Distribution of prostaglandin E2-sensitive adenylate cyclase along the rat nephron

To define sites of prostaglandin action of renal tubules, the distribution of adenylate cyclase sensitive to prostaglandin E₂ (PGE₂) was examined in single nephron segments dissected from rat kidney. Further, the interaction between PGE₂ and vasopressin on adenylate cyclase activity in nephron segments sensitive to vasopressin was evaluated. Procedures involved in isolating nephron segments were without effects on adenylate cyclase stimulation by PGE₂. PGE₂ stimulated adenylate cyclase activity of the thin descending limb of Henle (tDL), cortical collecting tubules (CCT), and medullary collecting tubules (MCT) at concentrations of 1.4 × 10^?5 to 2.8 × 10^?5 M. PGE₂ was without effects in other nephron segments tested including proximal convoluated tubules, proximal pars recta, the thin and thick ascending limb of Henle's loop, and distal and connecting tubules. PGE₂, at both high (2.8 × 10^?5 M) and low (2.8 × 10^?8 M) concentrations, did not inhibit adenylate cyclase activity stimulated by submaximal doses of vasopressin in medullary thick ascending limb of Henle (MTAL), CCT, and MCT. These data define the distribution of PGE₂-sensitive adenylate cyclase in the rat nephron, i.e., tDL, CCT, and MCT, and show the lack of direct inhibitory actions of PGE₂ on vasopressin sensitive adenylate cyclase in MTAL, CCT, and MCT.     

Inhibition by indomethacin and aspirin of 15-hydroxy-prostaglandin dehydrogenase in vitro

15-Hydroxyprostaglandin dehydrogenase from bovine lung was purified 7.4 times to a specific activity of 1.4 mU/mg of protein. The isoelectric point was estimated to 5.4 and the molecular weight by gelfiltration to 40,000. K_m for prostaglandin E₁ and for NAD⁺ were found to be 3.4 μM and 1.1 × 10^?4M respectively. The enzyme was inhibited by indomethacin and aspirin. The indomethacin inhibition was found to be non-competitive to prostaglandin E₁ having a K_i=1.4 × 10^?4M and a K_i^′=1.6 × 10^?5M.     

Effect of hormonal and neuronal agents on adenylate cyclase from smooth muscle of the gastric antrum

Smooth muscle adenylate cyclase of a membrane preparation of canine gastric antrum has been characterized, and the effect of hormonal and neuronal agents examined. The enzyme is active in the presence of Mg²⁺ or Mn²⁺, but is inhibited by Ca²⁺. The K_m is 0.5 mM ATP, similar to the K_m of skeletal muscle adenylate cyclase. The enzyme is activated by isoproterenol but not norepinephrine, consistent with a β₂-catecholamine receptor-adenylate cyclase interaction. Secretin activates the enzyme in concentrations as low as 1 · 10^?11 M, while glucagon was effective only at 1 · 10^?6 M. Prostaglandin E₁ and E₂ have a biphasic effect with activation of adenylate cyclase at 1 · 10^?5 M and a small but significant inhibition of enzyme activity at 1 · 10^?11 M.     

Kinetics of cyanide binding to chloroperoxidase in the presence of nitrate: detection of the influence of a heme-linked acid group by shift in the appa

The kinetics of the binding of cyanide to ferric chloroperoxidase have been studied at 25°C and ionic strength 0.11 M using a stopped-flow apparatus. The dissociation constant (K_CN) of the peroxidase-cyanide complex and both forward (k₊) and reverse (k_?) rate constants are independent of the H⁺ concentration over the pH range 2.7 to 7.1. The values obtained are k_cn = (9.5 ± 1.0) × 10^-5 M, k₊. = (5.2 ± 0.5) × 10⁴ M^?1 sec^?1 and k_- = (5.0± 1.4) sec^-1. In the presence of 0 06 M potassium nitrate the affinity of cyanide for chloroperoxidase decreases due to the inhibition of the forward reaction. The dissociation rate is not affected. The nitrate anion exerts its influence by binding to a protonated form of the enzyme, whereas the cyanide binds to the unprotonated form. Binding of nitrate results in an apparent shift towards higher pK_a values of the ionization of a crucial heme-linked acid group. Hence the influence of this group can be detected in the accessible pH range. Extrapolation to zero nitrate concentration yields a value of 3.1±0.3 for the pK_a of the heme-linked acid group.     

Noncompetitive Amine Uptake Inhibition by the New Antidepressant Pridefine

Abstract: Pridefine (AHR-1118) is a pyrrolidine derivative with clinically established antidepressant efficacy. Previous work from this laboratory indicates that pridefine is a reuptake blocker of catecholamines and serotonin with weak releasing activity. This study characterized the mode of amine uptake inhibition by pridefine as noncompetitive. The uptake experiments were performed utilizing ouabain instead of zero-degree controls to differentiate between the passive and active components of uptake. Furthermore, the passive component was resolved into diffusion and binding of substrate. Correction was made for the effects of ouabain on binding. Kinetic constants determined from Lineweaver-Burk plots were: K_m= 3 × 10^?7 M for NE, K_m= 9 × 10^?8 M for DA, and K_m= 3 × 10^?8 M for 5-HT. Dixon analyses of uptake at various pridefine concentrations indicated noncompetitive inhibition with K_i= 2.5 × 10^?6 M for NE uptake, K_i= 2.0 × 10^?6 M for DA uptake, and K_i= 1 × 10^?5 M for 5-HT uptake. These constants compare well with IC₅₀ values for the same transmitters: NE, IC₅₀= 2.4 × 10^?6 M; DA, IC₅₀= 2.8 × 10^?6 M; 5-HT, IC₅₀= 1.0 × 10^?5 M. The in vitro results indicate that pridefine is relatively specific as a catecholamine uptake blocker. It differs from tricyclic antidepressants which are reportedly competitive inhibitors of monoamine uptake. The possible mechanisms by which pridefine acts as a noncompetitive inhibitor are discussed.     

The periodic change in adenosine 3′,5′-monophosphate concentration in sea-urchin eggs

The level of adenosine 3′,5′-monophosphate (cyclic AMP) in the eggs of the sea urchin, Anthocidaris crassispina, was found to change periodically after fertilization. The minimum and maximum levels of cyclic AMP were 1.0·10^?7 M and 1.5·10^?6 M, respectively. The activity of adenylate cyclase in a 105 000 × g precipitate reached a plateau at 20 min after fertilization and stayed constant for at least 2 h. It was also found that 1.0 mM CaCl₂ increased the activity of adenylate cyclase in the same precipitate from unfertilized eggs. In contrast, phosphodiesterase activity changed periodically and correlated with cyclic AMP levels in the eggs. Up to a concentration of 1.5·10^?6 M cyclic AMP, phosphodiesterase activity was low, but it became activated when the level of cyclic AMP rose beyond this level. These results indicate that the change in the intracellular level of cyclic AMP is regulated mainly by the change in phosphodiesterase activity.     

Inhibition of guanylate cyclase and cyclic GMP phosphodiesterase by cholera toxin.

Results of cholera toxin exposure in rabbit small intestinal epithelial cells, following 4 to 6 hours of incubation, indicate that there is simultaneous dose-dependent activation of adenylate cyclase and deactivation of guanylate cyclase. In addition, cyclic GMP phosphodiesterase activity is repressed. These data indicate that cholera toxin interacts with a binding site of dissociation constant K_d=3.8±1.3 × 10^?9M to produce multiple coordinated events in the cells.     

Action of the adenosine triphosphate analog, adenylyl imidodiphosphate in mitochondria.

Adenylyl imidodiphosphate (AMP-PNP), and analog of adenosine triphosphate (ATP), is a potent competitive inhibitor of mitochondrial ATPase activity. It inhibits both the soluble oligomycin-insensitive ATPase (K_i = 9.2 × 10^?7 M) and the bound oligomycin-sensitive APTase (K_i = 1.3 × 10^?6 M). ATPase activity of inside-out submitochondrial preparations are more sensitive to AMP-PNP in the presence of an uncoupler (K_i = 2.0 × 10^?7 M). Mitochondrial ATP-dependent reactions (reversed electron transfer and potassium uptake) do not proceed if ATP is replaced with AMP-PNP; however, the analog does affect these systems. Oxidative phosphorylation of whole mitochondria and submitochondrial preparations were unaffected by AMP-PNP.     

Guanosine diphosphate binding,metabolism and regulation of follitropin-sensitive adenylate cyclase activity in Sertoli cell membranes

Nucleotides such as GTP and GDP appear to be involved in signal transduction via G protein modulation of adenylate cyclase activity. Studies on direct binding of [³H]GDP to membranes prepared from cultured immature rat Sertoli cells indicated that this process was reversible, approached steady state within 10 min, had a K_a of 4.5 ·10⁶M⁻¹ and was specific for guanine nucleotides. The non-hydrolyzable analog, guanosine 5′-O-[3-thio]triphosphate (GPPP[S]), was most effective as an inhibitor of [³H]GDP binding (ED₅₀ = 4.8·10⁻⁸M), whereas guanosine 5′-O-[2-thio]diphosphate (Gpp[S]) was less potent (ED₅₀ = 3.4·10⁻⁷M). Release of bound GDP was enhanced by follitropin (FSH) in the presence of Gppp[S], although not by FSH alone. Sertoli cell membranes possess guanine nucleotide hydrolase activity, where 95% of added nucleotide was rapidly degraded to guanosine. Binding kinetics were significantly influenced by nucleotide metabolism, which was prevented by controlling the Mg²⁺ concentration with EDTA and including App[NH]p to reduce nonspecific hydrolysis. Kinetic studies indicated that Gpp[S] inhibited (P < 0.05) Gppp[S]-stimulated adenylate cyclase activity (K_i = 1.8·10⁻⁷M), whereas basal activity remained unaffected. Addition of Gpp[S] to pre-activated enzyme (FSH plus GTP) resulted in a time-dependent decay of adenylate cyclase activity with a K_off value of 6 ± 1·min⁻¹. Using a two-stage pre-inculbation technique, adenylate cyclase activity was demonstrated to be sensitive to the nucleotide bound. When FSH was included, catalytic activity was not altered by the order of pre-incubation with the nucleotides. This suggested that the exchange of bound Gpp[S] for Gppp[S] was enhance by FSH. Activation and attenuation of FSH-sensitive adenylate cyclase activity is dependent on a nucleotide exchange mechanism which is driven by (1) the higher affinity of G for GTP than GDP, (2) enhanced release of GD when FSH is present and (3) GTP hydrolysis coupled to rapid metabolism of guanine nucleotides.     

Chalcone cyclase and flavonoid biosynthesis in grapefruit

A chalcone cyclase (CC), which acts unidirectionally upon the chalcone-flavanone equilibrium reaction, was isolated from immature grapefruit. The enzyme required neohesperidose at C-4′ of the chalcone A-ring and a free, unhindered hydroxyl group at C-4 of the B-ring for activity. The CC bound, but did not cyclize, prunin chalcone (K_i= 2.5 × 10^?5 M). The results suggest that the intermediates that form the B-ring of chalcones are hydroxylated prior to chalcone formation, that chalcones are glycosylated during their formation, and that methylation occurs after cyclization of the chalcones to flavanones.     

ONTOGENETIC DEVELOPMENT OF NEOSTRIATAL DOPAMINE RECEPTORS IN THE RAT 1

Abstract— The actions of dopamine and apomorphine on the kinetic properties of striatal adenylate cyclase were investigated during ontogenesis in the rat. The maximum stimulatory effect of dopamine (5 × 10^?5 M) was constant from birth to maturity (1 to 60 days of age). In contrast, the stimulatory effect elicited by apomorphine (5 × 10^?5 M) was almost the same as that of dopamine in 6-day-old rats, but it declined during maturation reaching 50% of the initial value at 60 days of age. The apparent K_m value for dopamine did not change during development, while the K_m for apomorphine was higher in the adult than in the newborn. Apomorphine appeared to have a greater affinity than dopamine for the striatal adenylate cyclase both in adult and newborn rats.     

Competitive substrate inhibition of amyloglucosidase from Rhizopus sp. by vanillin and curcumin

Kinetic studies of two glucosylation reactions catalyzed by an amyloglucosidase from Rhizopus sp. leading to the synthesis of vanillin-α/β-D-glucoside from D-glucose and vanillin and curcumin-bis-α-D-glucoside from D-glucose and curcumin were investigated in detail. Initial reaction rates were determined from kinetic runs involving different concentrations of D-glucose and vanillin (5?mM to 0.1?M) or D-glucose and curcumin (5?mM to 0.1?M). Graphical double reciprocal plots showed that the kinetics of the two enzyme catalyzed reactions exhibited Ping-Pong Bi-Bi mechanism where competitive substrate inhibition by vanillin/curcumin led to dead-end amyloglucosidase–vanillin/curcumin complexes at higher concentrations of vanillin/curcumin. An attempt to obtain the best fit of this kinetic model through computer simulation yielded in good approximation, the values of four important kinetic parameters, vanillin-α/β-D-glucoside: k_cat=35.0±3.2 10^?5M?h^?1·mg, K_i=10.5±1.1?mM, K_mD-glucose=60.0±6.2?mM, K_mvanillin=50.0±4.8?mM; curcumin-bis-α-D-glucoside: k_cat=6.07±0.58 10^?5M?h^?1·mg, K_i=3.0±0.28?mM, K_mD-glucose=10.0±0.9?mM, K_mcurcumin=4.6±0.5?mM.     

Catecholamine and guanine nucleotide activation of skeletal muscle adenylate cyclase

Activation of adenylate cyclase by guanine nucleotide and catecholamines was examined in plasma membranes prepared from rabbit skeletal muscle. The GTP analog, 5′-guanylyl imidodiphosphate caused a time and temperature-dependent activation of the enzyme which was persistent, the K_a was 0.05 μM. 5′-Guanylyl imidodiphosphate binding to the membranes was time and temperature dependent, K_D 0.07 μM. Beta adrenergic amines accelerated the rate of 5′-guanylyl imidodiphosphate activation of the enzyme with an order of potency isoproterenol ≈ soterenol ≈ salbutamol > epinephrine ? norepinephrine. Catecholamine activation was antagonized by propranolol and the β₂ antagonist butoxamine; the β₁ antagonist practolol was inactive. [³H]Dihydroalprenolol bound to the membranes and binding was antagonized by β adrenergic agonists with an order of potency similar to the activation of adenylate cyclase and was antagonized by butoxamine but not by practolol. The data are consistent with the idea that adenylate cyclase in skeletal muscle plasma membranes is coupled to adrenergic receptors of the β₂ type.     

In vitro cyclic AMP-mediated lipolytic activity of endorphins, enkephalins and naloxone

The maximum lipolytic activity (L_max) of β-endorphin is two and one half times that of Leu⁵-enkephalin and twice that of Met⁵-enkephalin, D-Ala², Met⁵-enkephalinamide, α-endorphin and γ-endorphin in the rabbit adipocyte. D-Met², Pro⁵-enkephalin-amide, however, has an L_max 1.6 times greater than that of Met⁵-enkephalin. The potencies (A₅₀) of Met⁵-enkephalin and its analogs and that of Leu⁵-enkephalin lie between 1.4 and 3 μM. The A₅₀ values for α-endorphin, β-endorphin and γ-endorphin are significantly less (1.2 × 10^?1 μM). Naloxone acts as an agonist in this system (A₅₀ = 2.5 μM; L_max 1.4 × Met⁵-enkephalin). All of the peptides and naloxone stimulated adenylate cyclase activity.     

Kinetics of formation of ferrioxamine B

Stopped-flow kinetic studies of the formation of ferrioxamine B were performed. Formation of the complex follows the rate law

where K_a is the acid dissociation constant of the iron(III) aquo species in 0.1 M formate buffer. At 25°C k₁ = 3.94 × 10²M^?1 sec^?1, k₂K_a = 1.18 × 10^?1 sec^?1, k₃ = 3.6 × 10^?1 sec^?1. Activation parameters for k₁ are ΔH^≠ = 11.7 kcal mole^?1 and ΔS^≠ = ?8 cal K^?1 mole^?1. An associative mechanism is proposed. Attachment of the first chelate ring is the slow step and favorably positions the second chelate ring for attachment. Coordination of two chelate rings favorably positions the third chelate ring for attachment. These results are compared to kinetics of formation of model complexes and to a previous study of the formation of ferrioxamine B in which attachment of the third chelate ring was proposed as the slow step     

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (K_D = 4.7 ± 1.0 × 10^?10 and 1.6 ± 0.3 × 10^?10M, respectively; B_max = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ET_A receptor, potently competed for ¹²⁵I-ET-1 binding to sections of the microvessels with IC₅₀ values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10^?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC₅₀ = 5.7 ± 2.5 × 10^?10 and 1.4 ± 0.2 × 10^?6M for tumor microvessels, 1.8 ± 0.6 × 10^?10 and 1.1 ± 0.3 × 10^?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ET_A subtype.     

Chymotrypsin inhibitor from potatoes: interaction with target enzymes

The interactions of chymotrypsin, subtilisin and trypsin with a low MW proteinase inhibitor from potatoes were investigated. The K_i value calculated for the binding of inhibitor to chymotrypsin was 1.6 ± 0.9 × 10^?10M, while the second-order rate constant for association was 6 × 10⁵ M^?1/sec. Although binding was not observed to chymotrypsin which had been treated with diisopropyl fluorophosphate or with l-tosylamide-2-phenylethyl chloromethyl ketone, the 3-methylhistidine-57 derivative bound inhibitor with a K_i value of 9.6 × 10^?9 M. The inhibitor also exhibited a tight association with subtilisin (K_i < 4 × 10^?9 M). In contrast, little inhibition of trypsin was observed, and this was believed to be due to low levels of a contaminant in our preparations. No evidence for reactive site cleavage was observed after incubation of the inhibitor with catalytic amounts of chymotrypsin or subtilisin at acid pH.     

Nitrofuran induced mutagenesis and error prone repair in Escherichia coli

The binding of cis(c)- and trans(t)-Pt(NH₃)₂Cl₂ to DNA at platinum/DNA-nucleotide ratios (R_i) of 0.1 or less has been studied by means of radioactive ^195mPt-labeled compounds. Kinetic data are consistent with the following scheme:

At 25°C and pH 5–6 in 5 mM NaClO₄, the values for the rate constants in the above scheme for the c-isomer are k₂ = 2.2 × 10^?5 sec^?1, k₇ = 0.32 (sec M)^?1, and k₈ = 143 (sec M)^?1; for the t-isomer the values are k₂ < 0.5 × 10^?5 sec^?1 and k₇ = 0.95 (sec M)^?1. Platinum-DNA adducts do not undergo detectable exchange after 3 days at 37°C, indicating the absence of a dynamic equillibrium. For both isomers the rate of binding is the same for single- and double-stranded DNA. The conclusions derived from Ag⁺ and H⁺ titration studies are consistent with binding at guanine N(7) for R_i < 0.1. The reaction rate is competitively inhibited by various salts and buffers and is suppressed by raising the pH (50% inhibition of initial rates at pH 7.3). At 37°C and pH 7 in 0.15 M NaCl, 6–8% of both the c- and t-isomers bind to DNA in 24 h, suggesting that both compounds should bind to DNA under biological conditions.     

The beta-adrenergic receptor in human lymphocytes: subclassification by the use of a new radio-ligand, (+/-)-125 Iodocyanopindolol

(±)?¹²⁵Iodocyanopindolol (ICYP), a new radio-ligand with high affinity and specificity to β-adrenoceptors was used to identify and characterized β-adrenergic receptors in human lymphocytes. Binding of ICYP was saturable with 1.56 ± 0.2 fmol ICYP specifically bound/10⁶ cells at maximal occupancy of the sites and of high affinity (K_D=57 ± 7.1pM, N=4. In contrast to ¹²⁵Iodohydroxybenzylpindolol ICYP-binding was not affected by phentolamine (up to 10^?4M) or serotin (up to 10^?5M). Analysis of inhibition of ICYP-binding via a pseudo-Scatchard-plot (“Hofstee-plot”) by β_1-selective (practocol, metaprolol) and β_2-selective (IPS 339, zinterol) adrenergic drugs resulted in linear plots suggesting the existence of a homogeneous population of β-adrenergic receptorsin human lymphocytes. From the resulting K_D-values for practolol (16.8 μM), metoprolol (4.11 μM), zinterol (0.08 μM) and IPS 339 (0.002 μM) is concluded that the β-adrenergic receptor present in human lymphocytes is of the β₂-subtype. According to its low non-specific binding and its high specificity to β-adrenergic receptors ICYP appears to be an ideal ligand for long-term studies on the regulation of β-adrenergic receptors of human lymphocytes.     

Genetic Recombination — Understanding the Mechanism : by H.L.K. Whitehouse Wiley; Chichester,New York, 1982

Interaction of peanut agglutinin with MeUmbβGalβ(1→3)GalNAc was followed with the stopped-flow technique. The mechanism is a simple bimolecular association with k₊ = 7.1 × 10³ M^?1. s^?1 and k_? = 0.24 s^?1 at 25°C. The very slow dissociation rate of the complex strongly supports earlier conclusions that the combining site of peanut agglutinin is complementary to the Galβ(1→3)GalNac structure.