Peptidoleukotriene binding in guinea pig uterine membrane preparations

Peptidoleukotrienes are known to be potent smooth muscle contractile agents in many tissues, including guinea pig uterus. In order to characterize the receptors at which the leukotrienes interact, guinea pig uteri were homogenized in 50nM Tris-HCl, pH 7.4 at 40°C and centrifuged at 1000xg fpr 10 min. The supernatant was centrifuged at 40,000 xg and the washed pellet was used to measure the binding of ³H-LTC₄ and ³H-LTD₄. Specific binding of ³H-LTD₄ was not detected, but specific, saturable binding of ³H-LTC₄ was measured at 40°C, was complete in 10 min. and was rapidly reversible on addition of unlabeled LTC₄. Binding was linear with protein concentration and stimulated by CaCl₂ and L-serine borate. Scatchard and kinetic analysis of binding in the presence of calcium suggested a K_d of 10–12 nM. LTC₄ was a more potent competitor of binding than LTD₄ (IC₅₀ − 40nM and 30 μM, respectively). FPL 55712 inhibited binding from 10–100 μM but stimulated binding at lower concentrations. Thus, the guinea pig uterus has specific receptors for LTC₄, but not LTD₄, that can be demonstrated by radioligand binding.     

Assay and characteristics of the iron binding moiety of reticulocyte endocytic vesicles

Summary A⁵⁹Fe assay was designed to detect an Fe(III) binding capacity in NP-40 solubilized proteins from rabbit reticulocyte endocytic vesicles. The iron binding capacity had an apparent molecular weight as determined by gel exclusion chromatography of 450,000 daltons. The iron binding moiety coincided with the major nontransferrin iron-containing material of endocytic vesicles labeled in vivo by incubation of cells with⁵⁹Fe,¹²⁵I-labeled transferrin. The material solubilized from vesicles with NP-40 exhibited two classes of saturable binding sites, one with an association constant for⁵⁹Fe-citrate of 3.63×10⁹ m ^–1 and with 6.6×10^–12 moles of iron bound per mg protein and the other with a constant of 3.96×10⁸ m ^–1 and 1.0×10^–12 moles of iron bound per mg protein. These affinities are sufficient to satisfy the sobulility characteristics of Fe(III) at pH 5.0. Most of the⁵⁹Fe bound both in vivo and in vitro to the iron binding moiety could be displaced with⁵⁶Fe and an equivalent amount of⁵⁹Fe could subsequently be rebound in vitro. The iron binding assay was adopted to vesicle proteins separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to nitrocellulose and revealed an iron binding activity of molecular weight approximately 95,000 daltons.     

[125I]calmodulin binding to synaptic plasma membrane from rat brain: Kinetic and arrhenius analysis

Binding of [¹²⁵I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the Ca²⁺-dependent membrane binding of [¹²⁵I]calmodulin was found to have a B_max of 284 pmol/mg protein and an apparent affinity with a K_d of 131 nM. Kinetic analysis indicates that at 37°C, the dissociation of [¹²⁵I]calmodulinmembrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7×10^–1 min^–1 and a half-time (t_1/2) of 1.8 min for the fast component, and a k of 4.8×10^–2 min^–1 and a t_1/2 of 14.5 min for the slow component. At 0°C, substantial dissociation still occurred, with a k of 4.5×10^–2 min^–1 and a t_1/2 of 15.3 min for the fast component, and a k of 5.5×10^–3 min^–1 and a t_1/2 of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associated and dissociate calmodulin. In Arrhenius analysis of temperature effects, [¹²⁵I]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (T_d) estimated to be 23.8°C, suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [¹²⁵I]calmodulin to the synaptic membrane was found to be increased by corticosterone (10^–7–10^–6 M), a steroid hormone, and decreased by ethanol (50–200 mM), a centrally acting drug. Our data on the characteristics of calmodulin binding to the SPM provide groundwork for future studies on physiological and pharmacological regulation of calmodulin translocation to and from the plasma membrane in synaptic terminals.Abbreviations used CaM calmodulin - SPM synaptic plasma membrane - ATPase adenosine triphosphatase - Tris tris(hydroxymethyl)aminomethane - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - SDS sodium dodecyl sulfate - TFP trifluoperazine - K_d dissociation constant - B_max maximum binding - k first-order rate constant - t_1/2 half-time - T_d transition temperature     

Ca binding to the human red cell membrane: characterization of membrane preparations and binding sites.

Inside out and right side out vesicles were used to study the sidedness of Ca binding to the human red cell membrane. It was shown that these vesicles exhibited only a limited permeability to Ca, enabling the independent characterization of Ca binding to the extracellular and cytoplasmic membrane surfaces...     

Calcium at the surface of cardiac plasma membrane vesicles: Cation binding,surface charge screening,and Na−Ca exchange

Summary Calcium binding and Na–Ca exchange activity were measured in isolated cardiac plasma membrane vesicles under various ionic conditions. A model was developed to describe the Ca binding characteristics of cardiac sarcolemmal vesicles using the Gouy-Chapman theory of the diffuse double layer with specific cation binding to phospholipid carboxyl and phosphate groups. The surface association constants used for Ca, Na, K and H binding to both of these groups were 7, 0.63, 0.3 and 3800m ^–1, respectively. This model allows the estimation of surface [Ca] under any specific ionic conditions. The effects of the divalent screening cation, dimethonium, on Ca binding and Na–Ca exchange were compared. Dimethonium had no significant effect on Ca binding at high ionic strength (150mm KCl), but strongly depressed Ca binding at low ionic strength. Dimethonium had no significant effect on Na–Ca exchange (Na-inside dependent Ca influx) at either high or low ionic strength. These results suggest that the Ca sites of the Na–Ca exchanger are in a physical environment where they are either not exposed to or not sensitive to surface [Ca].     

The interaction between wheat germ agglutinin and membrane incorporated glycophorin A. An optical binding study

A novel integrated optical technique is used to monitor the kinetics of incorporation of glycophorin A (GPA) from solution into a planar dimyristoylphosphatidylcholine-cholesterol bilayer membrane, and the subsequent binding of wheat germ agglutinin (WGA) to the membrane-incorporated GPA. The technique significantly improves the attainable accuracy of kinetic measurements. The number of bound molecules can be determined to a precision of ca ± 80 mol µm^–2. Our results show that GPA incorporates spontaneously into the bilayer. Binding of WGA to GPA is optimal in the presence of human serum albumin, and can be reversed byN-acetyl-d-glucosamine. The kinetics of the binding are consistent with the presence of two classes of kinetically distinguishable binding sites with association rates of 2.0×10⁴ and 9.6×10² M^–1 s^–1, and dissociation rates of 2.7×10^–3 s^–1 and <10^–5 s^–1, respectively. A stoichiometry of 4 WGA monomers per GPA monomer was determined as characteristic of the overall binding interaction.Abbreviations DMPC dimyristoylphosphatidylcholine - GlcNAc N-acetyl-d-glucosamine - GPA glycophorin A - HSA human serum albumin - NeuNAc N-acetyl-d-neuraminic acid - TE transverse electric - TM transverse magnetic - WGA wheat germ agglutinin     

Surface charge and calcium binding in sarcoplasmic reticulum membranes

Vesicles of fragmented sarcoplasmic reticulum membranes have been adsorbed on to 2.68 latex spheres. Observation of these vesicle containing spheres in the presence of an electric field allows a calculation of the electrophoretic mobility of the vesicles Following this determination, the net membrane surface charge has been estimated. The mobility of sarcoplasmic reticulum membranes exhibited a dependency on pH. At an ionic strength of 0.10 a mobility (pH=7.0) of –0.67±0.10/sec/volt/cm was observed. At pH=7.0 and /2=0.150 the net excess negative charge density was 2.0×10^–2 coul/m². This is equivalent to one charge per 10³ A² (assuming a uniform charge distribution). With an average vesicle volume of 2.8×10⁸ A³ and a surface area of 2×10⁶ A² the surface of one vesicle would contain a net of approximately 2×10³ negative charges. While the mobility did not change during uptake of calcium by the vesicles, both glutaraldehyde fixation and lecithin extraction by phospholipase C greatly altered the mobility of the vesicle membrane. Calcium binding and uptake both exhibited a dependence on pH.     

Opioid receptor binding in rat spinal cord

The interaction of various radioligands with spinal opioid receptors has been characterized under variable experimental conditions. Binding to , , and sites was measured in all (cervical, thoracic, lumbar) segments. The apparent affinity constant (K) of [³H]Ethylketocyclazocine (EKC) was similar in Tris, 2.09 (±1.06)×10⁸ M^–1, and phosphate buffer, 2.16 (±0.02)×10⁸ M^–1, when its interaction with and sites was blocked. Without blocking ligands, EKC binding was resolved in two components:K ₁=1.01 (±0.21)×10⁹ M^–1 andK ₂=0.95 (±0.61)×10⁷ M^–1. Likewise, the binding of [D-Ala², MePhe⁴, Gly(ol)⁵]enkephalin (DAGO) or [D-Ala², D-Leu⁵]-enkephalin (DADLE) alone was represented by a 2-site model. By adjusting the radioligand and receptor concentration or by the addition of blocking ligands, binding was represented by a 1-site model for DAGO,K=4.35 (±1.41)×10⁸ M^–1, and DADLE,K=2.44 (±0.08)×10⁸ M^–1.The abbreviations used are DADLE [D-Ala², D-Leu⁵]enkephalin - DAGO [D-Ala², MePhe⁴, Gly(ol)⁵]enkephalin - EKC ethylketocyclazocine - DYN dynorphin (1–17)     

High affinity DNA-microtubule associated protein interaction

We have isolated the MAP/tau proteins from twice-cycled chick brain microtubule preparations and demonstrated that they are responsible for the nitrocellulose DNA binding activity we and others have measured. Using the isolated MAP/tau proteins we then measured the apparent affinity constant K_app for the homologous chick DNA interaction and found evidence for two equilibrium affinity classes-a K_app = 6 × 10⁷ M^–1, responsible for the bulk of the DNA binding activity and a small (< 10%) higher affinity K_app = 10⁸ – 10⁹ M^–1, likely due to sequence specific binding protein species. Using the same chick brain MAP-tau protein, a heterologous interaction with D. melanogaster DNA, was found to possess just the lower affinity class-K_app = 2 × 10⁷ M^–1. Under stringent binding conditions we carried out equilibrium nitrocellulose filter binding experiments in a ternary reaction mixture at constant MAP/tau protein and ³⁵S radiolabelled chick DNA concentration using increasing and excess concentrations of competitor DNAs of different sources. The order of competitor strengths found was-chick DNA > mouse DNA > D. melanogaster = E. coli. DNA. These data and specifically the homologous DNA: protein case being the strongest competitor corroborate our previous studies using total microtubule protein and provide new evidence for a conserved interaction of a small DNA sequence class with MAP/tau protein species. Moreover, these data allow us to conclude that the conserved DNA sequence: MAP/tau protein interactions do not critically depend upon any energetic feature co-involving tubulin for their properties since tubulin is absent from these preparations.     

Water and salt permeability of gastric vesicles

Summary Volume-dependent changes in light scatter have been shown to be a linear function of the osmotic gradient imposed upon gastric vesicles purified from hog gastric mucosa. Observation of the light scattered 90° to incident, using the Durrum stop flow system D-110, indicates that the vesicles exposed to hypertonic medium undergo rapid shrinkage due to water loss from the vesicle interior. The rate constant for this water movement is 1.1±0.09 sec^–1 (n=10) and is linearly dependent on temperature between 16 and 36°C. The activation energy of 13.93±0.60 kcal mole^–1 (n=3), calculated from an Arrhenius plot, is inconsistent with water movement facilitated by a large-pore aqueous channel. A slower reswell phase, dependent on solute entry into the intravesicular space, follows the water-dependent shrink phase. KCl entry, studied because of the intravesicular requirement for active K⁺/H⁺ transport, exhibits two entry stages. The faster, described by a single exponential imposed upon a constantly sloping background, has a rate constant of 7.75±0.48×10^–3 sec^–1 (n=15). The slower phase, which typically accounts for 90% of the reswell process, demonstrates a rate constant of 1.94±0.23×10^–4 sec^–1 (n=15). In the presence of valinomycin or nigericin, two fast rate constants and one slow rate constant of swelling are observed. The rate constant of the faster reswell phase is increased from 7.75±0.48×10^–3 sec^–1 (n=15) to 15.74±3.7×10^–3 sec^–1 (n=5) and 17.23±3.4×10^–3 (n=3) by the addition of nigericin (1 g ml^–1) and valinomycin (4.5 m), respectively. The second part of the faster reswell phase is approximately that seen in the control population. Transport-dependent volume changes of significant magnitude can be demonstrated following the addition of ATP to vesicles equilibrated with 150mm KCl. The volume change is a function of HCl leak rate and is abolished by ionophores which eliminate the transport-dependent pH gradient. So ₄ ^–- substitution, which eliminates the overshoot phenomena observed in KCl medium, also eliminates the shrinkage resulting from ATP addition.     

Partial purification and characterization of guanine aminohydrolase fromtrypanosoma cruzi

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) catalyzes the hydrolytic deamination of guanine to xanthine. A rapid procedure for the partial purification of guanine deaminase fromTrypanosoma cruzi using granulated bed electrofocusing was developed. Supernatants of cell sonicates (40,000 g) were subjected to electrofocusing with a broad range ampholyte (pH 4–9). Sections of the gel were eluted and assayed for xanthine production. Active fractions were pooled, concentrated, and again subjected to electrofocusing with a pH 5–7 range ampholyte. This procedure resulted in over 240-fold purification. The compounds 4-amino-5-imidazolecarboxamide andN ⁶-methyladenine were found to be potent competitive inhibitors of the enzyme. Their respective K_i values were 3.5×10^–6 M and 9.5×10^–6 M. Irreversible inactivation of the enzyme was observed upon incubation withp-chloromercurophenylsulfonic acid andN-ethyl-maleamide at 5.0×10^–4 M. The enzyme was labile to heat; a substantial loss of activity occurred upon incubation at 55°C for 5 min. A broad pH range of activity (pH 7.5–8.5) was observed in Tris, citrate, and phosphate buffers.     

Glomerular binding of angiotensin II in the rainbow trout,Salmo gairdneri

Summary Isolated glomeruli of the rainbow trout have been exposed in vitro to¹²⁵I-angiotensin II (0.88 × 10^–9 M) and binding sites located by light-microscopic autoradiography. These studies provide evidence of specific binding of angiotensin II by glomeruli. Binding was significantly inhibited by excess (10^–5 M) unlabelled angiotensin II, but a high degree of non-specific binding also occurred. The mammalian competitive antagonist, saralasin (3 × 10^–7 M) did not influence¹²⁵I-angiotensin II binding to fish glomeruli. Intense binding of¹²⁵I-angiotensin II was noted at the vascular pole of some glomeruli.     

Effect of tris(hydroxymethyl)aminomethane on isolated sarcoplasmic reticulum vesicles.

The preincubation of isolated sarcoplasmic reticulum vesicles in Tris-Cl (pH 7.3) increases their (Ca²⁺ + Mg²⁺)-dependent adenosine triphosphatase activity and decreases their ATP-dependent Ca uptake capacity. These effects of Tris are dependent on the preincubation time and the Tris concentration; they are maximal below 10 μm Ca and decrease upon the increase of Ca concentration in the preincubation media, and they increase upon the increase of the preincubation pH. Differences in ATPase activity between preincubated and control vesicles are abolished by A23187 but not by carbonyl cyanide p-trifluoromethoxy phenyl hydrazone. The results suggest that: (i) Preincubation of the vesicles in Tris causes an increase of their permeability for Ca, or a membrane damage. (ii) Tris must diffuse within the vesicles to promote these effects. (iii) Ca prevents these effects by decreasing the membrane permeability for Tris. The basic findings were reproduced replacing Tris by imidazole.     

Dependence of sodium-calcium exchange current through the membrane of salivary gland cells ofChironomus on pH of the extracellular solution

The dependence of sodium-calcium exchange current (I _Na(Ca)) through the membrane of isolated secretory cells ofChironomus larva on pH of the extracellular solution was studied with the voltage-clamp technique with intracellular perfusion.I _Na(Ca) evoked by hyperpolarization of the membrane from –20 to –60 mV was recorded within physiological values of Na⁺ and Ca²⁺ gradients. It was established that acidification of extracellular solution from pH 7.2 to 4.0 gradually decreased the amplitude ofI _Na(Ca) with pK' — 3.72. In all cases at pH 3.0 an outward current of considerable amplitude emerged in response to membrane hyperpolarization. The reversal of the current occurred at pH around 3.25. A decrease inI _Na(Ca) was due to protonation of acid ionogenic groups (quite possibly, of the residues of aspartic or glutamic amino acids), which had been involved in binding of cations. Alkalization of extracellular solution from pH 7.2 to 10.0 produced a gradual increase in theI _Na(Ca) amplitude; pK' was in the pH range between 9 and 10. The increase inI _Na(Ca) in alkaline medium was probably due to the appearance of negatively charged cations at binding sites, which could be carried by deprotonated thiosulfate groups of cysteine residues. This was indicated by the possibility of initial decrease inI _Na(Ca) under the action of Hg²⁺ ions.Neirofiziologiya/Neurophysiology, Vol. 28, No. 4/5, pp. 193–196, July–October, 1996.     

Surface charges on the outer side of mollusc neuron membrane

Summary The shifts of current-voltage characteristics of sodium and calcium inward currents produced by changes in the concentration of divalent cations (Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺) and in pH of the extracellular solution have been measured on isolated neurons of the molluscHelix pomatia intracellularly perfused with potassium-free solutions. On the basis of these shifts and using Stern's theory (O. Stern, 1924.Z. Electrochem. 30508–516), the binding constants for the ions to charged groups of the outer side of the somatic membrane and the density of the surface charges produced by these groups have been calculated. For groups located in the vicinity of sodium channels we obtainedK _Ca=90±10,K _Sr=60±10,K _Ba=25±5 andK _Mg=16±5m ^–1 at pH=7.7 and for groups located in the vicinity of calcium channelsK _Ca=67±10,K _Sr=20±5 andK _Ba=19±5m ^–1 at pH=7.0. The same groups bind H⁺ ions with apparent pK=6.2±0.2 that corresponds toK _H=1.6×10⁶ m ^–1. The density of fixed charges near the sodium channels is 0.17±0.05 e/nm² (pH=7.7) and near the calcium channels is 0.23±0.05 electrons/nm² (pH=7.0). From the comparison of the obtained values with the data about binding constants of the same ions to different negatively charged phospholipids, a suggestion is made that just the phophatidylserine is responsible for the surface potential of the outer side of the somatic membrane. It was also shown that the presence of this potential results in a change in the concentration of carrier ions near the membrane which affects the maximal values of the corresponding transmembrane currents.     

A circadian rhythm of the luciferin binding protein fromGonyaulax polyedra

Summary It has previously been shown that a protein extracted fromGonyaulax polyedra strongly and specifically binds luciferin, the substrate of the bioluminescent reaction. This binding is markedly dependent on pH with tight binding at pH 8.0 and almost no binding at pH 6.5, as measured by two independent methods. A procedure for the determination of the dissociation constant (K_d) of the luciferin binding protein (LBP) is presented, and K_d is estimated to be7×10^–9 M at pH 8.0, assuming an overall quantum yield of 0.1 for the bioluminescent reaction. With cells grown in a 12 h light — 12 h dark cycle, 5 to 10 times more LBP activity can be extracted from dark phase cells than from light phase cells. This rhythm persists in a circadian fashion in cultures maintained in constant dim light.Supported in part by a grant from the National Institutes of Health to J.W.H. (GM 19536)     

Varietal differences in potassium uptake by excised roots ofNicotiana tabacum

Summary The rate of potassium uptake by two varieties of burley tobacco (Nicotiana tabacum) differed by as much as two-fold when excised roots were absorbing K from solutions of 2.5 × 10^–4 M KCl. With increasing substrate levels of KCl the difference between varieties decreased and no difference was observed at a KCl concentration of 1.6 × 10^–2 M. The K-absorption by excised roots of several varieties from solutions of 5 × 10^–4 M KCl was compared.Contribution of the Department of Agronomy, Kentucky Agr. Exp. Sta., Lexington, and published with the approval of the Experiment Station Director.     

Permeability parameters of the toad isolatedstratum corneum

Summary A technique for isolating thestratum corneum from the subjacent layers of the epithelium was developed which permits studying thestratum corneum as an isolated membrane mounted between half-chambers. The method basically consists of an osmotic shock induced by immersing a piece of skin in distilled water at 50°C for 2 min. When the membrane is bathed on each surface by NaCl-Ringer's solution, its electrical resistance is 14.1±1.3 cm² (n=10). This value is about 1/100 of the whole skin resistance in the presence of the same solution. The hydraulic filtration coefficient (L _p ) measured by a hydrostatic pressure method, with identical solutions on each side of the membrane, is 8.8×10^–5±1.5×10^–5 cm sec^–1 atm^–1 (n=10) in distilled water and 9.2×10^–5±1.4×10^–5 cm sec^–1 atm^–1 (n=10) in NaCl-Ringer's solution. These values are not statistically different and are within the range of 1/80 to 1/120 of the whole skinL _p . Thestratum corneum shows an amphoteric character when studied by KCl diffusion potentials at different pH's. The membrane presents an isoelectric pH of 4.6±0.3 (n=10). Above the isoelectric pH the potassium transport number is higher than the chloride transport number; below it, the reverse situation is valid. Divalent cations (Ca⁺⁺ or Cu⁺⁺) reduce membrane ionic discrimination when the membrane is negatively charged and are ineffective when the membrane fixed charges are protonated at low pH.     

Purple membrane vesicles: Morphology and proton translocation

Summary Purple membrane vesicles prepared by different techniques differ widely in their morphology and ability to establish a proton gradient in the light. The procedures used to prepare active vesicles do not completely dissociate the purple membrane and thus preserve a preferential orientation of the protein, while most of the lipid is exchanged for added lipid. Responses to illumination are largely determined by the size of the vesicles and the degree to which bacteriorhodopsin is preferentially oriented. Any attempt to compare the interaction of different lipids with bacteriorhodopsin by measuring the pH response must take these factors into account.With an improved technique we have obtained vesicles of rather uniform size and bacteriorhodopsin orientation, which accumulate protons with an initial rate of 160 ng H⁺ sec^–1 mg^–1 protein at light intensities of 10⁶ erg cm^–2 sec^–1. The kinetics of the process are complex and at present insufficiently understood.     

CL−-Fluxe,Akkumulation und Transport in abgeschnittenen Mais-Wurzeln

Summary Fluxes, accumulation and transport rates of Cl^- in excised corn roots were investigated.Flux equilibrium in 5×10^-4M KCl was not reached within an experimental period of 28 hr.Transport of Cl^- (5×10^-4M KCl) through the xylem of 4–6 cm long excised corn roots had a lag of 1–2 hr. From 6 to 28 hr rates of accumulation and transport were nearly constant (5×10^-4M KCl). The velocity of volume-flow within the xylem was 1.5–2 cm hr^-1 (5×10^-4M KCl). ³⁶Cl^--efflux through the cut end of roots preloaded in K³⁶Cl of low concentration exhibits a discontinuity which is explained by addition of two fluxes: efflux of ions concentrated in the xylem (and symplasm) plus efflux from the vacuoles through the xylem.Accumulation of Cl^- by excised roots approaches a maximum level (Jackson and Edwards, 1966). Influx rates remain constant while efflux rates increase with time. It is shown in this paper that the flux of Cl^- through the xylem becomes a large proportion of the influx across the plasmalemma. Flux rates suggest that more than 50% of the Cl^- ions transported to the xylem passed through a vacuole (5×10^-4M KCl; 20–28 hr).