Reversibility of the thermal denaturation of yeast superoxide dismutase

Phenobarbital-inducible cytochrome P450 variants have a higher affinity (app. K_s = 0.6 μM) for metyrapone than do non-phenobarbital-inducible variants (app. K_s = 80 μM). The difference in affinity has enabled us to quantitate, by metyrapone-induced difference spectral analysis, phenobarbital-inducible cytochromes P450 in liver microsomal membranes. The result obtained by this simple, rapid technique compare well with those found by more sophisticated techniques such as radioimmunoassay and immunoprecipitation.     

Specificity of nucleotide binding sites in isolated chloroplast coupling factor (CF1)

The binding of various nucleotides to chloroplast coupling factor CF₁ was studied by two dialysis techniques. It was found that the number of nucleoside diphosphate sites and their specificities for the base moiety is dependent on the magnesium concentration. In the presence of 50 μM added MgCl₂, the protein has a single strong site/mol protein with K_d = 0.5 μM for ADP and high specificity (K_d > 20 μM for ?ADP, GDP, CDP). In the presence of 5 mM MgCl₂, the protein has two independent tight ADP sites (K_d = 0.4 μM) of low specificity (K_d ≈ 0.8, 2, and 2 μrmM, respectively for ?ADP, GDP, and CDP). These results are compared with the specificity of the partial reactions for photophosphorylation.     

Stereospecific,reversible binding of D-[3H] glucose to crude membrane fractions prepared from rat brain

To a crude preparation of synaptic membranes prepared from rat brain, stereospecific, saturable, reversible binding was described of D-[³H]glucose. Binding showed a K_d = 0.45 μM and the fractional rate of dissociation was approximately eight times the fractional rate of association. D-[³H]glucose binding was displaced by 2-deoxyglucose and 3-0-methylglucose and it was abolished when membranes were denatured by heating.     

Conversion of high affinity acetylcholine receptor from Torpedo californica electroplax to an altered form.

Most Triton extracts of fresh electroplax membranes showed a single kind of acetylcholine binding, of high affinity, with K_d = 17 nM. Fresh membrane preparations without Triton also showed only high-affinity binding (K_d = 14 nM), but membrane preparations from electroplax stored for 5 months in liquid nitrogen gave equal amounts of low-affinity binding material (K_d = 0.2 μM). Heating Triton extracts at 40 °C for 40 min or treating with 10^?4mp-chloromercuribenzoate or 5,5′-dithiobis(2-nitrobenzoic acid) converted the preparation to more than 90% low-affinity binding. It is suggested that the high-affinity is the native form and the low-affinity is an oxidation product.     

Inhibitors of two enzymes which metabolize cytokinins

Compounds which inhibit the natural metabolic inactivation of cytokinins are of considerable physiological significance. In this study, inhibitors have been found for two enzymes which form glucose and alanine conjugates of cytokinin bases, namely, cytokinin 7-glucosyltransferase and β-(9-cytokinin)alanine synthase. The most effective inhibitors found for the former enzyme were the cytokinin analogues 3-methyl-7-n-pentylaminopyrazolo[4,3-d]pyrimidine, which acted competitively (K_i, 22 μM), and the diaminopurine, 6-benzylamino-2-(2-hydroxyethylamino)-9-methylpurine (K_i, 3.3 μM). However these compounds were ineffective as inhibitors of the cytokinin-alanine synthase which was inhibited competitively by IAA (K_i 70 μM) and related compounds, especially 5,7-dichloro-IAA (K_i 0.4 μM). Certain urea derivatives were moderately effective inhibitors of the enzymes (K_ica 100μM).     

Characterization of the Electrogenic Sodium Channel from Rat Brain Membranes Using Neurotoxin-Dependent 22Na Uptake

The sodium channel was studied in osmotically-sensitive membrane preparations from rat brain and in innervated and chronically denervated rat soleus and extensor digitorum longus muscles. These experiments were undertaken in order to define a set of parameters for sodium channel function at the subcellular level to be used as a measure of retention of channel integrity upon subsequent isolation of the channel. Various neurotoxins and drugs were employed to control the permeability of the brain membranes to ²²Na and the sodium-conductance properties of the muscles. Batrachotoxin (ED₅₀ = 0.2 μM), veratridine (ED₅₀ = 1 μM), or grayanotoxin I (ED₅₀ = 30 μM) stimulated ²²Na uptake in brain membranes is inhibited in an apparently uncompetitive manner by the sodium channel blocking agents tetrodotoxin and saxitoxin in a simple competitive manner by Ca²⁺ and in a partial or allosteric competitive manner by lidocaine and procaine. This ²²Na uptake assay, which can be equated to a measure of equilibrium toxin binding, shows dependence on the concentration of the membranes and is sensitive to pH, temperature, ionic strength, and the ionic composition of the media. Parallel biophysical studies on sodium channels in rat muscle show that the properties of the sodium channel are similarly affected by these agents.     

Glucose transport and metabolism in Gymnodinium breve

Florida's red tide organism, Gymnodinium breve, utilized exogenous glucose in the light for the synthesis of cellular components. Glucose was not taken up in the dark. Kinetic parameters for glucose uptake include a K_FD of 11 μM and a V_max of 1 × 10^?10 mol of glucose taken up/mg cellular protein/hr. Glucose uptake was competitively inhibited by phloridzin (K_i = 40 μM), mannose (K_i = 12O μM), and 2-deoxy-d-glucose (K_i = 190 μM) and non-competitively inhibited by galactose (K_i = 125 μM). Kinetics and inhibition of glucose uptake are consistent with a facilitated diffusion transport system.     

Investigation of the subcellular distribution of cyclic-AMP phosphodiesterase in rat hepatocytes,using a rapid immunological procedure for the isolation of plasma membrane

The distribution of cyclic-AMP phosphodiesterase was investigated in subcellular fractions prepared from homogenates of rat liver or isolated hepatocytes. When measured at 1 mM or 1 μM substrate concentration, approx. 35% or 50%, respectively, of enzyme activity was particulate. The soluble activity appeared to be predominantly a ‘high K_m’ form, whereas the particulate activity had both ‘high K_m’ and ‘low K_m’ components. The recovery of cyclic-AMP phosphodiesterase was measured using 1 μM substrate concentration, in plasma membrane-containing fractions prepared either by centrifugation or by the use of specific immunoadsorbents. The recovery of phosphodiesterase was lower than that of marker enzymes for plasma membrane, and comparable with the recovery of markers for intracellular membranes. It was concluded that regulation of both ‘high K_m’ and ‘low K_m’ phosphodiesterase could potentially make a significant contribution to the control of cyclic AMP concentration, even at μM levels, in the liver. The ‘low K_m’ enzyme, for which activation by hormones has been previously described, appears to be located predominantly in intracellylar membranes in hepatocytes.The immunological procedure for membrane isolation allowed the rapid preparation of plasma membranes in high yield. Liver cells were incubated with rabbit anti-(rat erythrocyte) serum and homogenized. The antibody-coated membrane fragments were then extracted onto an immunoadsorbent consisiting of sheep anti-(rabbit IgG) immunoglobulin covalently bound to aminocellulose. Plasma membrane was obtained in approx. 40% yield within 50 min of homogenizing cells.     

A specific uncoupler-binding protein in Tetrahymena pyriformis and Paracoccus denitrificans

The uncoupler of mitochondrial oxidative phosphorylation, 2-nitro-4-azido-carbonylcyanide phenylhydrazone (N₃CCP) which is capable of photoaffinity labeling has been used to examine the effect of uncouplers on the energy conserving membranes of Paracoccus denitrificans and Tetrahymena pyriformis. The N₃CCP uncouples respiration in P. denitrificans and T. pyriformis cells with $\text{U}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values of 1.05 μM and 0.24 μM, respectively. Binding studies show the presence of 0.65 ± 0.05 high affinity sites per cytochrome a with a K_d of 0.5 ± 0.1 μM in P. denitrificans membranes and 1.4 ± 0.2 sites per cytochrome a₂ with a K_d of 0.4 ± 0.1 μM in T. pyriformis membranes. Irradiation of [³H]-N₃CCP bound to the membranes leads to a covalent linking of the radioactive uncoupler to a peptide of 10–15 kdaltons as analyzed by SDS-polyacrylamide gel electrophoresis. It is concluded that these two microbial systems contain a specific high affinity uncoupler binding site very similar to that of mammalian mitochondria (Katre, N.V. and Wilson, D.F. (1978) Arch. Biochem. Biophys. 191, 647–656).     

Guanine nucleotides modulate cell surface cAMP-binding sites in membranes from Dictyostelium discoideum

D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (K_d = 15 nM, $\text{t}\text{1}\text{2}\text{= 15 s}$). A second class is fast dissociating ($\text{t}\text{1}\text{2}$ about 1 s) and is composed of high affinity binding sites H (K_d ≈ 60 nM), and low affinity binding sites L (K_d = ≈ 450 nM) which interconvert during the binding reaction. Guanine nucleotides affect these three binding types in membranes prepared by shearing D.discoideum cells through Nucleopore filters. The affinity of S for cAMP is reduced by guanine nucleotides from 13 nM to 25 nM, and the number of S-sites is reduced about 50%. The number of fast dissociating sites is not altered by guanine nucleotides, but these sites are mainly in the low affinity state. Half-maximal effects are obtained at about 1 μM GTP, 2 μM GDP and 10 μM Gpp(NH)p(guanyl-5′-yl-imidodiphosphate); ATP and ADP are without effect up to 1 mM. These results indicate that D.discoideum cells have a functionally active guanine nucleotide binding protein involved in the transduction of extracellular cAMP signals via cell surface cAMP receptors.     

[3H]adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cell line and rat brain membranes

Adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cells and rat brain membranes were investigated using [³H]adenosine as labelled ligand. Both the hybrid cells and brain membranes were found to have a high affinity binding site, K_d 0.8 and 3 nM respectively. The same ligand was used to demonstrate two lower affinity binding sites on brain membranes, K_ds 1.4 and 29.1 μM and a single low affinity site on the hybrid cells, K_d 2.6 μM. Structure activity studies of the low affinity binding site on hybrid cells showed this to be an ‘R’ adenosine receptor of the A₂ subtype. It is concluded that [³H]adenosine can be used to demonstrate both high and low affinity binding sites and that 108CC15 hybrid cells provide a valuable system for studying adenosine receptors.     

Cooperative interaction of reduced pyridine nucleotides with estrogen synthetase of human placental microsomes

The estrogen synthetase present in human placental microsomes appears to be dependent on the cooperative interaction of the reduced cofactors NADPH and NADH for optimal activity. Using steady-state concentrations of either cofactor, it was found that while the estrogen synthetase activity followed hyperbolic saturation kinetics with NADPH (K_mapp = 14 μM), the enzyme followed sigmoidal saturation kinetics when the cofactor was NADH, with the half-maximum velocity attained at a cofactor concentration of 1.1 mm. The maximum velocity obtained with NADPH as the cofactor was greater than with corresponding concentrations of NADH. Estrogen synthetase activity in the presence of NADH was not due to NADPH contamination. NADH, in the presence of small concentrations of NADPH (0.5 to 5 μm), stimulated significantly the rate of estrogen formation from androstenedione by placental microsomes and, in addition, the enzyme saturation kinetics changed from sigmoidal to hyperbolic, thus mimicking the effect of NADPH. Estrogen synthetase activity, measured in the presence of 1 mm NADH, was stimulated in a dose-dependent manner by NADPH (K_mapp = 0.4 μM NADPH) and, when the enzyme was measured in the presence of 5 μm NADPH, the activity was stimulated in a dose-dependent manner by NADH (K_mapp = 45 μM NADH). Estrogen synthetase activity measured in the presence of NADH, without and with NADPH (1 μm) remained linear both with time of incubation for approximately 15 min and with microsomal protein concentration up to 3 mg/ml. The apparent K_m of estrogen synthetase for androstenedione, when measured in the presence of NADH, was 1 μm. The synergistic interaction between NADH and NADPH in stimulating placental estrogen synthetase activity observed in vitro may, conceivably, take place in vivo in the intact placenta.     

Characterization and solubilization of the cytochalasin B binding component from human placental microsomes

Human placental microsomes exhibit uptake of d-[³H]glucose which is sensitive to inhibition by cytochalasin B (apparent K_i = 0.78 /gm M). Characterization of [³H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by d-glucose with an ED₅₀ = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a K_d = 0.15μM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60–70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [³H]cytochalasin B to the soluble protein displays a pattern of inhibition by d-glucose similar to that observed for intact membranes, and the measurement of an ED₅₀ = 37.5 mM d-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [³H]cytochalasin B incorporation into soluble protein (M_r range 42 000-68 000) is prevented by the presence of d-glucose. An identical photolabelling pattern is observed for incorporation of [³H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions.     

Growth phase-dependent active transport of pyridoxine in a fission yeast, Schizosaccharomyces pombe

Schizosaccharomyces pombe showed maximum pyridoxine uptake activity around 10 h after starting cultivation. High concentrations of thiamine and pyridoxine in the medium did not affect the activity or the time but changed intracellular levels of vitamin B₆ compounds. Pyridoxine was taken up by a saturable mechanism with two kinds of affinity (K_m 22.4 μM and 118 μM). The uptake depended on the energy produced anaerobically with an optimum pH of 4.5. The uptake was completely inhibited by amiloride, sodium azide or 2,4-dinitrophenol. The uptake system of the fission yeast was different in various respects from that of a budding yeast.     

Sulphate/proton cotransport in plasma-membrane vesicles isolated from roots of Brassica napus L.: increased transport in membranes isolated from sulphur-starved plants

The characteristics of sulphate uptake into right-side-out plasma-membrane vesicles isolated from roots of Brassica napus L., Metzger, cv. Drakkar, and purified by aqueous polymer two-phase partitioning, were investigated. Sulphate uptake into the vesicles was driven by an artificially imposed pH gradient (acid outside), and could be observed for 5–10 min before a plateau was reached and no further net uptake occurred. The uptake was partially inhibited in the presence of depolarizing agents and little uptake was observed in the absence of an imposed pH gradient. Uptake was strongly pH-dependent, being greatest at more acidic pH. After imposition of a pH gradient, the capacity for uptake decreased slowly (t1/2>10 min). The uptake had a high-affinity component which was strongly dependent on the external proton concentration (K _m=10μM at pH 5.0, 64 μM at pH 6.5). The K _m for protons varied from 0.4–1.9 μM as the sulphate concentration was reduced from 33 to 1 μM. A low-affinity component was observed which could be resolved at low temperatures (0 °C). Microsomal membranes that partitioned into the lower phase of the two-phase system gave no indication of high-affinity sulphate transport. Sulphate uptake into plasma-membrane vesicles isolated from sulphur-starved plant material was approximately twofold greater than that observed in those isolated from sulphate-fed plant material. Isolated vesicles therefore mirror the well-known in-vivo response of roots, indicating an increase in the number of transporters to be, at least in part, the underlying cause of derepression.     

A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site

The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by Y_Z. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[−Mn]) membranes. The rate of Mn(II) oxidation saturates at ≥10 μM in PSII(−Mn) membranes and ≥500 μM in EDC-treated PSII (−Mn) samples. Intact PSII(−Mn) membranes have only one site for Mn(II) oxidation via Y_Z (dissociation constant, K_d = 0.64 μM), while EDC-treated PSII(−Mn) samples have two sites (K_d = 1.52 and 22 μM; the latter is the low-affinity site). When PSII(−Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (K_d = 2.3 μM) for exogenous Mn(II) oxidation by Y_Z radical. In this case, the rate of electron donation via Y_Z saturated at a Mn(II) concentration ≥15 μM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HA_Z site is protected from the action of the modifier by the iron cations blocking the HA_Z site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HA_Z site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site.     

4-Azidophlorizin,a high affinity probe and photoaffinity label for the glucose transporter in brush border membranes

A new phlorizin derivative ($\text{2′-O-(β-}\text{d}\text{-}\text{glucopyranosyl}\text{)-4-}\text{azidophloretin}$, 4-azidophlorizin) has been synthesized and its affinity for the d-glucose, Na⁺ co-transport system in brush border vesicles from intestinal and renal membranes has been compared with that of phlorizin. The extent of the reversible interaction of the ligand with the transporter in dim light has been evaluated from three separate measurements: (1) K′_i, the constant for fully-competitive inhibition of (Na⁺, Δψ)-dependent d-glucose uptake, (2) K′_d, the dissociation constant of 4-azido[³H]phlorizin binding in the presence of an NaSCN inward gradient, and (3) K″_i, the constant for fully-competitive inhibition of the specific ((Na⁺, Δψ)-dependent, d-glucose protectable) high-affinity [³H]phlorizin binding. In experiments with vesicles derived from rat kidney, all three constants (K′_i, K′_d and K″_i) were essentially equal and ranged between 3.2 and 5.2 μM, that is, the azide derivative has almost the same affinity for this transporter as phlorizin itself. On the other hand, compared to phlorizin, the 4-azidophlorizin has a lower affinity for the transporter in vesicles prepared from rabbit; its K′_i values are some 15–20-times larger than those determined with rat membranes. However, the affinity of the azide for the sugar transporter in membranes from either the intestine or kidney of the same animal species (rabbit or rat) was essentially the same. In spite of the lower affinity for the transporter in either membrane system from the rabbit, results described elsewhere (Hosang, M., Gibbs, E.M., Diedrich, D.F. and Semenza, G. (1981) FEBS Lett., 130, 244–248) indicate that 4-azidophlorizin is an effective photoaffinity label in this species also. Photolysis of the azide yields a reactive intermediate which reacts with a 72 kDa protein in rabbit intestine brush borders. Covalent labeling of this protein occurred under conditions which suggests that it is (a component of) the glucose transporter.     

L-Carnitine transport in mouse renal and intestinal brush-border and basolateral membrane vesicles

We characterized the uptake of carnitine in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, isolated from mouse kidney and intestine. In kidney, carnitine uptake was Na⁺-dependent, showed a definite overshoot and was saturable for both membranes, but for intestine, it was Na⁺-dependent only in BLM. The uptake was temperature-dependent in BLM of both kidney and intestine. The BBM transporter in kidney had a high affinity for carnitine: apparent K_m=18.7 μM; V_max=7.85 pmol/mg protein/s. In kidney BLM, similar characteristics were obtained: apparent K_m=11.5 μM and V_max=3.76 pmol/mg protein/s. The carnitine uptake by both membranes was not affected within the physiological pH 6.5-8.5. Tetraethylammonium, verapamil, valproate and pyrilamine significantly inhibited the carnitine uptake by BBM but not by BLM. By Western blot analysis, the OCTN2 (a Na⁺-dependent high-affinity carnitine transporter) was localized in the kidney BBM, and not in BLM. Strong OCTN2 expression was observed in kidney and skeletal muscle, with no expression in intestine in accordance with our functional study. We conclude that different polarized carnitine transporters exist in kidney BBM and BLM. L-Carnitine uptake by mouse renal BBM vesicles involves a carrier-mediated system that is Na⁺-dependent and is inhibited significantly by specific drugs. The BBM transporter is likely to be OCTN2 as indicated by a strong reactivity with the anti-OCTN2 polyclonal antibody.     

Characterization of uptake and release processes ford- andl-aspartate in primary cultures of astrocytes and cerebellar granule cells

The uptake ofl-andd-aspartate was studied in astrocytes cultured from prefrontal cortex and in granule cells cultured from cerebellum. A high affinity uptake system forl- andd-aspartate was found in both cell types, and the two stereoisomers exhibited essentially the sameK _m - andV _max -values in bouth astrocytes (l-aspartate:K _m 77 μM;V _max 11.8 nmol×min^?1×mg^?1;d-aspartate:K _m 83 μM;V _max 14.0 nmol×min^?1×mg^?1) and granule cells (l-aspartate:K _m 32 μM;V _max 2.8 nmol ×min^?1×mg^?1;d-aspartate:K _m 26 μM;V _max 3.0 nmol×min^?1×mg^?1). To investigate whetherl-glutamate,l-aspartate andd-aspartate use the same uptake system a detailed kenetic analysis was performed. The uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, and no essential differences between the uptake characteristics of the amino acids were found. In addition to the uptake studies the release ofD-aspartate from cerebellar granule cells was investigated and compared withl-glutamate release. A Ca²⁺-dependent, K⁺-induced release was found for both amino acids.     

Taurine uptake in synaptosomal fractions of rat cerebral cortex.

Abstract— [³⁵S]Taurine was found to be accumulated in synaptosomal fractions of rat cerebral cortex. Kinetic analysis in the range of 1–800 μm -[³⁵S]taurine revealed at least two different uptake processes. A high affinity uptake with a K_m of 20 μM and a low affinity uptake with a K_m of about 450 μM. The high affinity component was dependent on temperature and energy, and virtually abolished in the absence of sodium. Examination of the influence of structural analogues and putative transmitter substances indicates that the high affinity uptake of taurine into synaptosomal fractions of rat cerebral cortex is unique and highly specific. No specific actions of several centrally acting drugs on taurine uptake could be observed.