Ionic Requirements for the Specific Binding of [3H]GBR 12783 to a Site Associated with the Dopamine Uptake Carrier

At 0°C, when Na⁺ was the only cation present in the incubation medium, increasing the Na⁺ concentration from 3 to 10 mM enhanced the affinity of [³H]l-[2-(di-phenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine ([³H]GBR 12783) for the specific binding site present in rat striatal membranes without affecting the 5_max. For higher Na⁺ concentrations, specific binding values plateaued and then slightly decreased at 130 mM Na⁺. In a 10 mM Na⁺ medium, the K_D and the B_max were, respectively, 0.23 nM and 12.9 pmol/mg of protein. In the presence of 0.4 nM [³H]GBR 12783, the half-maximal specific binding occurred at 5 mM Na⁺. A similar Na⁺ dependence was observed at 20°C. Scatchard plots indicated that K⁺, Ca²⁺, Mg²⁺, and Tris⁺ acted like competitive inhibitors of the specific binding of [³H]GBR 12783. The inhibitory potency of various cations (K⁺, Ca²⁺, Mg²⁺, Tris⁺, Li⁺ and choline) was enhanced when the Na⁺ concentration was decreased from 130 to 10 mM. In a 10 mM Na⁺ medium, the rank order of inhibitory potency was Ca²⁺ (0.13 mM) > Mg²⁺ > Tris⁺ > K⁺ (15 mM). The requirement for Na⁺ was rather specific, because none of the other cations acted as a substitute for Na⁺. No anionic requirement was found: Cl^-, Br^-, and F^- were equipotent. These results suggest that low Na⁺ concentrations are required for maximal binding; higher Na⁺ concentrations protect the specific binding site against the inhibitory effect of other cations.     

[3H]Imipramine Binding of Protein Nature in Human Platelets: Inhibition by 5-Hydroxytryptamine and 5-Hydroxytryptamine Uptake Inhibitors

Abstract: The nature of [³H]imipramine binding to human platelets was investigated. Desipramine and 5-hydroxytryptamine (5-HT) displaced the same amount of binding and the binding was sensitive to protease treatment. The nature of pharmacological inhibition of [³H]imipramine binding was investigated in saturation experiments. Increases in K d without changes in B _max were noted with the addition of 5-HT, desipramine, norzimeldine, or 5-methoxytryptoline. Reductions in B _max without alterations in K _D were obtained when citalopram or clomipramine was added. It is concluded that the [³H]imipramine binding site in human platelets is of protein nature and that this binding site contains the substrate recognition site for 5-HT uptake. In addition, [³H]imipramine and other 5-HT uptake inhibitors have bonds to other parts of the 5-HT uptake carrier or to the surrounding lipid membrane. This additional binding outside the substrate recognition site is not one single site but most likely represents sites that are specific for the chemical structure of each uptake inhibitor, respectively.     

Kinetic Modulation by GABAergic Agents of High- and Low-Affinity Binding of [3H]Methyl β-Carboline-3-Carboxylate

The kinetics of dissociation of [3H]methyl beta-carboline-3-carboxylate (beta-CCM) binding was studied in a synaptosomal membrane preparation of rat cerebral cortex. Dissociation was biphasic: a faster phase (10-30% contribution) was followed by a slower phase. Picrotoxin pretreatment at 22 degrees C enhanced the equilibrium binding of [3H]beta-CCM. The half-life of the slower phase of beta-CCM dissociation (t1/2II) was increased by 60 muM picrotoxin from 1.7 min to 3.3 min. The dissociation of [3H]beta-CCM was identical when initiated by an excess of either diazepam or beta-CCM. Quasi-equilibrium Scatchard analysis of [3H]beta-CCM binding was performed by a kinetic separation of the rapid and slow phases of dissociation. The slow and rapid phases represented beta-CCM binding sites of high and low affinity, respectively. The dissociation of [3H]beta-CCM (control t1/2II = 2.0 min) was decelerated by the gamma-aminobutyric acid (GABA) antagonist 3-alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (R 5135) (t1/2II = 2.5 min) and accelerated by GABA (t1/2II = 1.6 min). GABA inhibited both high- and low-affinity beta-CCM bindings.     

Substrate stabilization of lysophosphatidylcholine-solubilized plasma membrane H+-ATPase from oat roots

Plasma membrane vesicles with H⁺-ATPase activity were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots using an aqueous polymer two-phase system. Of several detergents tested, only lysophosphatidylcholine solubilized the H⁺-ATPase in an active form. Solubilization of the H⁺-ATPase with lysophosphatidylcholine was possible in the absence of glycerol, but the ATPase activity decreased about 4–5 times as rapidly in the absence as in the presence of 30% (w/v) glycerol. The solubilized enzyme was further stabilized by ATP and protons. Addition of 1 m M ATP to the plasma membranes halted inactivation of the H⁺-ATPase. Even in the absence of polyol compounds and ATP, the enzyme was stable for hours at relatively low pH with an optimum around pH 6.7 at room temperature. The curve for the stability of soluble H⁺-ATPase as a function of pH closely resembles the pH curve for the activity of the H⁺-ATPase. This suggests that binding of protons to transport sites may stabilize the soluble H⁺-ATPase in an enzymatically active form.     

Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Potassium Transport and Regulation of Intracellular pH in Elodea densa Leaves

The effects of extracellular K⁺ concentration ([K⁺]_o) on the pH of cell sap, “bulk cytoplasm” and vacuole have been investigated in Elodea densa leaves under conditions of either low or high activity of the plasmalemma electrogenic H⁺ pump. Cell sap pH was evaluated directly in the cell sap expressed after freezing and thawing. Cytoplasmic and vacuolar pH were calculated by the weak base and weak acid distribution method, DMO and benzylamine appearing to be a suitable acid and base, respectively, for this purpose in this material. When added to the basal medium (no rapidly permeating ions present), 5 mM K⁺ induced an increase in intracellular pH, larger for the cell sap and the vacuole (about 0.2 units), and smaller but still significant for the cytoplasm (0.07 units). This alkalinizing effect of K⁺ was thus associated with a significant decrease in the pH difference across the tonoplast. The alkalinizing effect of K⁺ was markedly and synergistically enhanced by the presence of fusicoccin, a condition inducing a marked activation of H⁺ extrusion and of K⁺ uptake. The correlation between these effects of [K⁺]_o on intracellular pH and those on H⁺ extrusion indicates that changes in extracellular K⁺ concentration, and thus in K⁺ influx, can influence cytoplasmic and vacuolar pH by modulating the rate of H⁺ extrusion by the plasmalemma H⁺ pump.     

Influence of the food plants on the degree of parasitism of larvae ofHeliothis armigera byCotesia kazak

Influence of the food plants ofHeliothis armigera (Hb.) on the degree of parasitism by exotic parasiteCotesia kazak Telenga (Hymenoptera: Braconidae) was studied in cages in the laboratory on 7 food plants such as cotton (Gossypium hirsutum L.), tomato (Lycopersicon esculentum Mill), okra [Abelmoschus esculentus (L.) Moench], Dolichos (dolichos lablab L.), pigeonpea [Cajanus cajan (L.) Millsp.], Cowpea (Vigna unquiculata (L.) and chickpea (Cicer arietium L.). To determine the preference of the parasite 2 test methods were employed. In single plant choice test cotton was most preferred. Next in order of preference were tomato and okra. Dolichos, pigeonpea, cowpea and chickpea were least preferred. In multiple choice test, however, cotton and okra were preferred followed by tomato. Parasites were seen visiting these plants very frequently and high parasitism was recorded on these plants. Chick pea, pigeon pea, cowpea and Dolichos were the least preferred food plants. There appears to be some difference in fecundity as affected by some food plants. Exposure on okra, cotton and tomato resulted in higher cocoon production as compared to pigeonpea, Dolichos, cowpea and chickpea. There was, however, no difference in sex-ratio and longevity of the progeny as affected by food plants. This exotic parasite should be released first in crops such as cotton, okra and tomato on whichH. armigera is a very serious pest in India and elsewhere. Contribution No. 140/86 of the Indian institute of Horticultural Research, Bangalore-560 089     

In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) fromEuglena gracilis [J. Biol. Chem.,260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as -N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as thein vitro substrate. Presently, histone H1 has been purified fromEuglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. TheEuglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higherR _f value than the other histones H1 in acid/urea gel electrophoresis. When theEuglena histone H1 was [methyl-³H]-labeledin vitro by a homologous enzyme (one of the twoEuglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C₁₈ columns on HPLC, we demonstrated thatEuglena histone H1 contains approximately 9 mol% of -N-methyllysines (1.40, 1.66, and 5.62 mol% for -N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of -N-methyllysines in histone H1.     

Opioid receptor antagonist affinity ligands: 6β-bromoacetamido-6-desoxynaltrexone and 6β-thioglycolamido-6-desoxynaltrexone

The present study, utilizing thioglycolamido as the reactive group, describes the synthesis and pharmacology of a new opioid antagonist affinity ligand, 6-thioglycolamido-6-desoxynaltrexone (TAN) and compares TAN with a related known compound, 6-bromoacetamido-6-desoxynaltrexone (BAN). Both compounds were tested for their reversible and irreversible inhibition of [³H]naloxone binding to calf brain membranes. Reversible binding of BAN and TAN had K_i values of 1×10^–9 and 1×10^–10 M, respectively as determined by log probit plots. Irreversible binding was determined after extensive washing to remove all non-covalently bound ligand. At a concentration of 5×10^–8 and 1×10^–8 M for BAN and TAN irreversible binding was inhibited 50% of the maximum value. A study of the time course of irreversible inhibition of [³H]naloxone binding revealed that maximal inhibition occurred within 5 min with a concentration of 1×10^–7 M of either agent. TAN but not BAN when administered systematically to mice produced an antinociceptive effect as measured by the writhing test. When administered intracerebraventricularly BAN did not block morphine-induced analgesia for more than 2 hr; whereas, with a single ED₅₀ dose of 20 nmoles of TAN i.c.v. morphine-induced analgesia was almost completely blocked for a period of over 24 hr, as determined by the tail flick test. Although the SH group of TAN were required for the covalent interaction with opioid receptors, the site of TAN's interaction appears to involve other than protein SH groups.     

Sigma receptors in schizophrenic cerebral cortices

Antipsychotics represent high affinity for sigma receptors and sigma-like drugs often have the psychotomimetic properties. Besides, the receptors are unevenly distributed in human brain. These findings suggest that sigma receptors might be involved in the pathophysiology of schizophrenia. Sigma receptors in rat and human brain were measured with [³H]-1, 3, di-o-tolylguanidine (DTG) and non-specific binding of [³H]DTG was determined in the presence of 10^–5M haloperidol. Monovalent and divalent cations strongly inhibited [³H]DTG binding. Glutamate, aspartate and glycine also decreased the binding to human cerebral membranes. With post-mortem brain samples from 12 schizophrenics and 10 controls, sigma receptors were measured in 17 areas of cerebral cortex. Sigma receptors binding showed the regional differences in the cortex, but no significant differences between schizophrenics and controls were observed except the superior parietal cortex where the binding significantly increased in the schizophrenic group. These results suggest that sigma receptors in cerebral cortices might not be directly concerned with the pathophysiological role in schizophrenia.Dedicated to Dr. Morris Aprison. Received too late for publication in special issue.