OX40–OX40 ligand interaction may activate phospholipase C signal transduction pathway in human umbilical vein endothelial cells

Studies have recently supported the emerging role of OX40/OX40L interaction in atherosclerosis. The mechanism of OX40/OX40L interaction may be related to a variety of signal pathways. The most important signal pathway involves the activation of phospholipase C (PLC) which induces diacylglycerol–protein kinase C (DAG–PKC) and the inositol trisphosphate (IP₃)–intracellular free calcium ([Ca²⁺]_i) pathway. The aim of this work was to investigate whether OX40–OX40L interaction can stimulate the PLC signal pathway in human umbilical vein endothelial cells (HUVEC). The DAG and IP₃ level in HUVEC were measured by radio-enzymatic assay. The activity of PKC was detected by its ability to transfer phosphate from [γ-³²P]ATP to lysine-rich histone. [Ca²⁺]_i concentrations were measured by flow cytometric analysis. Results showed that the DAG level was markedly increased in a concentration-dependent, biphasic manner in HUVEC induced by OX40. The early phase was rapid, peaking at 30 s. The late phase reached the maximum level at 15 min and decayed slowly. OX40 increased PKC activity in a dose-dependent manner with two peaks at 40–50 s and 12–16 min, then decreased slowly, yet maintained a high level for at least 30 min. PKC activity was mainly in cytosol at rest and translocated from cytosol to membrane when stimulated by OX40. Similarly, OX40-induced rapid IP₃ formation coincided with the peak of DAG level. Moreover, OX40 also induced peak [Ca²⁺]_i responses including the rapid transient phase and the sustained phase. Anti-OX40L antibody significantly suppressed OX40-induced DAG–PKC and IP₃–[Ca²⁺]_i signal pathway activation in HUVEC. In conclusion, the data suggested that OX40–OX40L interaction induced a robust stimulation of phospholipase C signal transduction pathway in HUVEC.     

ICChI, a glycosylated chitinase from the latex of Ipomoea carnea

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14–15%), has a molecular mass of 34.94 kDa (MALDI–TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0–9.0, 80 °C and the optimal activity is observed at pH 6.0 and 60 °C. Using p-nitrophenyl-N-acetyl-β-d-glucosaminide, the kinetic parameters K_m, V_max, K_cat and specificity constant of the enzyme were calculated as 0.5 mM, 2.5 × 10⁻⁸ mol min⁻¹ μg enzyme⁻¹, 29.0 s⁻¹ and 58.0 mM⁻¹ s⁻¹ respectively. The extinction coefficient was estimated as 20.56 M⁻¹ cm⁻¹. The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G–E–I–A–I–Y–W–G–Q–N–G–G–E–G–S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields.     

Synthesis and evaluation of phosphopeptides containing iminodiacetate groups as binding ligands of the Src SH2 domain

Phosphopeptide pTyr-Glu-Glu-Ile (pYEEI) has been introduced as an optimal Src SH2 domain ligand. Peptides, Ac-K(IDA)pYEEIEK(IDA) (1), Ac-KpYEEIEK (2), Ac-K(IDA)pYEEIEK (3), and Ac-KpYEEIEK(IDA) (4), containing 0–2 iminodiacetate (IDA) groups at the N- and C-terminal lysine residues were synthesized and evaluated as the Src SH2 domain binding ligands. Fluorescence polarization assays showed that peptide 1 had a higher binding affinity (K_d = 0.6 μM) to the Src SH2 domain when compared with Ac-pYEEI (K_d = 1.7 μM), an optimal Src SH2 domain ligand, and peptides 2–4 (K_d = 2.9–52.7 μM). The binding affinity of peptide 1 to the SH2 domain was reduced by more than 2-fold (K_d = 1.6 μM) upon addition of Ni²⁺ (300 μM), possibly due to modest structural effect of Ni²⁺ on the protein as shown by circular dichroism experimental results. The binding affinity of 1 was restored in the presence of EDTA (300 μM) (K_d = 0.79 μM). These studies suggest that peptides containing IDA groups may be used for designing novel SH2 domain binding ligands.     

Synthesis and pharmacological evaluation of 6-naltrexamine analogs for alcohol cessation

A series of substituted aryl amide derivatives of 6-naltrexamine, 3 designed to be metabolically stable were synthesized and used to characterize the structural requirements for their potency to binding and functional activity of human mu (μ), delta (δ) and kappa (κ) opioid and nociceptin (NOP) receptors. Binding assays showed that 4–10 had subnanomolar K_i values for μ and κ opioid receptors. Functional assays for stimulation of [³⁵S]GTPγS binding showed that several compounds acted as partial or inverse agonists and antagonists of the μ and δ, κ opioid or NOP receptors. The compounds showed considerable stability in the presence of rat, mouse or human liver preparations and NADPH. The inhibitory activity on the functional activity of human cytochrome P450s was examined to determine any potential inhibition by 4–9. Only modest inhibition of CYP3A4, CYP2C9 and CYP2C19 was observed for a few of the analogs. As a representative example, radiolabeled 6 was examined in vivo and showed reasonable brain penetration. The inhibition of ethanol self-administration in rats trained to self-administer a 10% (w/v) ethanol solution, utilizing operant techniques showed 5–8 to have very potent efficacy (ED₅₀ values 19–50 μg/kg).     

HLA class I binding of synthetic nonamer peptides carrying major anchor residue motifs of HLA-B27 (B*2705)-binding peptides

Eight nonamer peptides that comply with the major anchor residue motifs (the combination of amino acid residues at positions 2 and 9), R-K and R-R, of HLA-B27 (B^*2705)-binding peptides were synthesized and tested for their direct binding to HLA class I alpha chains by the HLA class I alpha chain refolding assay previously described. One was a known B27 (B^*2705)-binding heat shock protein peptide, HSP89 (201–209), and the other seven were derived from the sequence of wild-type P53, a human tumor suppressor protein. A total of 36 HLA class I allospecificities were tested. HSP89 (201–209) and two P53 peptides, P53 (362–370) and P53 (378–386), all possessing the motif R-K, bound strongly to B27 (B^*2705) alpha chains. A weak binding was seen for P53 (272–280) and P53 (334–342), both showing the motif R-R. Most of these B27-binding peptides were found to bind to A3 alpha chains as well. In addition, P53 (173–181) and P53 (334–342), both with the R-R motif, showed substantial binding with A31 alpha chains. All the peptides, carrying the motif R-K also showed weak binding with A31 alpha chains. The remaining two peptides, P53 (201–209) and P53 (282–290), with the motif R-R, did not show significant bininding with any of the alpha chains tested. This study demonstrates both the specificity of peptide binding to a given HLA allelic product and the occurence of cross-peptide-binding between the allelic products of different HLA loci. Correspondence to: N. Tanigaki.     

The influence of N-glycosylation on biochemical properties of Amy1, an α-amylase from the yeast Cryptococcus flavus

The yeast Cryptococcus flavus secretes a glycosylated α-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5 μg mL⁻¹ affected C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after C. flavus growth in the presence of 0.5 μg mL⁻¹ TM. SDS–PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5 kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The K_M values of native and non-glycosylated α-amylases were 0.052 and 0.098 mg mL⁻¹, and V_max values were 0.038 and 0.047 mg min⁻¹, respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg²⁺ and Cu²⁺ suggesting that N-glycosylation is involved in the folding of Amy1.     

Characterization of chicken cystatin binding to rat renal brush-border membranes

Chicken cystatin, a homologue of human cystatin C, like other low-molecular-weight proteins is metabolized by renal proximal tubule cells. However, the precise mechanism(s) of this process has not been elucidated yet. To characterize chicken cystatin binding to renal brush-border membranes, the incubation of fluorescein labelled protein with rat cortical homogenate was performed. Saturation-dependent and reversible binding with low affinity (K_d = 3.67–4.07 μM) and high capacity (B_max = 2.32–2.79 nmol/mg) was observed. Bovine albumin was the most potent competitor (K_i = 0.7 μM) among other megalin/cubilin ligands tested. The presence of Ca^+ 2 ions was necessary to effective cystatin binding by brush-border membranes. Obtained data strongly support the hypothesis that chicken cystatin is a novel ligand for megalin/cubilin receptors tandem on proximal tubular cells.     

η-N-succinimidato complexes of iron, molybdenum and tungsten as reversible inhibitors of papain

Recently we have found that the metallocarbonyl complexes (η⁵-C₅H₅)M(CO)_x(η¹-N-maleimidato) (M = Fe, Mo, W; x = 2 or 3) bearing a maleimide function were irreversible inhibitors of the enzyme papain. To get further insight into the binding mechanism of these compounds we synthesized the related complexes (η⁵-C₅H₅)M(CO)_x(η¹-N-succinimidato) (M = Fe, Mo, W; x = 2 or 3) that lacked the ethylenic bond responsible for alkylation of the cysteine 25 thiol group in the papain‘s catalytic pocket. We performed kinetic studies of the interaction of the synthesized complexes towards papain. We found that they act as reversible inhibitors of the enzyme with IC₅₀ values in the range 480–1700 μM. Docking experiments confirmed binding of these complexes to the enzyme’s catalytic pocket.     

Intermedin1–53 inhibits rat cardiac fibroblast activation induced by angiotensin II

Intermedin (IMD) is a novel peptide related to calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM). Proteolytic processing of a larger precursor of IMD yields a biologically active C-terminal fragment IMD_1–53. We aimed to observe the cardioprotective antifibrotic effects of IMD_1–53 and its mechanism. Radioimmunoassay and Western blot analysis was used to determine IMD content in angiotensin II (AngII)-treated rat cardiac fibroblasts (CFs). Real-time PCR was used to measure mRNA levels of IMD and the IMD receptor components calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP) 1, 2 and 3. AngII was a powerful stimulator of CF activation. It decreased the production and secretion of IMD and increased the mRNA levels of the IMD receptor components CRLR, RAMP2 and RAMP3, but not IMD and RAMP1. Moreover, IMD_1–53 (10^− 8 or 10^− 7 mol/l) exerted a 25% and 45% respective inhibition in [³H]-thymidine incorporation and 16% and 36% respective inhibition in [³H]-proline incorporation in rat CFs incubated with AngII, and the actions of IMD_1–53 could be blocked by CGRP_8–37 and ADM_22–52. Immunofluorescence and Western blot analysis revealed that IMD_1–53 inhibited the increase of alpha-SMA in CFs induced by AngII, and the above effects of IMD_1–53 were similar to or more potent than those of an equivalent dose of ADM. Otherwise, IMD_1–53 resulted in dose-dependent increases of cAMP production in CFs, and co-incubated with H89 blocked the inhibition effect of IMD_1–53 on AngII-induced [³H]-thymidine, [³H]-proline incorporation and alpha-SMA expression. Collectively, these results show that IMD and its receptor components could be involved in an onset of cardiac fibrosis, and like ADM, IMD_1–53 exerts an antifibrotic effect in CFs, and the effect can be mediated by cAMP–PKA pathway and implicated with the ADM and CGRP receptors.     

Kinetic analyses for bindings of concanavalin A to dispersed and condensed mannose surfaces on a quartz crystal microbalance

A biotinylated mannotriose (Man3-bio) was dispersively immobilized in the matrix of biotinylated lactose (Gal-Glc-bio) on a streptavidin-covered, 27-MHz quartz crystal microbalance (QCM), and binding kinetics of concanavalin A (Con A) to Man3-bio in the Gal-Glc-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. Association constants (K_a) and binding and dissociation rate constants (k_on and k_off) could be determined separately as the 1:1 and 1:2 bindings of Con A to Man3-bio on the surface. When Man3-bio was immobilized with content of 1 to 5 mol% in the matrix, the 1:1 binding of Con A to Man3-bio was obtained as K_a = (4 ± 1) × 10⁶ M⁻¹, k_on = (4 ± 1) × 10⁴ M⁻¹ s⁻¹, and k_off = (12 ± 2) × 10^–3 s⁻¹. On the contrary, when Man3-bio was immobilized with content of 20 to 100 mol% in the matrix, the 1:2 binding of Con A to Man3-bio was obtained as K_a = (14 ± 2) × 10⁶ M⁻¹, k_on = (14 ± 2) × 10⁴ M⁻¹ s⁻¹, and k_off = (7 ± 2) × 10^–3 s⁻¹. Thus, K_a for the 1:2 binding was 10 times larger than that for the 1:1 binding, with a three times larger binding rate constant (k_on) and a three times smaller dissociation rate constant (k_off). This is the first example to obtain separate kinetic parameters for the 1:1 and 1:2 bindings of lectins to carbohydrates on the surface.     

The effects of the deep-sea environment on transmembrane signaling

Membrane-associated processes may be particularly susceptible to perturbation by the high hydrostatic pressures and low temperatures of the deep ocean. Transmembrane signaling by guanyl nucleotide binding protein (G protein) coupled receptors (GPCRs) is affected at a number of steps: (1) agonist activation of the GPCR; (2) the interaction of the receptor with the heterotrimeric G protein; (3) the G protein GTPase cycle; and (4) the activation and function of the effector element, adenylyl cyclase. The effects of low temperature and high hydrostatic pressures on the A₁ adenosine receptor–inhibitory G protein (G_i)–adenylyl cyclase signaling complex were examined in teleost fishes from three families, Scorpaenidae, Macrouridae and Moridae. In a comparison of teleost fishes, rat and chicken, species with body temperatures from 1 to 40 °C, at atmospheric pressure, A₁ adenosine receptor agonist binding is conserved at the body temperature of the species. In the marine teleost fishes examined, increased pressure decreases agonist efficacy. There are differences among species in the effects of increased hydrostatic pressure on G protein interactions with receptors, GTP binding to G protein α subunits and the intrinsic GTPase activity of α subunits. Adenylyl cyclase activity and modulation are affected by increased pressure in all the species examined, except Antimora rostrata which was unaffected by pressure changes. At pressures approximating those which the species experience in situ adenylyl cyclase activity retains its sensitivity to modulators. To understand the physiological consequences of impaired cell signaling several prototypical human diseases are discussed.     

Kinetics of alkaline phosphatase activity and phosphorus availability for phytoplankton and bacterioplankton in lake plu\see (North German Eutrophic Lake)

Annual studies of kinetics of alkaline phosphatase (APA) activity and phosphorus availability for microplankton in the photic zone of an eutrophic lake are reported. The total APA activity of microplankton varied strongly. V_max was highest during summer P depletion, and in autumn and winter total APA activity was low. The total APA specific activity of the microplankton was also highest (average 3.55 pmole PO₄ ^3– ng ATP^–1 min^–1) when ambient orthophosphate concentrations were very low. Both V_max and specific APA activity were not dependent on the biomass of microplankton; they were strongly affected by P available for microplankton. A differential filtration technique was used for separation of microplankton into two size classes, i.e., algal, larger than 3m, and bacterial fraction with size 0.2–3.0m. The algal size fraction had lower specific APA activity (average 1.224 pmole PO₄ ^3– ng ATP^–1 min^–1) and higher K_M values (38.8mole × liter^–1) than microorganisms which were smaller than 3m (2.011 pmole PO₄ ^3– ng ATP^–1 min^–1 and 25.4mole liter^–1, respectively). The K_M values of free, dissolved APA (36.8mole liter^–1) indicated that free APA was probably released by algae. Phytoplankton were major APA activity producers in the photic zone of the lake from March to November, and their activity constituted, on the average, 48.6% of the total APA activity in the water. Bacteria were the dominant APA activity producers in winter (41.3–44.9%); however, during other periods they contributed significantly (average 21.7%) to total APA activity. When surplus constituted less than 10% of particulate P in seston, phytoplankton produced high specific APA activity, and when surplus P was higher than 15%, the specific APA activity of phytoplankton size fraction rapidly decreased. APA of the bacterial size fraction of the seston was not affected by P concentrations. Orthophosphate was a competitive inhibitor of APA produced by microorganisms of the size fraction larger than 3.0m, and increasing concentrations of inorganic phosphate caused an increase in K_M values. The hypothetical metabolic-coupling between phytoplankton and bacterioplankton in the phosphorus cycle in conjunction with carbon metabolism in the lake is discussed.     

Use of a real-time remote monitoring network (RTRM) and shipborne sampling to characterize a dinoflagellate bloom in the Neuse Estuary, North Carolina, USA

The spatial-temporal distribution of a dinoflagellate bloom dominated or co-dominated by Prorocentrum minimum was examined during autumn through early spring in a warm temperate, eutrophic estuary. The developing bloom was first detected from a web-based alert provided by a network of real-time remote monitoring (RTRM) platforms indicating elevated dissolved oxygen and pH levels in upper reaches of the estuary. RTRM data were used to augment shipboard sampling, allowing for an in-depth characterization of bloom initiation, development, movement, and dissipation. Prolonged drought conditions leading to elevated salinities, and relatively high nutrient concentrations from upstream inputs and other sources, likely pre-disposed the upper estuary for bloom development. Over a 7-month period (October 2001–April 2002), the bloom moved toward the northern shore of the mesohaline estuary, intensified under favorable conditions, and finally dissipated after a major storm. Bloom location and transport were influenced by prevailing wind structure and periods of elevated rainfall. Chlorophyll a within bloom areas averaged 106 ± 13 μg L⁻¹ (mean ± 1 S.E.; maximum, 803 μg L⁻¹), in comparison to 20 ± 1 μg L⁻¹ outside the bloom. There were significant positive relationships between dinoflagellate abundance and TN and TP. Ammonium, NO₃⁻, and SRP concentrations did not decrease within the main bloom, suggesting that upstream inputs and other sources provided nutrient-replete conditions. In addition, PAM fluorometric measurements (09:00–13:00 h) of maximal PSII quantum yield (F_v/F_m) were consistently 0.6–0.8 within the bloom until late March, providing little evidence of photo-physiological stress as would have been expected under nutrient-limiting conditions. Nitrogen uptake kinetics were estimated for P. minimum during the period when that species was dominant (October–December 2001), based on literature values for N uptake by an earlier P. minimum bloom (winter 1999) in the Neuse Estuary. The analysis suggests that NH₄⁺ was the major N species that supported the bloom. Considering the chlorophyll a concentrations during October and December and the estimated N uptake rates, phytoplankton biomass was estimated to have doubled once per day. Bloom displacement (January–February) coincided with higher diversity of heterotrophic dinoflagellate species as P. minimum abundance decreased. This research shows the value of RTRM in bloom detection and tracking, and advances understanding of dinoflagellate bloom dynamics in eutrophic estuaries.     

New catalyst design for polymerization of norbornene esters by reducing intramolecular interaction

We have used density functional theory to study palladium-based catalysts commonly used for the polymerization of norbornene derivatives with an ester group. Exo–exo, exo–endo, and endo–endo isomers of catalyst complexes were investigated; the endo–endo isomer was the most stable and inactive due to an intramolecular interaction between Pd and O of the carbonyl group. Phosphine groups are effective in minimizing the Pd–O interaction in the endo–endo isomer and P(C₆H₁₁)₃ was found to be the most efficient reagent. The intramolecular Pd–O interactions were estimated using model complexes, and it was demonstrated that they play a crucial role in stabilizing the endo–endo isomer.Figure Methyl ester norbornene complex for endo–endo isomers with P(C₆H₁₁)₃     

Al avoidance and Al tolerance ofMucuna pruriens var.utilis: Effects of a heterogeneous root environment and the nitrogen form in the root environment

InMucuna pruriens var.utilis, grown with nitrate-N in a hydroponic split-root system, an Al avoidance reaction of root growth was observed, which was ascribed to local P stress in the Al containing compartment. The Al avoidance reaction was similar to the avoidance ofMucuna roots of acid subsoil in the field where roots grew preferentially in the topsoil. In the present paper the effect of different N forms (NO₃ ^– and NH₄ ⁺) on the reactions ofMucuna to Al were studied, since in acid soils N is present as a mixture of NO₃ ^– and NH₄ ⁺. No interaction between the N form and Al toxicity was found. A hydroponic split-root experiment with NH₄NO₃ nutrition, which is comparable to the situation in the field, showed that under these conditions Al avoidance did not occur. It is concluded that a relation between the Al avoidance reaction ofMucuna and P stress is still likely.Abbreviations D_r root diameter - L_pr total root length per plant - L_rw specific root length - NRA nitrate reductase activity - S/R shoot: root ratio     

Purification and characterization of chitinase from the stomach of silver croaker Pennahia argentatus

A chitinase was purified from the stomach of a fish, the silver croaker Pennahia argentatus, by ammonium sulfate fractionation and column chromatography using Chitopearl Basic BL-03, CM-Toyopearl 650S, and Butyl-Toyopearl 650S. The molecular mass and isoelectric point were estimated at 42 kDa and 6.7, respectively. The N-terminal amino acid sequence showed a high level of homology with family 18 chitinases. The optimum pH of silver croaker chitinase toward p-nitrophenyl N-acetylchitobioside (pNp-(GlcNAc)₂) and colloidal chitin were observed to be pH 2.5 and 4.0, respectively, while chitinase activity increased about 1.5- to 3-fold with the presence of NaCl. N-Acetylchitooligosaccharide ((GlcNAc)_n, n = 2–6) hydrolysis products and their anomer formation ratios were analyzed by HPLC using a TSK-GEL Amide-80 column. Since the silver croaker chitinase hydrolyzed (GlcNAc)_4–6 and produced (GlcNAc)_2–4, it was judged to be an endo-type chitinase. Meanwhile, an increase in β-anomers was recognized in the hydrolysis products, the same as with family 18 chitinases. This enzyme hydrolyzed (GlcNAc)₅ to produce (GlcNAc)₂ (79.2%) and (GlcNAc)₃ (20.8%). Chitinase activity towards various substrates in the order pNp-(GlcNAc)_n (n = 2–4) was pNp-(GlcNAc)₂ >> pNp-(GlcNAc)₄ > pNp-(GlcNAc)₃. From these results, silver croaker chitinase was judged to be an enzyme that preferentially hydrolyzes the 2nd glycosidic link from the non-reducing end of (GlcNAc)_n. The chitinase also showed wide substrate specificity for degrading α-chitin of shrimp and crab shell and β-chitin of squid pen. This coincides well with the feeding habit of the silver croaker, which feeds mainly on these animals.     

Phosphorus removal by the Ceratophyllum/periphyton complex in a south Florida (USA) freshwater marsh

Removal of phosphorus (P) by Ceratophyllum demersum L. and associated epiphytic periphyton was quantified by measuring the disappearance of soluble reactive P (SRP) from microcosms during 1-h in situ incubations conducted over a 1-year period. Initial P concentrations in these incubations ranged from 30 to >10,000 μg P L⁻¹. Phosphorus removal was proportional to initial P concentrations and was weakly correlated with solar irradiance and water temperature. Removal rates (0.6–32.8 mg P m⁻² d⁻¹) and k_v coefficients (0.68–1.93 h⁻¹) from experiments run at low initial P concentrations (up to 200 μg P L⁻¹) were comparable to results reported for other macrophytes. Removal rates from experiments run at the highest (>10,000 μg P L⁻¹) initial P concentrations (5300 and 11,100 mg P m⁻² d⁻¹) most likely represented luxury nutrient consumption and were not thought to be sustainable long term. We were unable to determine a V_max for P removal, suggesting that the nutrient-storage capability of the C. demersum/periphyton complex was not saturated during our short-term incubations. Based on N:P molar ratios, the marsh was P limited, while the C. demersum/periphyton complex was either N limited or in balance for N and P throughout this study. However, despite its tissue stoichiometry, the C. demersum/periphyton complex always exhibited an affinity for P. It appeared that the biochemical mechanisms, which mediate P removal, at least on a short-term basis, were more influenced by increases in ambient P levels than by tissue nutrient stoichiometry.     

Characteristics of antibody inhibition of rat kidney (Na+−K+)-ATPase

Summary Antibodies which were raised against highly purified membrane-bound (Na⁺–K⁺)-ATPase from the outer medulla of rat kidneys inhibit the (Na⁺–K⁺)-ATPase activity up to 95%. The antibody inhibition is reversible. The time course of enzyme inhibition and reactivation is biphasic in semilogarithmic plots.In the purified membrane-bound (Na⁺–K⁺)-ATPase negative cooperativity was observed (a) for the ATP dependence of the (Na⁺–K⁺)-ATPase activity (n=0.86), (b) for the ATP binding to the enzyme (n=0.58), and (c) for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity (n=0.77). By measuring the Na⁺ dependence of the (Na⁺–K⁺-ATPase reaction, a positive homotropic cooperativity (n=1.67) was found.As reactivation of the antibody-inhibited enzyme proceeds very slowly (t _0.5=5.2hr), it was possible to measure characteristics of the antibody-(Na⁺–K⁺)-ATPase complex: The antibodies exerted similar effects on the ATP dependence of the (Na⁺–K⁺)-ATPase reaction and on the ATP binding of the enzyme.V _max of the (Na⁺–K⁺)-ATPase reaction and the number of ATP binding sites were reduced whileK _{0.5 ATP} for the (Na⁺–K⁺)-ATPase activity and for the ATP binding were increased by the antibodies. The Hill coefficients for the ATP binding and for the ATP dependence of the enzyme activity were not significantly altered by the antibodies. The antibodies increased theK _0.5 value for the Na⁺ stimulation of the (Na⁺–K⁺)-ATPase activity, but they did not alter the homotropic interactions between the Na⁺-binding sites. The negative cooperativity which was observed for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity was abolished by the antibodies.The data are tentatively explained by the following model: The antibodies bind to the (Na⁺–K⁺)-ATPase from the inner membrane side, reduce the ATP binding symmetrically at the ATP binding sites and reduce thereby also the (Na⁺–K⁺)-ATPase activity of the enzyme. The antibodies may inhibit the ATP binding by a direct interaction or by means of a conformational change at the ATP binding sites. This may possibly also lead to the alteration of the Na⁺ dependence of the (Na⁺–K⁺)-ATPase activity and to the observed alteration of the dose response to the ouabain inhibition.