Chemical modification of the actin binding site of rabbit muscle aldolase by diethylpyrocarbonate

Summary To extend the available information on the significance of the interactions between glycolytic enzymes and the actin component of the cellular ultrastructure, investigations into the compositional characteristics of the actin binding site on one of the major glycolytic enzymes, aldolase, have been undertaken. As the electrostatic nature of the association has been previously reported indicative of a cationic region on the enzyme involved in the binding, these studies have investigated the possibility of the involvement of histidine residues in this binding region. By the use of the histidine specific reagent, diethylpyrocarbonate, we have been able to establish a difference in nature of an actin binding domain and the active site domain which does contain an essential histidine. The results have been discussed in relation to the significance of this finding with respect to the binding of aldolase to subcellular structure.     

Interaction of plasma proteins with negatively charged sites on the pulmonary capillary endothelium of the rat

Summary The endothelial glycocalyx, a polyanionic structure which may regulate the passage of solutes and water through the endothelium, readily binds cationic ferritin (CF). In normal, nonexchange-transfused rats, however, only 7.5% and 6.0% of the luminal plasma membrane and 7.5% and 5.0% of vesicle diaphragms on the thick and thin side of pulmonary capillaries, respectively, bound cationic ferritin. With the graded removal of circulating proteins by exchange transfusion with fluorocarbon emulsion, up to 89 and 82% of the luminal surface, and 76 and 73% of vesicle diaphragms on the thick and thin sides, respectively, bound CF. Although the extent of binding on the thin side was consistently less than on the thick side, the difference was not statistically significant. The extensive binding of CF to the glycocalyx in totally exchange-transfused rats was completely reversible upon addition of lyophilized rat serum protein to the perfusate. These data suggest that in vivo anionic sites of the endothelial glycocalyx are partially masked by adsorbed plasma proteins.     

Commissural ring nerve: A female-specific neurosecretory tract supplied by bifurcating median neurons in the cockroach Periplaneta americana (L.) and the cricket Teleogryllus commodus (Walker)

Summary In the American cockroach, Periplaneta americana, and the Australian field cricket, Teleogryllus commodus, the two nerves supplying the bases of the cerci are joined by a branch that crosses behind the last abdominal ganglion. This commissural ring nerve is restricted to females, and it contains many axons filled with granular and agranular vesicles. The axons stem from somata located within the ganglion. There are one (Periplaneta) or two (Teleogryllus) groups of median neurons with bilaterally symmetrical bifurcations, and a group of postero-ventral neurons on each side. In T. commodus, these neurons are distinct from others associated with the cerci. In the two species, the ring nerve neurons contribute to a neuropile near the root of each cereal nerve. The bifurcating median neurons arborize on both sides before entering the ring nerve, while the postero-ventral ones branch more extensively ipsilateral to their somata. The possibilities are discussed that the bifurcating neurons may be homologous to dorsal unpaired median neurons, and that the ring nerve may be a neurohemal area.     

A re-examination of the Na+-independent binding of [3H]β-alanine to rat brain stem-spinal cord

The Na⁺-independent binding of [³H]-alanine to rat brain stem plus spinal cord was reinvestigated, in order to study in more detail the characteristics of previously described -alanine binding processes. Binding was absent when amino acid-free postnuclear supernatants or crude synaptic membranes were used. Experiments performed with several other Na⁺-free preparations showed a sole binding component, irrespective of the preparation used. Biochemical characterization of this Na⁺-independent binding, using frozen/thawed/washed synaptosomal-mitochodrial fractions, showed that binding reached a plateau between 7 min and 13 min, increasing thereafter. Binding was linear with fraction protein over a range of 200–415 g/ml incubation medium. Binding was completely inhibited by glycine, alanine, -aminobutyric acid, -aminoisobutyric acid, hypotaurine and strychnine, and to a lesser extent by 2,2-dimethyl--alanine, brucine and gelsemine. It was insensitive to taurine, -aminobutyric acid (GABA), 2-guanidinoethanesulfonic acid (GES), carnosine, and bicuculline methiodide. Binding was reversible, saturable (K _D 20 M), and heat sensitive.     

Chemical modification of active sites in relation to the catalytic mechanism of F1

Recent studies of chemically modified F₁-ATPases have provided new information that requires a revision of our thinking on their catalytic mechanism. One of the subunits in F₁-ATPase is distinguishable from the other two both structurally and functionally. The catalytic site and regulatory site of the same subunit are probably sufficiently close to each other, and the interaction between the various catalytic and regulatory sites are probably sufficiently strong to raise the uni-site rate of ATP hydrolysis by several orders of magnitude to that of promoted (multi-site) ATP hydrolysis. Although all three subunits in F₁ possess weak uni-site ATPase activity, only one of them () catalyzes promoted ATP hydrolysis. But all three subunits catalyze ATP synthesis driven by the proton flux. Internal rotation of the ₃₃ or ₃ moiety relative to the remainder of the F₀F₁ complex did not occur during oxidative phosphorylation by reconstituted submitochondrial particles.     

[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.     

Levels and Distribution of the Calcium-Modulated Proteins S100 and Calmodulin in Rat C6 Glioma Cells

To understand better the mechanisms involved in the transduction of a calcium signal into an intracellular response via multiple calcium-modulated proteins, we have examined the calcium-modulated proteins, S100 and calmodulin, and their intracellular targets in rat C6 glioma cells. Subconfluent, confluent, and postconfluent C6 cells contain predominantly, if not exclusively, the S100 beta polypeptide. The level of S100 beta in C6 cells increases approximately 20-fold from subconfluency to postconfluency whereas the level of calmodulin increases only about two-fold. The subcellular distribution of S100 beta and calmodulin in mitotic cells is similar. However, the subcellular distribution of these proteins in interphase cells is different and appears to change with cell density. Gel overlay analysis demonstrated that the S100- and calmodulin-binding protein profiles are significantly different and that some of the binding proteins appear to change in intensity with cell density. These data demonstrate that S100 beta is the predominant S100 polypeptide in C6 cells and suggest that changes in S100 beta and S100 beta-binding proteins may be involved in regulating S100-mediated intracellular processes in C6 cells. Our studies also suggest that the levels of S100 and calmodulin may be differentially regulated in C6 cells.     

Melatonin Binding Sites

The distribution and characterization of specific melatonin binding sites were studied using 125I-melatonin. Autoradiography revealed only three sites of specific melatonin binding in brain: the suprachiasmatic nuclei, the median eminence, and the small part of choroid plexus at the caudal end of the fourth ventricle. Two other sites were detected outside the CNS: the anterior pituitary and the retina. The specific binding of 125I-melatonin was saturable and reversible. The dissociation constant (KD) of the binding sites was 60 pM. The concentration of the binding sites (Bmax) in the median eminence was 26 fmol/mg protein, and in the pituitary 3 fmol/mg protein. Specificity of the binding sites was tested by displacement of 125I-melatonin. The order of potency--melatonin much less than N-acetyl-5-hydroxytryptamine less than 5-methoxytryptamine much less than 5-hydroxytryptamine = 3,4-dihydroxyphenylethylamine = noradrenaline--shows high specificity of the binding sites for melatonin.     

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.     

Interaction of l-Prolyl-l-Leucyl Glycinamide with Dopamine D2 Receptor: Evidence for Modulation of Agonist Affinity States in Bovine Striatal Membranes

The role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) in modulating the agonist binding to bovine striatal dopamine D2 receptor was investigated using a selective high-affinity agonist, n-propylnorapomorphine (NPA). PLG caused an enhancement in [3H]NPA binding in striatal membranes in a dose-dependent manner, the maximum effect being observed at 10(-7)-10(-6) M concentration of the tripeptide. The Scatchard analysis of [3H]NPA binding to membranes preincubated with 10(-6) M PLG revealed a significant increase in the affinity of the agonist binding sites. In contrast, there was no effect of PLG on the binding pattern of the antagonist [3H]spiroperidol. The antagonist versus agonist competition curves analyzed for agonist high- and low-affinity states of the receptor displayed an increase in the population and affinity of the high-affinity form of the receptor with PLG treatment. The low-affinity sites concomitantly decreased with relatively small change in the affinity for the agonists. Almost similar results were obtained when either NPA or apomorphine was used in the competition experiments. A partial antagonistic effect of PLG on 5'-guanylylimidodiphosphate [Gpp(NH)p]-induced inhibition of high-affinity agonist binding was also observed, as the ratio of high- to low-affinity forms of the receptor was significantly higher in the PLG-treated membranes compared to the controls. Direct [3H]NPA binding experiments demonstrated that PLG attenuated the Gpp(NH)p-induced inhibition of agonist binding by increasing the EC50 of the nucleotide (concentration that inhibits 50% of the specific binding). No effect of PLG on high-affinity [3H]NPA binding, however, could be observed when the striatal membranes were preincubated with Gpp(NH)p.(ABSTRACT TRUNCATED AT 250 WORDS)