Dynamics of microparticles inside lipid vesicles: movement in confined spaces

Microparticles and nanoparticles used in drug delivery frequently depend on their movement in confined spaces such as cells. Liposomes containing small numbers of 1-µm diameter polystyrene particles were used to study the dynamics of their movement within the confined space of the liposome interior. The analysis of the trajectories of single and multiple entrapped particles revealed that the particles were largely localized toward the periphery of the liposome with a rare presence in the centre. Interparticle interactions were studied by calculating interparticle distances, ranging from close to zero to around 8 µm with a mean of ～4 µm. The diffusion coefficient of a single entrapped particle was D?=?0.27?×?10^?9 cm² s^?1 when compared with 5.1?×?10^?9 cm² s^?1 free in water. When more than one particle was entrapped, the calculated diffusion coefficients were D?=?0.61?×?10^?9 cm² s^?1 for two particles, D?=?1.26?×?10^?9 cm² s^?1 for three particles, and D?=?1.3?×?10^?9 cm² s^?1 for multiple particles). Particle movement was found to be distinctly faster at the periphery (average velocity 21.4 μm s^?1) than at the centre of the vesicle (average velocity 14.2 μm s^?1). These results demonstrate the significance of particle–particle interactions as well as particle–surface interactions, which is evident here in some systems by particle aggregation close to the liposome membrane.     

Stimulation of DNA synthesis and pyrimidine deoxyribonucleoside transport systems in mouse glioma and mouse neuroblastoma cells by inorganic mercury

Biochemical studies on effects of methylmercury and inorganic mercury on mouse glioma and neuroblastoma cells were carried out. Inorganic mercury accelerated DNA synthesis of mouse glioma cells at 10^?6 to 10^?5m specifically in the middle log phase of the growth. Inorganic mercury facilitated the thymidine and deoxycytidine transports within a narrow range of concentration (1 to 2 × 10^?5m), but no stimulation of transport was observed in the cases of purine deoxyribonucleosides. Inorganic mercury markedly inhibited both DNA synthesis and the transport systems of the precursors above 5 × 10^?5m. On the other hand, methylmercury showed hardly any accelerating effects and markedly inhibited both the synthesis of DNA and the precursors' transport above 5 × 10^?5m. In the case of mouse neuroblastoma cells, inorganic mercury stimulated DNA synthesis at 1 to 5 × 10^?6m and also thymidine transport system at 1 to 2 × 10^?5m.     

Interactions of an acidic cyclooctapeptide with metal ions: microcalorimetric and fluorescence analyses

Abstract: The metal‐ion binding preferences of an acidic amphipathic cyclopeptide, cyclo[d ‐Leu‐Leu‐d ‐Leu‐Trp‐(d ‐Glu‐Glu)₂] (CP), was studied by isothermal titration calorimetry and steady‐state fluorescence spectroscopy. CP adopted a partial beta structure, and variable temperature circular dichroism showed small secondary structural changes over the temperature range from 5 °C to 95 °C. The peptide did not bind alkali or alkaline earth metal ions but exhibited selectivity for some divalent transition metal ions (with association constants K_Cu2+ 4.5 × 10⁴ m ^?1, K_Zn2+ 1.6 × 10⁵ m ^?1, K_Cd2+ 1.3 × 10⁴ m ^?1, K_{Hg2+ (1)} 2.2 × 10⁶ m ^?1, and K_{Hg2+ (2)} 6.5 × 10³ m ^?1), for Pb²⁺ (2.0 × 10⁵ m ^?1), and a trivalent Group III metal, Al³⁺ (1.6 × 10⁵ m ^?1). The thermodynamic data show that the interaction between CP and these metal ions are spontaneous and entropically driven. A large range of binding enthalpies coupled with a smaller range of binding free energies of CP for these metal ions indicate an entropy–enthalpy compensation dependent on the ionic size of participating metal ions. The interaction of Pb²⁺, Hg²⁺, and Cu²⁺ with CP in aqueous solution specifically modulates the fluorescence emission properties of CP. The results of this study show that CP exhibits selectivity in metal‐ion binding, which is reflected in its fluorescence spectra. The observed trends can be useful for the design of heavy‐metal sensors based on fluorophore‐tagged acidic cyclopeptides.     

Interaction of Hydroflumethiazide and 2,4-Disulfamyl-5-trifluoromethylaniline with Cyclic AMP Phosphodiesterase and Carbonic Anhydrase

Abstract: The inhibitory effect of hydroflumethiazide (HFT) and its metabolite, 2,4-disulfamyl-5-trifluoro-methylaniline (DTA) on cyclic AMP phosphodiesterase and the binding of HFT and DTA to carbonic anhydrase was studied in vitro. Significant inhibition of rat kidney low-K_m cyclic AMP phosphodiesterase was observed with DTA concentration above 2.5 × 10^?4 mol/1 and with HFT concentration above 1 × 10^?4 mol/1. 50% inhibition was observed at a DTA concentration of 1 × 10^?3 mol/1. Binding of DTA and HFT to commercially obtained bovine erythrocyte carbonic anhydrase was demonstrated by equilibrium dialysis. Data were consistent with one class of binding sites. The product of n (number of binding sites) and K_ass (association constant) was 5 × 10⁵ M for DTA and 3.3 × 10⁴ M for HFT at 2°. In human blood in vitro at 37°, the equilibrium erythrocyte/plasma concentration ratio was 18 for DTA and 1.6 for HFT. It is concluded that HFT and DTA have approximately the same potency as cyclic AMP phosphodiesterase inhibitors, whereas DTA is more extensively bound by erythrocyte carbonic anhydrase.     

Anticoagulant, anti-aggregation and antithrombotic effects of a novel hexapeptide

Objectives Hexapeptide is a novel synthetic oligopeptide with a structure similar to that of eptifibatide. This study was designed to investigate the anticoagulant, anti‐aggregation, disaggregation and anti‐thrombogenesis effects of hexapeptide. Methods The effects of antiplatelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid (AA) and thrombin, and the effect of disaggregation of platelet aggregation induced by ADP were determined. The anticoagulation indexes were determined by different kits. Key findings Hexapeptide 1 × 10^?5–1 × 10^?4m could significantly prolong rabbit blood clotting time, thrombin time, prothrombin time and activated partial thromboplastin enzyme time, and reduce the length, wet weight, dry weight and the index of thrombus in a concentration‐dependent manner. Hexapeptide 1 × 10^?4m decreased platelet adhesion rate by 40.2%. The platelet aggregation inhibition of hexapeptide in dogs and humans was more obvious than in rabbits and rats. The aggregation inhibition rate of 1 × 10^?5m hexapeptide in dogs, rabbits, rats and humans induced by ADP was 93.9 ± 1.3%, 66.2 ± 1.4%, 76.1 ± 3.2% and 99.8 ± 0.2%, respectively; the 50% inhibitory concentration (IC50) of hexapeptide was 7.24 × 10^?8, 3.24 × 10^?6, 6.61 × 10^?6 and 8.91 × 10^?8m , respectively. For the aggregation inhibition rate of hexapeptide in dogs, rabbits and humans induced by AA, the IC50 was 1.29 × 10^?9, 1.32 × 10^?6 and 9.33 × 10^?8m , respectively; the IC50 of aggregation inhibition rates induced by thrombin was 2.88 × 10^?6, >1 × 10^?5 and 4.17 × 10^?6m , respectively. The disaggregation rate of 1 × 10^?4m hexapeptide in dog induced by ADP was 68.8 ± 7.4%. Conclusions Hexapeptide has anticoagulant, antiplatelet aggregation, disaggregation and antithrombotic effects in vitro.     

Angiotensin II binding to zona glomerulosa cells from rabbit adrenal glands.

Tritiated angiotensin II binds in a highly specific manner to zona glomerulosa cells prepared from the adrenal cortex of male rabbits. The reaction is a time-dependent process which obeys second-order kinetics (k₁ = 2.4 × 10⁵ M^?1 sec^?1) and reaches saturation in 5–7 min. Dissociation of the angiotensin II-cell complex is rapid (t_{1 2} = 100 sec) and obeys first-order kinetics for the first 3 min (k_?1 = 6.9 × 10^?3 sec^?1).Increased binding was observed with NaCl concentrations from 0 to 40 × 10^?3 M; however, concentrations from 40 to 140 × 10^?3 M decreased binding. Neither MgCl₂ nor Cll₂ at concentrations of 0 to 4 × 10^?3 M alter the binding of angiotensin II to zona glomerulosa cells. A significant decrease in binding was observed with increasing concentrations of KCl (0 to 140 × 10^?3 M).Temperature studies indicate that initially binding is more rapid at 37° (37° > 25° > 0°). However, binding of angiotensin II decreases after 3 min at 37° and after 7 min at 25°. Binding at 0° did not reach a plateau in the 15-min period studied.     

Serum albumin and lipoproteins as the quinidine binding molecules in normal human sera.

To substantiate the binding of quinidine in human sera and predict variations of binding dissociation constants and number of binding sites were determined for separate serum proteins. Human sera were fractionated by gel filtration and ultracentrifugation, and binding was evaluated by equilibrium dialysis at pH 7·30 at 20° and 37° in a Krebs-Ringer phosphate buffer. Quinidine was bound to all serum lipoproteins and to serum albumin. The binding was influenced by the buffer composition. In sodium phosphate buffer there were two separate binding sites for quinidine on LDL and HDL, while there was only one detectable binding site on VLDL and HDL in a Krebs-Ringer phosphate buffer. On LDL also there appeared to be one binding site but it exhibited a positive cooperative binding effect at lower concentrations of quinidine. This effect was assumed to be caused by inorganic ions of the Krebs-Ringer phosphate buffer. At a therapeutic level of quinidine in normal human serum the concentration of quinidine bound to serum proteins was 1·062 × 10^?5 M. Calculated from the evaluated binding parameters VLDL contributed with 0·101 × 10^?5 M of this binding, LDL with 0·143 × 10^?5 M, HDL with 0·083 × 10^?5 M and albumin with 0·699 × 10^?5 M.     

Interaction of 3′ -O-caffeoyl d-quinic acid with human serum albumin

The interaction of chlorogenic acid (CGA) with human serum albumin (HSA) was studied from the viewpoint of thermodynamics and mechanism of binding at pH 6.0. The association constants (K_a) for the HSA-CGA interaction at 10, 25 and 40° C were 6.0 × 10⁴, 9.0 × 10³ and 2 × 10⁴ M^?1, resulting in AG of -6.21, -5.80, -6.32 kcal/mol, respectively. These high K_a -values showed that the interaction between CGA and HSA is strong, endothermic and entropically driven. Binding of chlorogenic acid induces conformational change in HSA as indicated by quenching of fluorescence emission intensity along with a red shift in the emission maxima from 338 to 350 mm. This suggested the involvement of the lone tryptophan residue in the region of binding. Far-ultraviolet circular dichroic data showed a decrease in the α-helical content of HSA from 56 to 50% upon binding of CGA. These data are also supported by the decrease in the apparent Tm of HSA by 4°C upon binding of CGA causing destabilization of the HSA molecule. The kinetics of the interaction involves a single step in the binding, and the kinetic curve attains equilibrium within 180 ± 5 s. Data on caffeic acid (CA) and quinic acid (QA), which are the hydrolysis products of the bidentate CGA molecule, indicate that CA interacts more strongly than CGA. CA binds with an association constant of 8 × 10⁴ M^?1and with a maximum number of binding sites of four. Microcalorimetric investigation of the interaction of these ligands with HSA suggests that the strength of binding follows the order CA?CGA?>QA with a single class of binding sites. The effect of temperature on the binding of CGA to HSA showed that the interaction is dominated by hydrophobic forces and hydrogen bonding.     

A comparison of the binding constant (Kd) of 125I-labelled 3-(4-iodophenoxy)-1-isopropylamino-propan-2-ol obtained on β-adrenoceptors in guinea-pig myocardial membranes,with its dissociation constants (Kb) obtained on guinea-pig isolated atria and trachea

The dissociation constant of binding (K_D) of ¹²⁵I-labelled 3-(4-iodophenoxy)-1-isopropyl-aminopropan-2-ol (IIP) to guinea-pig myocardial membrane preparations was 2·2 × 10^?8m. In pharmacological experiments with the non-labelled material and 60 min contact time, IIP produced a parallel shift in the orciprenaline concentration-response line on guinea-pig isolated tracheal and atrial preparations. The dissociation constant (Kb ) of IIP was 2·9 × 10^?8m on atria (pA₂ 7·54) and 3·3 × 10^?8m on trachea (pA₂ 7·48). These values indicate that IIP is not a selective β-adrenoceptor blocking drug. In addition, agreement was found between the affinity constant of this antagonist for β-adrenoceptors as determined by a direct binding study and an indirect pharmacological study.     

The influence of cholinergic blockade on the uptake of alpha-aminoisobutyric acid by isolated human placental villi

Isolated villus from human term placenta contains about 167 nmol of acetylcholine (ACh) per gram of wet tissue and releases about 35 pmol of ACh/g/min when it is suspended in Krebs-Ringer bicarbonate buffer at 37°C. Chronic doses of nicotine, or smoking, which modify ACh output from human placental villus, are known to retard fetal intrauterine growth. Since one of the functions of placental villi is nutrient transport, and since it has not been possible to obtain ACh-free villi, the effects of cholinergic blockade by high concentrations of ACh (2 × 10^?3m), phospholine (5 × 10^?4m), nicotine (2.5 × 10^?3m), and atropine (10^?4 to 5 × 10^?4m) on active uptake of α-aminoisobutyric acid (AIB) were studied to explain their antigrowth effects. High concentrations of ACh and nicotine decreased the rate of uptake of AIB by 34 and 41%, respectively. Atropine inhibited the AIB uptake by 29 and 61% at concentrations of 10^?4 and 5 × 10^?4m, respectively. If all Ach were released from the syncytiotrophoblast, the concentration of ACh in the medium would be about 1.67 × 10^?5m. At the highest concentration of atropine used, the concentration of the active antagonist, d(?)S-hyoscyamine, in the medium is 2.5 × 10^?4m, which depressed AIB uptake by about 61%. Phospholine (an irreversible cholinesterase inhibitor) at 7 × 10^?6m increased AIB uptake by 20%, but it decreased AIB uptake at higher concentrations, with or without exogenous ACh. Mecamylamine (10^?4m) and d-tubocurarine (10^?4m) did not influence the AIB uptake. Among the pharmacological agents studied, nicotine increased the release of endogenous placental ACh, while atropine decreased ACh release. These observations indicate that endogenously released ACh exhibits a muscarinic effect on the placental villus and facilitates the uptake of amino acids. Blockade of the facilitating effects of ACh on amino acid uptake by placenta for long periods during pregnancy may result in a retardation of fetal growth.     

Cortisol effects on the glycosaminoglycan synthesis and molecular weight distribution in vitro

The effect of cortisol at different concentrations on the incorporation rate of [³H]glucosamine and [³⁵S]sulphate into glycosaminoglycans (GAGs) in human fibroblast culture medium was studied. The mol. wt distribution of the synthesized GAGs was determined by Sepharose 2B chromatography. Two sensitivity levels of GAGs to cortisol were observed: at a low cortisol concentration ( 1 × 10^?7 M) only the hyaluronic acid synthesis decreased and no changes were observed in the synthesis of sulphated GAGs or glycoproteins. At a high steroid concentration (1 × 10^?3 M) both the synthesis of hyaluronic acid and sulphated GAGs drastically decreased. The mol. wt distribution of medium GAGs did not change at cortisol concentrations 1 × 10^?9 M-1 × 10^?5 M. The possible role of cortisol in the metabolism of hyaluronic acid in vivo is discussed.     

In vitro studies on bee brain acetylcholinesterase-organophosphorus complex utilizing oxime drugs.

The oxime drugs pyridine-2-aldoxime methiodide (2-PAM) and diacetyl monoxime (DAM) were tested in vitro for their effect on the recovery of paraoxon- or malaoxon-inhibited acetylcholinesterase (AChE) from honey bee brain. Concentrations of 3.78 × 10^?10?3.78 × 10^?7m 2-PAM produced 0.9–100% activity recovery, and 9.9 × 10^?12?9.9 × 10^?7m DAM produced 11.8–95.3% activity recovery of paraoxon-inhibited bee brain AChE. Pyridine-2-aldoxime methiodide at concentrations of 3.78 × 10^?9?1.89 × 10^?6m also produced 0.7–88.5% activity recovery of malaoxon-inhibited bee brain AChE.     

In vitro hydroxylation of diphenylhydantoin and its inhibition by other commonly used anticonvulsant drugs

Using a crude 9,000 g rat liver musomal preparation, in vitro studies were carried out on (a) the metabolism (hydroxylation) of diphenylhydantoin (DPH), (b) the effect of other commonly used anticonvulsants on this hydroxylation. DPH hydroxylation exhibited saturation kinetics at a DPH substrate concentration of approximately 10^?4M. Mean K_m and V_m values for the reaction were 9.3 × 10^?5 M and 23.3 μg/m1 respectively. The linear Hill plot with an interaction coefficient of 1.0 suggests that there is no cooperativity between different DPH molecules during DPH receptor binding process. The anticonvulsants ethosuximide, sodium phenobarbitone, sodium valproate and sulthiame all exhibited inhibition of DPH hydroxylation to varying degrees. K_i, inhibition constants for the four anticonvulsants were respectively 1.1 × 10^?2, 9 × 10^?4, 1.8 × 10^?2 and 8.8 × 10^?4M. Inhibition of DPH hydroxylation by sodium phenobarbitone and sulthiame was strong and competitive in nature. Ethosuximide showed a weak competitive type of inhibition and sodium valproate a weak uncompetitive type of inhibition.     

Effects of edrophonium on end-plate currents in frog skeletal muscle

The effects of edrophonium on end-plate currents measured at frog neuromuscular junctions were assessed using the two microelectrode voltage clamp technique. Low concentrations of edrophonium (10^?5 M and 2.5 × 10^?5 M) increased the peak amplitude and the kinetic parameters of end-plate currents measured in curare-Ringer and in 5 mM Ca²⁺–10 mM Mg²⁺-Ringer solutions. At the highest concentration tested (6 × 10^?5 M), edrophonium decreased the peak amplitude and maximum rate of rise but had little effect on the time to peak. Time to half decay was the only parameter studied which continued to increase as edrophonium concentration was increased from 10^?5 M to 6 × 10^?5 M. Normalized values of the parameters for both 10^?5 M and 2.5 × 10^?5 M edrophonium revealed no significant difference between end plates bathed in curare-Ringer and those bathed in 5 mM Ca²⁺–10 mM Mg²⁺-Ringer.It is suggested that the low concentration of edrophonium inhibit the postjunctional acetylcholinesterase thereby causing an increase in transmitter concentration in the end-plate region, but that the highest concentration (6 × 10^?5 M) has a direct desensitizing action as well as the anticholinesterase action on the end-plate membrane. This desensitizing action is present although no depolarization of the end-plate membrane is observable.     

Conformational dynamics and kinetics of peptide antagonist interactions with interleukin-1 receptor. Fluorescence studies using the NBD-labelled peptide AF12415

Interleukin-1 plays a key role in the inflammatory response provoked by various disease states and inhibition of its action can bring therapeutic benefits. Steady-state and time-resolved studies of the intrinsic tryptophan fluorescence of the free soluble Type I form of interleukin-1 receptor (IL-1R) reveal that the rotational motions of the three major domains are strongly associated. Bound peptide antagonists are buried in hydrophobic regions, but a flexible association permits access to species from the aqueous phase. Ligand binding does not lead to rigidification of the receptor structure. The kinetics and mechanism of complex formation and dissociation, involving IL-1R with receptor antagonist protein (IL-1ra) and with peptides AF11733 (15 aa) and AF10961 (21 aa) were determined with the aid of peptide AF12415 (15 aa) labeled at its N-terminus by the NBD fluorophore, which exhibits a five-fold increase in emission intensity at 540 nm on binding of the peptide to IL-1R. The magnitude of the ON rate constant, typically 1 × 10⁶ M^?1 s^?1, implies the existence of an intermediate ‘encounter complex’ involving interactions of low specificity. Readjustments of the initial encounter complex leads to a final complex where very specific interactions dominate. The first-order rate constant for this latter process is the most sensitive indicator of the true peptide affinity for the receptor binding site, and thus provides a better criterion than the apparent OFF rate (typically 2 × 10^?3 s^?1) for discrimination of peptide antagonists. © Munksgaard 1997.     

THE USE OF THE RAT RIGHT VENTRICLE STRIP IN STUDIES ON NORADRENERGIC TRANSMISSION

• 1 The possibility of using contractility studies with the rat right ventricle strip to assess the effects of drugs on all aspects of noradrenergic transmission has been examined.
• 2 The force of the contractile responses to field stimulation at 0.5 and 2, but not 5 Hz, markedly decreased with time. About 55% of the tissues responded directly to (?)-isoprenaline, 1 × 10^?6M (123 tissues from 220 tested) in the absence of other stimuli. In these tissues the force of the contractile responses remained constant and the rate increased with time. Thus it is essential to provide time controls in studies with rat right ventricle.
• 3 Under conditions in which the responses to (?)-isoprenaline, 1 × 10^?6M, alone were unaffected the responses to field stimulation at 5 Hz were inhibited by 66 and 86% by 9 × 10^?6M tetrodotoxin and 1 × 10^?5M guanethidine, respectively. (+)-Propranolol and timolol (both at 1 × 10^?6M), but not phentolamine, 1 × 10^?6M, inhibited responses to (?)-isoprenaline, 1 × 10^?6M alone and responses to field stimulation at 5 Hz. This demonstrates that the responses to field stimulation are largely due to activation of noradrenergic nerves, the released noradrenaline acting at postsynaptic β-adrenoreceptors.
• 4 Although nortriptyline is a potent inhibitor of the neuronal uptake of noradrenaline, at 1 × 10^?6M it had no effect on the contractile responses to field stimulation at 5 Hz and inhibited responses to (?)-isoprenaline, 1 × 10^?6M, alone and at a higher concentration (1 × 10^?5M) nortriptyline abolished both responses. It is suggested that the rat right ventricle preparation may be useful in examining the effects of drugs on noradrenergic transmission in the heart.

     

Antilipolytic action of suloctidil in rat adipose tissue.

(1) Suloctidil (1-(4-isopropylthiophenyl)-2-n-octylamino-propanol) inhibited the in vitro lipolysis due to the action of isoproterenol, ACTH, and dibutyryl cAMP in rat epididymal adipose tissue fragments. Fifty per cent inhibition occurred at a 2 × 10^?4M suloctidil concentration. The drug was not affecting cAMP and ATP levels. (2) Suloctidil inhibited non-competitively the hormonally stimulated activity of a crude preparation of triglyceride lipase, with a K_iapp of 2.5 × 10^?4M. No effect was observed on lipoprotein lipase. (3) It is suggested that the antilipolytic action of the drug lies, at least partly, beyond the adenylate cyclase step, i.e. directly on the hormone sensitive triglyceride lipase. This effect is compared with that of nicotinic acid, clofibrate, procaine, and β-adrenergic blocking     

Concentration-dependent effects of fatty acids on warfarin binding to albumin

The interrelationships between fatty acid and warfarin binding to albumin were investigated. Various molar ratios of palmitic acid and oleic acid were added to defatted human albumin in the presence of warfarin, and the warfarin binding association constants, K_a, were calculated. Warfarin association constants increased from 0.84 × 10⁵ M ^?1 to 3.66 × 10⁵ M ^?1 as the oleic acid concentration increased from zero to three moles per mole of albumin and from 1.19 × 10⁵ M ^?1 to 3.13 × 10⁵ M ^?1 as the palmitic acid concentration increased from zero to two moles per mole of albumin. Larger amounts of either fatty acid progressively decreased the amount of warfarin bound in a noncompetitive fashion. In addition, two proteolytic fragments were utilized to define the location of the warfarin binding site on albumin. The warfarin site was located between loops 5 and 6 on the albumin molecule in close proximity to the secondary and tertiary binding sites of palmitic acid.     

Analysis of cytidine deaminase and tetrahydrouridine interaction by use of ligand techniques

Ligand techniques, as employed in radio-immunossay and radioreceptor assay, offer a sensitive and precise method for characterizing the interaction of enzymes and tight-binding inhibitors. Tetrahydrouridine (H₄U) inhibition of human liver cytidine deaminase (EC 3.5.4.5) has been examined by direct measurement of enzyme-inhibitor (EI) binding and release. With partially purified enzyme from human liver, the EI complex was found to have a dissociation constant (K_D) of 4.5 × 10^?8M at 37°, in close agreement with estimates based on inhibition of enzyme activity by H₄U. The presence of steady state conditions during competitive binding analysis was confirmed by direct measurement of the rate constants for EI binding at 25° and 37° (K^on 1.7 × 10⁴ and 5.6 x ×0⁴M^?1sec^?1 respectively). The rate constant for EI release at 37° was also determined experimentally (K^off = 4.0 × 10^?3sec^?1), and was in close agreement with the K^off value calculated from the experimentally determined K_D and K^on values (K_D = K^off/K^on). Scatchard analysis of H₄U-enzyme binding, both in the presence and in the absence of 10^?3M cytidine, showed no variation in total enzyme concentration (E_T) but a decrease in apparent inhibitor affinity for enzyme, suggesting that cytidine and H₄U compete for the same binding sites on cytidine deaminase, and confirming the competitive inhibition suggested by Lineweaver-Burk analysis. The turnover number for cytidine deaminase based on per H₄U binding sites was 3.9 × 10³ min^?1. Thermodynamic constants for cytidine deaminase-tetrahydrouridine binding were derived from data on the temperature dependence of binding and included an enthalpy change (ΔH) = ?12.7 kcal/mole, entropy change (ΔS) = ?8.68 cal/deg/mole and Gibbs free energy change (ΔG) = ?9.96 kcal/mole at 37.1°. This study indicates that ligand techniques can be applied to the difficult problem of characterizing the interaction of enzymes and tight-binding inhibitors.     

Actions of dopamine and apomorphine on the vasoconstrictor responses of perfused mesenteric arteries of mouse,rat and rabbit

Dopamine (10^?7-10^?6 m) and apomorphine (5 × 10^?7 ? 5 × 10^?6 m) inhibited the vasoconstrictor responses of the perfused mesenteric artery preparations of rat, rabbit and mouse to adrenergic nerve stimulation but did not affect responses to added noradrenaline. The inhibitory effects of both dopamine and apomorphine were prevented by haloperidol (3 × 10^?7 m) but not by yohimbine (3 × 10^?8 m) in rat and rabbit mesenteric artery preparations. In contrast, yohimbine (3 × 10^?8 m), but not haloperidol, antagonized the inhibitory effect of dopamine and apomorphine in mouse mesenteric artery preparations. In higher concentrations, dopamine (10^?6 ? 10^?4 m) produced a direct vascoconstrictor effect, which involved post-junctional α-adrenoceptors in all three species. However, in preparations contracted with 10^?7 m 5-hydroxytryptamine and in the presence of phentolamine (3 × 10^?7 m) and propranolol (10^?6 m), dopamine (10^?6 ? 10^?4 m) produced a direct relaxant effect in rabbit mesenteric artery preparations but not in those of rat and mouse. It is suggested that inhibition of neurogenic vasoconstrictor responses, by dopamine and apomorphine, may be mediated through a specific prejunctional inhibitory dopamine receptor in the mesenteric artery of rat and rabbit whereas in the mouse they involve activation of α-adrenoceptors.