The isolation and preliminary characterization of apotransketolase from human erythrocytes

Human erythrocyte apotransketolase (EC 2.2.1.1) has been isolated with greater than 400 fold purification, and free of glyceraldehyde-3-phosphate dehydrogenase. The preparation has an absolute requirement for thiamin pyrophosphate in order to exhibit enzyme activity. Neither thiamin nor thiamin monophosphate could substitute for this requirement, nor were they inhibitory separately or together at concentrations of 1 mM. The K_m for thiamin pyrophosphate was 0.4 μM. The K_m for ribose-5-phosphate was 3 × 10^?4M and for xylulose-5-phosphate 1.8 × 10^?4M.     

Immobilization of d-glucose oxidase onto a hydrogen peroxide permselective membrane and application for an enzyme electrode

A hydrogen peroxide permselective membrane with asymmetric structure was prepared and d-glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized d-glucose oxidase membrane was 0.34 units cm^?2 and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized d-glucose oxidase membrane was 1.6 × 10^?3 mol l^?1 and that of free enzyme was 4.8 × 10^?2 mol l^?1. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized d-glucose oxidase membrane. The enzyme electrode responded linearly to d-glucose over the concentration 0–1000 mg dl^?1 within 10 s. When the enzyme electrode was applied to the determination of d-glucose in human serum, within day precision (CV) was 1.29% for d-glucose concentration with a mean value of 106.8 mg dl^?1. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized d-glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of d-glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.     

Interaction of influenza virus proteins with planar bilayer lipid membranes II. Effects of rimantadine and amantadine

The dependence of the surface potential difference (ΔU), transversal elasticity module (E₁) and membrane conductivity (G₀) on the concentrations of the antiviral drugs, rimantadine and amantadine was studied in the planar bilayer lipid membrane system. The method used was based on independent measurements of the second and third harmonics of the membrane capacitance current. The binding constants of bilayer lipid membranes obtained from the drug adsorption isotherms were 2.1 · 10⁵ M^?1 and 1.3 · 10⁴ M^?1 for rimantadine and amantadine, respectively. The changes in G₀ took place only after drug adsorption saturation had been achieved. The influence of rimantadine and amantadine on the interaction of bilayer lipid membranes with matrix protein from influenza virus was also investigated. The presence of 70 μg/ml rimantadine in the bathing solution resulted in an increase in the concentration of M-protein at which the adsorption and conductance changes were observed. The effects of amantadine were similar to those of rimantadine but required a higher critical concentration of amantadine. The results obtained suggest that the antiviral properties of rimantadine and amantadine may be related to the interaction of these drugs with the cell membrane, which can affect virus penetration into the cell as well as maturation of the viral particle at the cell membrane.     

Carbonized Chicken Eggshell Membranes with 3D Architectures as High‐Performance Electrode Materials for Supercapacitors

Supercapacitor electrode materials are synthesized by carbonizing a common livestock biowaste in the form of chicken eggshell membranes. The carbonized eggshell membrane (CESM) is a three‐dimensional macroporous carbon film composed of interwoven connected carbon fibers containing around 10 wt% oxygen and 8 wt% nitrogen. Despite a relatively low surface area of 221 m² g^?1, exceptional specific capacitances of 297 F g^?1 and 284 F g^?1 are achieved in basic and acidic electrolytes, respectively, in a 3‐electrode system. Furthermore, the electrodes demonstrate excellent cycling stability: only 3% capacitance fading is observed after 10 000 cycles at a current density of 4 A g^?1. These very attractive electrochemical properties are discussed in the context of the unique structure and chemistry of the material.     

Magneto-electro-fusion of human erythrocytes

In inhomogeneous (static) magnetic fields close contact between ‘magnetic’ human erythrocytes was established. The cells were made magnetic by incubating them in a medium containing small Fe₃O₄-particles which adsorbed to the outer membrane surface. Fusion was induced by applying two electric field pulses (field strength: 8.5 kV · cm^?1; duration: 60 μs) to the magnetically collected cells. This procedure allowed the use of electrically conductive media (3 · 10^?1 Ω^?1 · cm^?1). Fusion of red blood cells occured very often. If cell suspensions of high density were used fusion resulted in the formation of giant red blood cells with osmotically intact membranes.     

Conformational changes in erythrocyte membranes by prostaglandins as measured by circular dichroism

Membranes were prepared from fresh, washed human erythrocytes by hemolysis and washing with 5 mm sodium phosphate buffer (pH 7.4). The mean residue ellipticity, [θ], of erythrocyte membrane circular dichroism was altered by prostaglandin E₁ or prostaglandin F_2α at 37 °C when observed from 250 nm to 190 nm. The decrease in negativity of [θ] with 10^?6m prostaglandin E₁ was 12.7% at 222 nm and 17.7% at 208 nm, and with 10^?6m prostaglandin F_2α 22.5% and 34.2%, respectively (P < 0.01). Similar changes in [θ] were observed at lower concentrations of prostaglandins. No strict relationship between amount of change of [θ] and prostaglandin concentrations of 3 × 10^?5m to 3 × 10^?12m was evident. A persistent alteration of [θ] with prostaglandin was observed at 37 °C. Transient change of [θ] occurred at 25 °C with prostaglandin. No change of [θ] was observed at 15 or 20 °C. Buffer or palmitic acid were without effect on membrane [θ]. Phosphatidyl inositol or methyl arachidonate caused an increase in negativity of membrane spectra. The observed alterations of membrane [θ] did not arise from changes in light scattering as the OD₇₀₀–OD₂₀₀ of membranes was not changed by prostaglandin. Effects of prostaglandin were not dependent on light path length. The prostaglandin E₁ antagonist, 7-oxa-13-prostynoic acid, at 10^?7m produced no change of [θ] of membrane spectra and prevented the otherwise demonstrable effects of 10^?10m prostaglandin E₁ on [θ]. The decrease in negativity of [θ] at 222 nm is indicative of a decrease in ellipticity of membrane protein. These studies suggest that prostaglandins may act by inducing a conformational change in membrane protein.     

Membranes as targets of ultraviolet radiation

In non-photosynthetic cells, evidence for UV (ultraviolet radiation) damage to membranes comes from electron microscopy, chemical analysis and observations of transport processes. Specific perturbations in transport across membranes occur quickly after a relatively low fluence of UV. As an example, irradiation of suspension-cultured rose cells with 500-2000 J m^?2 (at 254 nm) causes an appearance of K⁺ in the extracellular medium at the rate of 5 × 10^?10μmol cell^?1 min^?1 for 30 to 60 min and more slowly thereafter. The early, rapid phase of appearance of K⁺ reflects both an increase in efflux and a decrease in influx. The appearance of K⁺ is matched by an appearance of HCO^?₃ in the medium. The HCO^?₃ comes from respiratory CO₂, which hydrates and dissociates in the cytoplasm, leading to a decrease in cytoplasmic pH. Overall, these results not only demonstrate UV damage to membrane function, but also suggest several ways by which UV may alter the general metabolic state of the cell. A demonstration of direct effects of UV on membrane components requires a purified system. At lower fluence, < 1800 J m^?2 (254 nm), the ATPase of membrane vesicle preparations is inactivated in a two-phase process that suggests the presence of enzymes with different UV sensitivities. The existence of two non-mitochondrial enzymes in rose cell vesicles has been confirmed by solubilizing the vesicle proteins with 1% cholic acid and separating the components on G-150 Sephadex. One component of relatively high molecular weight is especially sensitive. The fact that it is still sensitive when it is dissolved in cholic acid strongly suggests that its sensitivity is intrinsic and does not depend on sensitization by other membrane components. The action spectrum for the inactivation of the ATPase has a major peak at 290 nm and extends into the UV-C and UV-A regions. The physiological effects of UV-stimulated membrane changes are uncertain. There is little evidence that the UV damage to membranes is responsible for cell death. A UV-induced loss of K⁺ from guard cells may result in lower stomatal conductance. UV-stimulated membrane changes may play a role in the UV-induced synthesis of anthocyanins.     

Flow Injection Analysis of Lactose in Milk Using a Chemically Modified Lactose Electrode

β-Galactosidase and glucose oxidase were immobilized with bovine serum albumin using glutaraldehyde on to a glassy carbon electrode silanized with 3-aminopropyltriethoxysilane. The laboratory-constructed lactose electrode was used for flow injection analysis to determine the lactose content in milk. Electrochemical interference could be detected by a non-enzymatic electrode (W₂) and the current was subtracted from that of the enzymatic electrode (W₁), providing an accurate measurement of the hydrogen peroxide that was enzymatically generated. The peak current was linearly related to the lactose concentration in the range 10^?4~ 1.5 × 10^?3 M (original concentration) and 40 samples/hr could be analyzed. The relative standard deviation for 10 assays was less than 2%. The proposed method was compared with the chloramine T method and the values determined by both methods were in good agreement.     

Guanine nucleotide regulatory protein alterations in young Milan hypertensive strain rats

Vascular smooth muscle cell membranes from prehypertensive rats of the Milan hypertensive strain (MHS) were used to examine adenylyl cyclase activity and its regulation by guanine nucleotide regulatory proteins (G-proteins). Basal adenylyl cyclase activity was similar in MHS and Milan normontensive strain (MNS) membranes. Forsokolin (10^?4 M) produced a significantly greater stimulatory response in MHS membranes, but this was not observed with NaF (10^?2 M). Isoporterenol (10^?4 M) caused a significantly decreased stimulation of adenylyl cyclase activity in MHS membranes, while prostaglandin E₁ (10^?5 M) produced similar responses in the two strains. G_i function and GTP responses, as observed by biphasic effects of GTP on isoproterenol-stimulated membranes, were similar in both strains. The levels of G_i2α and G_qα/G₁₁α were similar in the two strains, while the levels of G_sα (44 and 42 kDa forms) and the β-subunit were significantly reduced by ～20% in MHS membranes. The α-subunit of G_i3 was dramatically reduced by ～80% in MHS membranes. The affinities of β-adrenergic receptors for the antagonist, cyanophindolol, were similar in the two strains; however, the number of β-adrenoceptors was substantially reduced in MHS membranes. These findings may be of relevance to altered vascular reactivity and transmembrane ion distribution observed in the MHS.     

Effect of guanyl nucleotides on the stimulation of adenyl cyclase activity in human thyroid plasma membranes by thyroid-stimulating hormone and prostaglandin E2

Thyroid homogenates and thyroid plasma membranes were prepared from human thyroid and the effects of thyroid-stimulating hormone (thyrotropin), NaF, and prostaglandins E₁ and E₂ on adenyl cyclase activity in these preparations were studied. The basal level of adenyl cyclase activity in plasma membranes was 5–8 times greater than that of the original homogenates. Adenyl cyclase activity in plasma membranes was stimulated 4.7-fold by 100 munits/ml of thyrotropin and 5-fold by 10 mM of NaF, but the activity in the homogenates was only stimulated 2-fold by either thyrotropin or NaF. Prostaglandin E₁ (10^?6?10^?3 M) and prostaglandin E₂ (10^?7?10^?4 M) failed to stimulate adenyl cyclase activity in plasma membranes, but they did stimulate adenyl cyclase activity in the homogenates. A marked stimulatory effect of prostaglandin E₂ (10^?5 M) on adenyl cyclase activity in plasma membranes resumed in the presence of GTP (10^?7?10^?4 M), although GTP itself only slightly stimulated enzyme activity. GDP and GMP were also effective in this respect, although their potencies varied from compound to compound. GTP potentiated slightly the action of thyrotropin on adenyl cyclase in plasma membranes, but it significantly depressed an increase of enzyme activity produced by NaF. Since GTP did not affect the ATP-regenerating system, it seems that GTP, GDP or GMP was required for the manifestation of prostaglandin E₂ action on adenyl cyclases of human thyroid plasma membranes.     

A th-1 Mutant of Arabidopsis thaliana Is Defective for a Thiamin-Phosphate-Synthesizing Enzyme: Thiamin Phosphate Pyrophosphorylase

We have examined the activity of the thiamin phosphate pyrophosphorylase in Arabidopsis thaliana wild type and in a mutant (th-1) which requires exogenous thiamin for growth. Mutant and wild-type plants grown in 1 × 10⁻⁷ molar thiamin were used for the examination of the production of thiamin and thiamin monophosphate (TMP) using 4-methyl-5-hydroxyethylthiazole phosphate and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate as substrates. While the wild-type strain formed both thiamin and TMP, the th-1 mutant did not. When TMP was added to the extracts, the th-1 mutant, as well as wild type, produced thiamin. Accordingly, it was concluded that the th-1 mutant was defective in the activity of TMP pyrophosphorylase. Some of the characteristics of the enzyme from the wild-type plant were examined. The optimum temperature for the reaction is 45°C, and the K_m values for the substrates are 2.7 × 10⁻⁶ molar for 4-methyl-5-hydroxyethylthiazole phosphate and 1.8 × 10⁻⁶ molar for 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate.     

Polysulfonated Fluoro‐oxyPBI Membranes for PEMFCs: An Efficient Strategy to Achieve Good Fuel Cell Performances with Low H3PO4 Doping Levels

Polybenzimidazoles (PBIs) are promising materials to replace Nafion as the electrolyte in polymer electrolyte membrane fuel cells (PEMFCs). The challenge with these materials is to achieve a good compromise between the H₃PO₄ doping level and membrane stability. This can be obtained by a proper monomer design, which can lead to better performing membrane electrode assemblies (MEAs), in terms of durability, acid leaching, and electrode safety. Here the easy and inexpensive synthesis of hexafluoropropylidene oxyPBI (F₆‐oxyPBI) and bisulfonated hexafluoropropylidene oxyPBI (F₆‐oxyPBI‐2SO₃H) is reported. The membranes based on F₆‐oxyPBI‐2SO₃H are more stable in an oxidative environment and more mechanically resistant than standard PBI and F₆‐oxyPBI. Whereas the attainable doping levels are low because of fluorine‐induced hydrophobicity, polysulfonation allows high proton conductivity, and fuel cell performances better than those reported for MEAs with F₆PBI‐ or PBI membranes with much higher doping levels. In the case of MEA with a F₆‐oxyPBI‐2SO₃H membrane, a peak power density of 360 mW cm^?2 is measured. Fuel cell performances of 604 mV at 0.2 A cm^?2 are maintained for 800 h without membrane degradation. Low H₂ permeability is measured, which remains almost unaffected during a 1000 h life‐test.     

Raman spectroscopic investigations of sarcoplasmic reticulum membranes

Raman spectra are presented for sarcoplasmic reticulum membranes. Interpretation of the 1000–1130 cm^?1 region of the spectrum indicates that the sarcoplasmic reticulum membrane may be more fluid than erythrocyte membranes that have been examined by the same technique. The fluidity of the membrane also manifests itself in the amide I portion of the membrane spectrum with a strong 1658 cm^?1 band characteristic of CC stretching in hydrocarbon side chains exhibiting cis conformation. This band is unaltered in intensity and position in H₂O and in ²H₂O thus obscuring amide I protein conformation. Of particular interest is the appearance of strong, resonantly enhanced bands at 1160 and 1527 cm^?1 attributable to membrane-associated carotenoids.     

Kinetic and Equilibrium Studies of (-)125Iodocyanopindolol Binding to β-Adrenoceptors on Human Lymphocytes: Evidence for the Existence of two Classes of Binding Sites

Abstract

Saturation experiments were performed on intact human peripheral mononuclear leucocytes (MNL) and MNL membranes with (-)¹²⁵Iodocyanopindolol (¹²⁵ICYP) over a large concentration range (1.5-600pmol/l). The corresponding Scatchard plots were curvilinear suggesting two saturable classes of binding sites: A high affinity binding site (B_max1=1000±400 sites/cell, K_d1= 2.1±0.9 pmol/l for intact MNL and B_max1=550±190 sites/cell, K_d1=4.1±0.9 pmol/l for MNL membranes)and a low affinity binding site (B_max2=9150±3590 binding sites/cell, K_d2=440±50 pmol/l for intact MNL and B_max2=11560±4690 sites/cell, K_d2=410±70 pmol/l for MNL membranes). Dissociation of (-)¹²⁵ICYP from MNL was biphasic consisting of a slow dissociating component (dissociation rate constant k_-1=(0.5±0.2)x10^?3 min^?1 for intact MNL and k_-1=(1.0±0.1)x10^?3min^?1 for MNL membranes) and a fast dissociating component (k_-2=(80±20)x10^?3min^?1 for intact MNL and k_-2=(60±10)x10^?3min^?1 for MNL membranes). In dissociation experiments started after equilibration with various (-)¹²⁵ICYP concentrations k_-1 and k_-2 were independent of the equilibrium concentration, whereas the percentual occupancy of the slow and the fast dissociating component varied and was similar to the estimated fractional occupancy of either binding site at the same (-)¹²⁵ICYP concentrations in saturation experiments. The association rate constant was in the same order of magnitude for both binding sites. These results suggest two independent classes of binding sites for (-)¹²⁵ICYP on MNL.     

Properties of α-bungarotoxin binding sites in foetal human brain

Maximum levels of binding of α-bungarotoxin to foetal human brain membranes were found to remain essentially constant at 30–50 fmol/mg protein (1.1–1.5 pmol/g wet weight in whole brain) between gestational ages of 10 and 24 weeks. Equilibrium binding of α-bungarotoxin to both membranes and to detergent extracts showed saturable specific binding to a single class of sites with K_d (app) values of 3.5 × 10^?9 M and 2.4 × 10^?9 M respectively. Association rate constants, determined from time courses of binding of α-bungarotoxin to membranes and detergent extracts, were 2.3 × 10⁵ M^?1 sec^?1 and 2.6 × 10⁵ M^?1 sec^?1 respectively. Dissociation of α-bungarotoxin from both membrane and detergent extracts showed a rapid initial rate with $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx 15 min which, in the case of the detergent extract, was followed by a slower dissociation accounting for the remaining 20% of the bound ligand. Competition studies with a number of cholinergic ligands indicated that the α-bungarotoxin-binding sites in foetal brain display a predominantly nicotinic profile.     

Superoxide anion permeability of phospholipid membranes and chloroplast thylakoids

The permeability of phospholipid membranes to the superoxide anion (O₂^?) was determined using soybean phospholipid vesicles containing FMN in the internal space. The efflux of O₂^? generated by the illumination of FMN was so slow that more than 90% of the radicals were spontaneously disproportionated within the vesicles before they could react with cytochrome c at the membrane exterior. The amount of diffused O₂^? was proportional to the intravesicular concentration of O₂^? over a range from 1 to 10 μm which was deduced from its disproportionation rate. The permeability coefficient of the phospholipid bilayer for O₂^? was estimated to be 2.1 × 10^?6 cm s^?1 at pH 7.3 and 25 ° C. Superoxide dismutase trapped inside vesicles was not reactive with extravesicular O₂^? unless Triton X-100 was added. O₂^? generated outside spinach chloroplast thylakoids did not interact with superoxide dismutase or cytochrome c which had been enclosed in the thylakoids. Thus, chloroplast thylakoids also showed little permeability to O₂^?.     

The membraneless bioelectrochemical reactor stimulates hydrogen fermentation by inhibiting methanogenic archaea

The membraneless bioelectrochemical reactor (Ml-BER) is useful for dark hydrogen fermentation. The effect of the electrochemical reaction on microorganisms in the Ml-BER was investigated using glucose as the substrate and compared with organisms in a membraneless non-bioelectrochemical reactor (Ml-NBER) and bioelectrochemical reactor (BER) with a proton exchange membrane. The potentials on the working electrode of the Ml-BER and BER with membrane were regulated to ?0.9 V (versus Ag/AgCl) to avoid water electrolysis with a carbon electrode. The Ml-BER showed suppressed methane production (19.8?±?9.1 mg-C·L^?1·day^?1) and increased hydrogen production (12.6?±?3.1 mg-H·L^?1·day^?1) at pH_out 6.2?±?0.1, and the major intermediate was butyrate (24.9?±?2.4 mM), suggesting efficient hydrogen fermentation. In contrast, the Ml-NBER showed high methane production (239.3?±?17.9 mg-C·L^?1·day^?1) and low hydrogen production (0.2?±?0.0 mg-H·L^?1·day^?1) at pH_out 6.3?±?0.1. In the cathodic chamber of the BER with membrane, methane production was high (276.3?±?20.4 mg-C·L^?1·day^?1) (pH_out, 7.2?±?0.1). In the anodic chamber of the BER with membrane (anode-BER), gas production was low because of high lactate production (43.6?±?1.7 mM) at pH_out 5.0?±?0.1. Methanogenic archaea were not detected in the Ml-BER and anode-BER. However, Methanosarcina sp. and Methanobacterium sp. were found in Ml-NBER. Prokaryotic copy numbers in the Ml-BER and Ml-NBER were similar, as were the bacterial community structures. Thus, the electrochemical reaction in the Ml-BER affected hydrogenotrophic and acetoclastic methanogens, but not the bacterial community.     

Semi‐permeable membrane retention of synovial fluid lubricants hyaluronan and proteoglycan 4 for a biomimetic bioreactor

Synovial fluid (SF) contains lubricant macromolecules, hyaluronan (HA), and proteoglycan 4 (PRG4). The synovium not only contributes lubricants to SF through secretion by synoviocyte lining cells, but also concentrates lubricants in SF due to its semi‐permeable nature. A membrane that recapitulates these synovium functions may be useful in a bioreactor system for generating a bioengineered fluid (BF) similar to native SF. The objectives were to analyze expanded polytetrafluoroethylene membranes with pore sizes of 50 nm, 90 nm, 170 nm, and 3 µm in terms of (1) HA and PRG4 secretion rates by adherent synoviocytes, and (2) the extent of HA and PRG4 retention with or without synoviocytes adherent on the membrane. Experiment 1: Synoviocytes were cultured on tissue culture (TC) plastic or membranes ± IL‐1β + TGF‐β1 + TNF‐α, a cytokine combination that stimulates lubricant synthesis. HA and PRG4 secretion rates were assessed by analysis of medium. Experiment 2: Bioreactors were fabricated to provide a BF compartment enclosed by membranes ± adherent synoviocytes, and an external compartment of nutrient fluid (NF). A solution with HA (1 mg/mL, MW ranging from 30 to 4,000 kDa) or PRG4 (50 µg/mL) was added to the BF compartment, and HA and PRG4 loss into the NF compartment after 2, 8, and 24 h was determined. Lubricant loss kinetics were analyzed to estimate membrane permeability. Experiment 1: Cytokine‐regulated HA and PRG4 secretion rates on membranes were comparable to those on TC plastic. Experiment 2: Transport of HA and PRG4 across membranes was lowest with 50 nm membranes and highest with 3 µm membranes, and transport of high MW HA was decreased by adherent synoviocytes (for 50 and 90 nm membranes). The permeability to HA mixtures for 50 nm membranes was ～20 × 10^?8 cm/s (? cells) and ～5 × 10^?8 cm/s (+ cells), for 90 nm membranes was ～35 × 10^?8 cm/s (? cells) and ～19 × 10^?8 cm/s (+ cells), for 170 nm membranes was ～74 × 10^?8 cm/s (± cells), and for 3 µm membranes was ～139 × 10^?8 cm/s (± cells). The permeability of 450 kDa HA was ～40× lower than that of 30 kDa HA for 50 nm membranes, but only ～2.5× lower for 3 µm membranes. The permeability of 4,000 kDa HA was ～250× lower than that of 30 kDa HA for 50 nm membranes, but only ～4× lower for 3 µm membranes. The permeability for PRG4 was ～4 × 10^?8 cm/s for 50 nm membranes, ～48 × 10^?8 cm/s for 90 nm membranes, ～144 × 10^?8 cm/s for 170 nm membranes, and ～336 × 10^?8 cm/s for 3 µm membranes. The associated loss across membranes after 24 h ranged from 3% to 92% for HA, and from 3% to 93% for PRG4. These results suggest that semi‐permeable membranes may be used in a bioreactor system to modulate lubricant retention in a bioengineered SF, and that synoviocytes adherent on the membranes may serve as both a lubricant source and a barrier for lubricant transport. Biotechnol. Bioeng. 2010; 106: 149–160. © 2009 Wiley Periodicals, Inc.     

Cationic dependency of high affinity prostaglandin F2α receptors in bovine corpus luteum cell membranes

The specific binding of [³H] Prostaglandin (PG) F₂α to bovine corpus luteum cell membranes prepared in homogenizing buffer containing either 1 mM EDTA (H-EDTA) or 1 mM Ca²⁺ (HCa²⁺) was examined. The membranes prepared in H-EDTA buffer bound less [³H] PGF₂α and had a single class of PGF₂α receptors with an apparent dissociation constant (Kd) of 2.7 × 10^?8M. The addition of Ca²⁺ to these membranes resulted in increased binding with the appearance of new PGF₂α receptors of Kd = 4.3 × 10^?9M. The membranes prepared in HCa²⁺ buffer contained two classes of receptors with Kds = 2.9 × 10^?9M and 2.9 × 10^?8M. The removal of Ca²⁺ from these membranes resulted in lower binding as well as a complete disappearance of receptors of Kd = 2.9 × 10^?9M. These results suggest the dependency of high affinity PGF₂α receptors, in bovine corpus luteum cell membranes, on cations.     

Characterization of specific binding sites for vasoactive intestinal peptide in rat intestinal epithelial cell membranes

Specific binding sites for vasoactive intestinal peptide were characterized in plasma membranes from rat intestinal epithelial cells. At 30°C, the interaction of ¹²⁵I-labelled peptide with intestinal membranes was rapid, reversible, specific and saturable. At equilibrium, the binding of ¹²⁵I-labelled peptide was competitively inhibited by native peptide in the 3 · 10^?11?3 · 10^?7 M range concentration. Scatchard analysis of binding data suggested the presence of two distinct classes of vasoactive intestinal peptide binding sites: a class with a high affinity K_d = 0.28 nM) and a low capacity (0.8 pmol peptide/mg membrane protein) and a class with a low affinity (K_d = 152 nM) and a high capacity (161 pmol peptide/mg membrane protein). Secretin competitively inhibited binding of ¹²⁵I-labelled peptide but its potency was 1/1000 that of native peptide. Glucagon and the gastric inhibitory peptide were ineffective. The guanine nucleotides, GTP and Gpp(NH)p inhibited markedly the interaction of ¹²⁵I-labelled peptide with its binding sites, by increasing the rate of dissociation of peptide bound to membranes. The other nucleotides triphosphate tested (ATP, ITP, UTP, CTP) were also effective in inhibiting binding of ¹²⁵I-labelled peptide to membranes but their potencies were 1/100-1/1000 that of guanine nucleotides.The specificity and affinity of the vasoactive intestinal peptide-binding sites in plasma membranes prepared from rat intestinal epithelial cells, which is in agreement with an adenylate cyclase highly sensitive to the peptide recently characterized in these membranes (Amiranoff, B., Laburthe, M., Dupont, C. and Rosselin, G. (1978) Biochim. Biophys. Acta 544, 474–481) further argue for a physiological role of the peptide in the regulation of intestinal epithelial function.