were present at approximately equal initial concentrations. The dissociation kinetics of the single-domain ACE complexes with the inhibitor were apparently first-order, and the dissociation rate constants were similar: (0.055 ± 0.001) and (0.041 ± 0.001) sec^–1 for the N-domain and for testicular ACE, respectively.     

Intraparticle mass-transfer resistance and apparent time stability of immobilized yeast alcohol dehydrogenase

The stability and, consequently, the lifetime of immobilized enzymes (IME) are important factors in practical applications of IME, especially so far as design and operation of the enzyme reactors are concerned. In this paper a model is presented which describes the effect of intraparticle diffusion on time stability behaviour of IME, and which has been verified experimentally by the two-substrate enzymic reaction. As a model reaction the ethanol oxidation catalysed by immobilized yeast alcohol dehydrogenase was chosen. The reaction was performed in the batch-recycle reactor at 303 K and pH-value 8.9, under the conditions of high ethanol concentration and low coenzyme (NAD⁺) concentration, so that NAD⁺ was the limiting substrate. The values of the apparent and intrinsic deactivation constant as well as the apparent relative lifetime of the enzyme were calculated.The results show that the diffusional resistance influences the time stability of the IME catalyst and that IME appears to be more stabilized under the larger diffusion resistance.List of Symbols C _A, C_B, C_E mol · m^–3 concentration of coenzyme NAD⁺, ethanol and enzyme, respectively - C _p mol · m³ concentration of reaction product NADH - d _p mm particle diameter - D _eff m² · s^–1 effective volume diffusivity of NAD⁺ within porous matrix - k _d s^–1 intrinsic deactivation constant - K _A, K_A, K_B mol · m^–3 kinetic constant defined by Eq. (1) - K _A ^x mol · m^–3 kinetic constant defined by Eq. (5) - r _A mol · m^–3 · s^–1 intrinsic reaction rate - R m particle radius - R _v mol · m^–3 · s^–1 observed reaction rate per unit volume of immobilized enzyme - t _E s enzyme deactivation time - t _r s reaction time - V mol · m^–3 · s^–1 maximum reaction rate in Eq. (1) - V ^x mol · m^–3 · s^–1 parameter defined by Eq. (4) - V _f m³ total volume of fluid in reactor - w _s kg mass of immobilized enzyme bed - factor defined by Eqs. (19) and (20) - kg · m^–3 density of immobilized enzyme bed - unstableness factor - effectiveness factor - Thiele modulus - relative half-lifetime of immobilized enzyme Index o values obtained with fresh immobilized enzyme     

Effects of constitutive expression of nitrate reductase in transgenic Nicotiana plumbaginifolia L. in response to varying nitrogen supply

Transformed Nicotiana plumbaginifolia plants with constitutive expression of nitrate reductase (NR) activity were grown at different levels of nitrogen nutrition. The gradients in foliar NO ₃ ^– content and maximum extractable NR activity observed with leaf order on the shoot, from base to apex, were much decreased as a result of N-deficiency in both the transformed plants and wild type controls grown under identical conditions. Constitutive expression of NR did not influence the foliar protein and chlorophyll contents under any circumstances. A reciprocal relationship between the observed maximal extractable NR activity of the leaves and their NO ₃ ^– content was observed in plants grown in nitrogen replete conditions at low irradiance (170 mol photons·m^–2 ·s^–1). This relationship disappeared at higher irradiance (450 mol photons·m^–2·S^–1) because the maximal extractable NR activity in the leaves of the wild type plants in these conditions increased to a level that was similar to, or greater than that found in constitutive NR-expressors. Much more NO ₃ ^– accumulated in the leaves of plants grown at 450 mol photons·m^–2·s^–1 than in those grown at 170 mol photons·m^–2·s^–1 in N-replete conditions. The foliar NO ₃ ^– level and maximal NR activity decreased with the imposition of N-deficiency in all plant types such that after prolonged exposure to nitrogen depletion very little NO ₃ ^– was found in the leaves and NR activity had decreased to almost zero. The activity of NR decreased under conditions of nitrogen deficiency. This regulation is multifactoral since there is no regulation of NR gene expression by NO ₃ ^– in the constitutive NR-expressors. We conclude that the NR protein is specifically targetted for destruction under nitrogen deficiency. Consequently, constitutive expression of NR activity does not benefit the plant in terms of increased biomass production in conditions of limiting nitrogen.Abbreviations Chl chlorophyll - N nitrogen - NR NADH-nitrate reductase - WT wild type     

The effect of carbon dioxide on the growth kinetics of fructose-limited chemostat cultures of Acetobacterium woodii DSM 1030

The growth of the anaerobic acetogenic bacterium Acetobacterium woodii DSM 1030 was investigated in fructose-limited chemostat cultures. A defined medium was developed which contained fructose, mineral salts, cysteine · HCl and Ca pantothenate (1 mg · 1^–1) supplied in a vitamin supplement. Growth at high dilution rates was dependent on the presence of CO₂ in the gas phase. The ^max was found to be 0.16 h^–1 and the fructose maintenance requirement was 0.1 to 0.13 mmol fructose · (g dry wt)^–1 · h^–1. A growth yield of 61 g dry wt · (mol fructose)^–1, corrected for the cell maintenance requirement and for incorporation of fructose carbon into cell biomass, was determined from the fructose consumption. A corresponding growth yield of 69 g dry wt · (mol fructose)^–1 was calculated from the acetate production assuming that fructose fermentation was homoacetogenic. A Y_ATP of 12.2 to 13.8 g dry wt · (mol ATP)^–1 was calculated from these growth yields using a value of 5 mol ATP · (mol fructose)^–1 as an estimate of the amount of ATP synthesised from fructose fermentation. The addition of yeast extract (0.5 g · 1^–1) to the medium did not influence the ^max or cell yield. After prolonged growth under fructose-limited conditions the requirement of the culture for CO₂ in the gas phase was reduced.Abbreviations YE yeast extract - IC inorganic carbon - D fermenter dilution rate : h^–1 - M_X maintenance requirement for X: mmol X · (g dry wt)^–1 · h^–1 - X may be fructose (Fruct), fructose consumed in energy metabolism (Fruct [E]), acetate (Ac) - ATP CO₂, NH _inf4 ^sup+ or Pi - qX specific rate of utilisation or consumption of X: mmol X · (g dry wt)^–1 · h^–1 - V fermenter volume: litre - rC · Cell, fermenter cell carbon production: mmol C · h^–1 - Y_X yield of cells on X: g dry wt · (mol X)^–1 - Y _infx ^supmax the yield corrected for cell maintenance: g dry wt · (mol X)^–1 - S_ATP stoichiometry of ATP synthesis from fructose: mol ATP · (mol frucose)^–1 - x cell concentration: g dry wt · 1^–1 - specific growth rate : h^–1 - ^max maximum specific growth rate: h^–1     

Function and regulation of the avian caecal bulb: influence of dietary NaCl and aldosterone on water and electrolyte fluxes in the hen (Gallus domesticus) perfused in vivo

Summary The function of the caecal bulb, and its adaptation to chronic high- or low-Na⁺ intake, was investigated by in vivo perfusion of anaesthetised birds. Effects of acute aldosterone injection (125 g·kg^–1 body mass) were also measured.Evidence was found for primary active net absorption of Na⁺, inducing parallel Na-linked absorption of water and Cl^– and secretion of K⁺. Around 20–35% of total Cl^– absorption and K⁺ secretion were independent of Na⁺ fluxes, and these components appear to be driven by passive processes with apparent conductances of 6.3×10^–3 (G _Cl) and 1.1×10^–3 (G _K) S·cm^–2.Acetate (40mM) stimulated Na⁺ fluxes (8.5–9.9 Eq·cm^–2·h^–1) and Na-linked water fluxes (27–44 l·cm^–2·h^–1). Increased coupling ratios (2.9–4.6 l·Eq^–1) and other data indicate that these effects may be due to increased osmotic permeabilities of barriers involved in the Na-linked water transfer pathway.Low-Na⁺ maintenance enhanced EPD (49–69 mV, serosa positive) and all net fluxes:J _Na (6.8–11.6);J _K (–3.2––4.3);J _Cl (4.3–5.6 Eq·cm serosal area^–2·h^–1);J _v (28–43 l·cm^–2·h^–1) (mucosal-serosal fluxes positive).Acute aldosterone enhancedJ _Na (10.8–14.0 Eq·cm^–2·h^–1) and EPD (54–66 mV) by 3 h after injection, but had no effect on the Na-linked components ofJ _K orJ _Cl.Abbreviations ECPD, EPD Electrochemical or electrical potential difference - G _Cl ,G _K ionic conductances (Cl^–, K⁺) - J _v ,J _ion net volume or ion flux rate, mucosa-serosa positive;P _d (Cl) diffusive permeability coefficient (of Cl^–) - SEDM standard error of difference between means