Cerebral microvessels contain a beta 2-adrenergic receptor

Cerebral microvessels isolated from cat forebrain contain a specific β-adrenergic-sensitive adenylate cyclase. Among various compounds tested, the most potent activator of enzyme activity is isoproterenol (k_a = 1.4 × 10^?7M), followed in order by epinephrine (k_a= 1.5 × 10^?6M), norepinephrine (k_a= 1.4 × 10^?5M) and phenylephrine (k_a> 3 × 10^?4M). Isoproterenol-stimulated enzyme activity is blocked by propranolol (k_i= 2.4 × 10^?9M, IPS 339 (k_i= 4 × 10^?9M), H35/25 (k_i = 1.2 × 10^?7M), atenolol (k_i= 5.9 × 10^?6M) and practolol (k_i= 1.8 × 10^?5M). These agonist and antagonist properties are quite similar to those demonstrated by β₂-adrenergic receptors and β₂-stimulated adenylate cyclase present in other tissues and indicate that the majority of adenylate cyclase-associated adrenergic receptors in cerebral microvessels are β₂. The findings are relevant to physiological studies of cerebral blood flow and vascular permeability.     

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.     

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.     

Effect of indomethacin on cyclic AMP phosphodiesterase activity in myometrium from pregnant rhesus monkeys

Our results indicate that indomethacin inhibits cyclic AMP phosphodiesterase in the myometrium of the pregnant rhesus monkey under in vitro as well as in vivo conditions. Kinetic data on extracts of myometrium from pregnant rhesus monkeys indicated two cyclic AMP phosphodiesterase activities. The apparent K_m value for the high affinity enzyme averaged 3.9 μM and for the low affinity enzyme 23 μM; the V_max values averaged 0.56 and 1.4 nmoles cyclic AMP hydrolized per mg protein min^?1 respectively. When indomethacin was added to the myometrial extracts, the activity of the high K_m phosphodiesterase was competitively inhibited, with an average K_i of 200 μM; the low K_m enzyme was noncompetitively inhibited with an average K_i of 110 μM. Experiments on myometrial slices demonstrated that 10 μM indomethacin potentiated the effect of PGE₁ and epinephrine on cyclic AMP levels, presumably by inhibiting the phosphodiesterase activity. The uterine relaxing effect of indomethacin is generally attributed to the inhibition of prostaglandin synthetase activity. However, treatment of pregnant rhesus monkeys with therapeutic doses of indomethacin resulted in a significant inhibition of myometrial cyclic AMP phosphodiesterase activity in association with uterine relaxation and prolongation of gestation.     

Selective inhibition of serotonin uptake by trazodone,a new antidepressant agent

The inhibitory effect of trazodone, a non tricyclic antidepressant, on 5-HT and catecholamine uptake into the synaptosomal preparation from the rat brain was compared with that of chlorimipramine. The inhibition of 5-HT uptake by trazodone is competitive with a K_i of 1.6 × 10^?6 M. Trazodone inhibits ³H-5-HT, ³H-NE and ³H-DA uptake with an IC₅₀ of 1.4 × 10^?6, 3.1 × 10^?4 and 5.2 × 10^?4 M, respectively. Therefore trazodone is 220 and 370 times more potent in inhibiting 5-HT than NE and DA uptake, respectively. The respective IC₅₀ values of chlorimipramine were 0.9 × 10^?7, 3.6 × 10^?6 and 4.0 × 10^?6 M for ³H-5-HT, ³H-NE and ³H-DA.     

Lunularic acid decarboxylase from the liverwort Conocephalum conicum

Some properties of a preparation of an enzyme, lunularic acid decarboxylase, from the liverwort Conocephalum conicum are described. The enzyme is normally bound and could be solubilized with Triton X-100; at least some of the bound decarboxylase activity appears to be associated with chloroplasts. For lunularic acid the enzyme has K_m 8.7 × 10^?5 M (pH 7.8 and 30°). Some substrate analogues have been tested but no other substrate was found. Pinosylvic acid is a competitive inhibitor for the enzyme, K_i 1.2 × 10^?4 M (pH 7.8 and 30°). No product inhibition was observed. Lunularic acid decarboxylase activity has also been observed with a cell-free system from Lunularia cruciata.     

Purification and properties of agmatine iminohydrolase from groundnut cotyledons

Agmatine iminohydrolase was purified ca 375-fold from groundnut cotyledons. The enzyme exhibited an optimum pH between 5.5 and 8.5 and the energy of activation was 22 kcal/mol. The K_m for agmatine was (7.57 ± 0.77) × 10^?4 M. The enzyme was inhibited by tryptamine, putrescine, cadaverine, spermidine and spermine. Inhibition by cadaverine and spermidine was competitive. The K_i values for cadaverine and spermidine were 4.1 × 10^?3 and 7.5 × 10^?4 M, respectively.     

Effect of some Cardiac and Respiratory Drugs on Succinate-Cytochrome c Reductase

Succinate-cytochrome c reductase was inhibited in vitro and in vivo by phenobarbitone, aminophylline and neostigmine using both 2,6-dichlorophenolindophenol (DCIP) and cytochrome c (cyt c) as substrates. The enzyme was also activated by gallamine towards both substrates. In vitro, phenobarbitone and aminophylline inhibited the enzyme with respect to the reduction of DCIP and cyt c in a non-competitive manner with K_i values of 1.5 × 10^?5 and 5.7 × 10^?5 M, respectively. Moreover, neostigmine competitively inhibited the enzyme towards both substrates with K_i values of 1.36 × 10^?5 and 1.50 × 10^?5 M, respectively.     

Kinetic properties of rat hepatic prolactin receptors

Binding of ¹²⁵I-labelled ovine prolactin to female rat liver membranes underequilibrium conditions showed an apparent K_d of 200 pM, and a Hill coefficient of 1.0. The association rate was second order, with a rate constant K₁, of 2.1 × 10⁷, 1.4 × 10⁷, 1.2 × 10⁷ and 4 × 10⁶ M^?1. min^?1 at 37, 30, 24 and 4° respectively. At 24° there were two components to the dissociation; a faster phase with K_?1=1.26 × 10^?2. min^?1 ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=55}\text{minutes}$) and a slower phase with K_?1=1.103 × 10^?3. min^?1. The apparent K_d (from K_?1K₁) was 1.05 nM for the faster phase and 87.5 pM for the slower phase. These data suggest that there is a conformational change following hormone binding which results in an increased receptor affinity, which effectively prevents release of bound hormone.     

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.     

Histrionicotoxin and alkylguanidine interactions with the solubilized and membrane-bound muscarinic acetylcholine receptor from porcine atria

The interaction of alkylguanidines and decahydrohistrionicotoxin with the membrane-bound and solubilized muscarinic acetylcholine receptor (mAcChR) from porcine atria was described. Alkylguanidines with alkyl chain lengths from one to ten carbons displaced l-[³H]quinuclidinyl benzilate (l-[³H]QNB) competitively from a single class of sites for the membrane-bound mAcChR. From a plot of ?ln K_i versus alkyl carbon chain number, a value of ?(473 ± 30) cal/mol was estimated as the energetic contribution per methylene group to the total binding energy. The binding of alkylguanidines to the digitonin/cholate solubilized mAcChR was complex in nature resulting in titration curves that did not obey the law of mass action for simple competitive inhibition at higher alkyl carbon numbers and a sigmoidal plot of ?ln K_i versus carbon number. Decahydrohistrionicotoxin bound in a competitive manner versus l-[³H]QNB to both the membrane-bound (K_i = (6.9 ± 1.4) × 10^?6 M) and the solubilized (K_i = (1.5 ± 0.3) × 10^?5 M) preparations.     

Phosphate transport in Ehrlich ascites tumor cells and the effect of arsenate

The effect of extracellular Pi and arsenate on Pi-transport in Ehrlich ascites tumor cells has been studied. Pi-transport can be described by Michaelis-Menten kinetics; the maximal flux equal to 44 mmoles (kg cell water)^?1 hour^?1 and K_m equal to 3.3 × 10^?4 M . Arsenate is a competitive inhibitor of Pi-transport with an inhibition constant (K_i) equal to 2.41 × 10^?3 M . The data support the hypothesis that cellular Pi is regulated by the cell membrane through the mediation of a carrier system.     

Titration of Photophosphorylation, Oxidative Phosphorylation and Glycolysis in Scenedesmus with Desaspidin: Regulation between Pathways and Sites for Photophosphorylation

Narrow concentration intervals were used, covering 10^?6– 10^?4M desaspidin. The interaction with glycolysis involves three steps, the inhibitor constants (K_i:s) being in turn 2.7 × 10^?5M, 1.3 × 10^?4M, and high. About 18% of total glycolysis is inhibited in each of the two first steps, and 65% left for the third reaction. After compensation for glycolysis, oxidative phosphorylation may show a sudden jump to about 10% inhibition at 1.5 × 10^?5M desaspidin, the possible K_i of the reaction starting here being very high. Correcting for glycolysis, desaspidin affects total Photophosphorylation in two steps, with the K_i values of 7.8 × 10^?5M and 4.6 × 10^?4M respectively. Inhibition in the first step is about 27% of the total photophosphorylation. By applying 10^?6M DCMU[/3-(3, 4-dichlorophenyl)-l, l-dimethy lurea], one can abolish non-cyclic photophosphorylation. Desaspidin then reacts in a single step with a K_i of 1.4 × 10^?4M. At 5 × 10^?5M DCMU, also the pseudocyclic photophosphorylation is abolished. The remaining, true cyclic photophosphorylation has a single K_i of 2.3 × 10^?5M for desaspidin. Under non-cyclic conditions, the true cyclic process contributes about 25% to total Photophosphorylation. Under pseudocyclic conditions, no cyclic photophosphorylation occurs. Under true cyclic conditions, the non-cyclic and pseudocyclic processes are inoperative. This indicates a regulative system, so that either (1) the (non-cyclic + true cyclic), (2) only the pseudocyclic, or (3) only the true cyclic systems can be traced, dependent on the level of DCMU applied. There are two sites for non-cyclic Photophosphorylation, one of them common to the pseudocyclic pathway. Cyclic photophosphorylation has a third site, different from the other two.     

An inhibitory effect of arachidonic acid on subsequent responses of canine cerebral arteries to serotonin and other agonists

Arachidonic acid (AA) at 10^?4M and 10^?3M produced a phasic contraction in isolated canine basilar arteries that peaked rapidly and then slowly declined. This contraction was evidently due to the conversion of AA to prostanoids because it was blocked by cyclooxygenase inhibitors and because 11, 14, 17 eicosatrienoic acid (10^?3M), which is not a cyclooxygenase substrate, failed to produce a contraction. When the artery was exposed to 10^?3M AA for 20 min and washed, subsequent contractile responses to 10^?6M serotonin (5-HT) were only 10% of control. Contractions produced by prostaglandin E₂ (10^?5M), uridine triphosphate (10^?4M) and potassium (5.5×10^?4M) were inhibited to a lesser degree than 5-HT, the response to potassium being the least affected (66% of control). This damaging effect of 10^?3M AA did not occur if the artery was washed at peak contraction nor with 10^?4M AA. Autooxidation products were evidently not responsible for the damage because prior oxygenation (90 min) of 10^?4M AA had no such effect. Pretreatment with superoxide dismutase or ascorbate did not prevent the inhibition, suggesting that free radical reactions were not involved. Pretreatment with indomethacin (3×10^?4M) or meclofenamate (10^?4M) also failed to prevent the inhibitory phenomenon. Saponin, a detergent, produced similar inhibitory effects but 11, 14, 17 eicosatrienoic acid or oleate (10^?3M) did not. The arteries partially recovered from the inhibition with time. In conclusion, AA produced contraction in basilar arteries by inducing prostaglandin synthesis but can produce secondarily by an unidentified mechanism an inhibition of the contractile responses evoked by various agonists that is both time and concentration dependent.     

Mechaism of Arthrobacter sialophilus neuraminidase: The binding of substrates and transition-state analogs

2-Deoxy-2,3-dehydro-N-acetylneuraminic acid and its methyl ester are competitive inhibitors of Arthrobacter sialophilus neuraminidase with K_i = 1.4 × 10^?6$\text{M}$ and 4.8 × 10^?5$\text{M}$, respectively. The K_m for the substrate, N-acetylneuraminlactose, is 1.0 × 10^?3$\text{M}$. These data, taken together with the conformation of these compounds, indicate that these compounds are transition-state analogs of the enzyme. These results also suggest that the substrate upon binding to neuraminidase is distorted to a conformation approaching that of a half-chair.     

Membrane transport of L-triiodthyronine by human red cell ghosts.

L-T₃ transport has been investigated in human red cell ghosts. Determination of initial T₃ uptake revealed two separate saturable uptake systems, one with a K_m of 1.6 × 10^?8M, the other with a K_m of 3.3 × 10^?6M. Binding experiments resulted in two dissociation constants, 1.4 × 10^?7M.and 2.6 × 10^?6M. Uptake was dependent on the ghost volume, indicating an intravesicular location of T₃. The T₃ was concentrated 6 times by the ghosts. Ouabain reduced the uptake by the low K_m system, but was without effect on the high K_m system. Thus evidence is provided both of binding of T₃ to the ghost membrane and of its uphill transport across the membrane.     

Further properties of the polyamine oxidase of barley leaves

Polyamine analogues have been studied as potential inhibitors or substrates of barley leaf polyamine oxidase. NH₂(CH₂)₃NH(CH₂)₁₀NH₂ was particularly effective as an inhibitor of spermine oxidation at pH 4·5 (K_i = 5 × 10^?6 M). Methylglyoxal-bis(guanylhydrazone) inhibited spermine oxidation only slightly (K_i = 10^?4 M). Activity with the polyamine analogues as substrates was generally 10% or less of the activity with spermine. The K_m for oxygen was 3 × 10^?4 M. The K_m for spermine oxidation was independent of oxygen concentration. Using the N-methyl-2-benzothiazolone hydrazine reagent, 1-(3-aminopropyl)pyrroline was shown to be formed stoichiometrically by the enzyme on oxidation of spermine. The enzyme will not function as a dehydrogenase in the presence of oxygen with either potassium ferricyanide or dichlorophenolindophenol as electron acceptors. Activity in the leaves increased with age, up to 4 weeks. In the leaves of 11-week-old plants activity was lower than in leaves of 1-week-old plants. The enzyme was mainly associated with an easily-sedimented particulate fraction, and relatively small proportions were found in the cell wall or soluble fractions.     

Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis

An extracellular acid phosphatase secreted into the medium during growth of Tetrahymena pryiformis strain W was purified about 900-fold by (NH₄)₂SO₄ precipitation, gel filtration and ion exchange chromatography. The purified acid phosphatase was homogenous as judged by polycrylamide gel electrophoresis and was found to be a glycoprotein. Its carbohydrate content was about 10% of the total protein content. The native enzyme has a molecular weight of 120 000 as determined by gel filtration and 61 000 as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The acid phosphatase thus appears to consist of two subunits of equal size. The amino acid analysis revealed a relatively high content of asparic acid, glutamic acid and leucine. The purified acid phosphatase from Tetrahymena had a rather broad substrate specificity; it hydrolyzed organic phosphates, nucleotide phosphates and hexose phosphates, but had no diesterase activity. The K_m values determined with p-nitrophenyl phosphate, adenosine 5′-phosphate and glucose 6-phosphate were 3.1·10^?4 M, 3.9·10^?4 M and 1.6·10^?3 M, respectively. The optima pH for hydrolysis of three substrates were similar (pH 4.6). Hg²⁺ and Fe³⁺ at 5 mM were inhibitory for the purified acid phosphatase, and fluoride, L-(+)-tartaric acid and molybdate also inhibited its cavity at low concentrations. The enzyme was competitively inhibited by NaF (K_i=5.6·10^?4 M) and by L-(+)-tartaric acid (K_i = 8.5·10^?5 M), while it was inhibited noncompetitively by molybdate K_i = 5.0·10^?6 M). The extracellular acid phosphatase purified from Tetrahymena was indistinguishable from the intracellular enzyme in optimum pH, K_m, thermal stability and inhibition by NaF.     

The inhibition of L-3-hydroxyacyl-CoA dehydrogenase by acetoacetyl-CoA and the possible effect of this inhibitor on fatty acid oxidation

Acetoacetyl-CoA was found to strongly inhibit the dehydrogenation of L-3-hydroxybutyryl-CoA catalyzed by L-3-hydroxyacyl-CoA dehydrogenase from pig heart. The inhibition constant (K_i) was determined to be 7.7 × 10^?6 M, a value which is similar to the K_m value of 12 × 10^?6 M obtained for acetoacetyl-CoA in its NADH-dependent reduction catalyzed by the same enzyme. A suggested ordered BiBi mechanism for this enzyme, with NAD binding to the enzyme first, explains the observed noncompetitive nature of this inhibition. The possible effect of this inhibition on fatty acid oxidation is discussed.     

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.