Evidence for a single glutamate receptor of the ionotropic kainate/quisqualate type

The kainate (KA) and the quisqualate (QUIS) receptors that activate cation channels in the central nervous system have previously been defined as two of the major glutamate receptor types. In amphibian brain, an exceptionally rich source of these sites, they can be coextracted by octylglucoside and shown to behave as one entity in all analyses made in solution. When partly purified by lectin affinity, ion-exchange chromatography or by sucrose gradient centrifugation, the two activities comigrate in a 1:1 ratio. When the QUIS component is bound to an immobilized specific QUIS agonist, the KA component is extracted in parallel with it. There are equivalent numbers of the QUIS and KA sites and the two sites show a single affinity series for the binding of glutamatergic agonists. We deduce that KA or QUIS select different conformations of a single KA/QUIS receptor binding site, leading thus to the different channel-opening events that have been reported for these two agonists.     

Two distinct (-)nicotine binding sites in goldfish brain. Identification and characterization of putative neuronal nicotinic acetylcholine receptor subtypes

The binding of (-)-[3H]nicotine to membrane fragments and a detergent solubilized fraction of goldfish brain was characterized. (-)-[3H]nicotine binding was not displaced by alpha-bungarotoxin, but was displaced by (-)nicotine and carbamoylcholine with Ki of approximately 8.6 and 86 nM, respectively. Preincubation of solubilized membrane extract with alpha-bungarotoxin-coupled Sepharose resulted in the loss of approximately 50% of the (-)-[3H]nicotine binding protein from the eluent and an increase in (-)-[3H]nicotine binding to the gel compared to control, non-alpha-bungarotoxin Sepharose. 125I-alpha-bungarotoxin binding protein in the eluent from the same preincubation experiments was totally removed. In addition, incubation of the solubilized tissue extracts with alpha-bungarotoxin-coupled Sepharose resulted in an increase in the affinity for (-)-[3H]nicotine in the eluent (mean KD = 3.1) compared to control solubilized tissue extracts (KD = 6.4 nM). Specific (-)-[3H]nicotine binding sites could be eluted from the alpha-bungarotoxin-coupled Sepharose with carbamoylcholine and D-tubocurarine. Similar to previously reported 125I-alpha-bungarotoxin binding data, eye removal resulted in an approximately 40% decrease in (-)-[3H]nicotine binding in the contralateral tectum compared to that in the ipsilateral tectum. These data indicate that at least two distinct subtypes of (-)nicotine binding sites may be present in goldfish brain, one which binds alpha-bungarotoxin and (-)nicotine and another which binds only (-)nicotine.     

Single-step unimolecular non-first-order enzyme deactivation kinetics

A two-parameter deactivation model is proposed to describe the kinetics of activity stabilization for some enzymes. The single-step unimolecular mechanism exhibits non-first-order deactivation kinetics since the final enzyme state, E(1) is not completely inactivated. The usefulness of the model is demonstrated by applying it to the inactivation of different enzymes. The influence of the concentration of active ester, ionic strength, and pH on the model parameters is examined during the inactivation of electric eel acetylcholinesterase.(25) In general, inactivators would decrease the level of activity stabilization, alpha(1), and increase the first-order inactivation rate constant, k(1). The effect of protecting agents would be to increase alpha(1) and to decrease k(1).     

Sympathetic and metabolic correlates of hypoxic moderation of spontaneous hypertension in the rat

Exposure to hypobaric hypoxia (H; simulated altitude = 3658 m) was initiated in 5-week-old, male spontaneously hypertensive (SHR) and Wistar-Kyoto (WKy) normotensive rats while normoxic controls (N) for both groups were maintained under laboratory conditions. Significant attenuation of systolic arterial blood pressure was evident in SHR-H relative to SHR-N (125 +/- 6 vs 145 +/- 5 mm Hg; P less than 0.05) but not in WKy-H relative to WKy-N (WKy-H, 116 +/- 2 vs WKy-N, 117 +/- 5 mm Hg). Hypoxia significantly decreased metabolic efficiency in both normotensive and hypertensive rats, although being both more severe and accompanied by significantly impaired growth rate in SHR-H. Urinary excretion of norepinephrine in the SHR was elevated relative to WKy, irrespective of altitude treatment, while hypoxia elicited similar increases in urinary excretion of norepinephrine in both SHR and WKy. Myocardial and adrenal contents of norepinephrine were significantly reduced following 3 days of simulated altitude exposure in both strains of rats. Tissue contents of norepinephrine in hypoxic rats returned to normoxic levels by 21 days of simulated altitude. Both urine and tissue indices provided consistent indirect evidence that changes in sympathetic neuronal activity in response to hypoxia were similar in normotensive and hypertensive rats. These findings suggest that prior reports of reduced alpha-adrenergic responsiveness in vasculature from hypoxia-exposed SHR reflect a postsynaptic event that is regulated independently of norepinephrine release from sympathetic nerve terminals.     

The disappearance of isocitrate lyase from the green alga Chlorella fusca studied by immunoprecipitation

When acetate-adapted cultures of Chlorella fusca were transferred to nitrogen-free medium containing glucose, isocitrate lyase activity was lost over a period of about 25 h. Using a combination of in vivo isotope labelling and immunoprecipitation with anti-isocitrate lyase IgG it was shown that: 1. The onset of loss of enzyme activity preceeded the complete cessation of enzyme synthesis. 2. Disappearance of isocitrate lyase activity was accompanied by loss of enzyme protein, without accumulation of antigenic protein distinguishable from the normal subunit polypeptide of the enzyme, as judged by SDS gel electrophoresis of immunoprecipitated samples from supernatant cell-free extracts. 3. SDS gel electrophoresis of immunoprecipitated isocitrate lyase revealed the presence of antigenic protein bands of M_r about twice that of the normal subunit polypeptide, but the appearance of these apparent dimer forms did not obviously correlate with enzyme degradation. 4. Isoelectric focusing of immunoprecipitated isocitrate lyase showed that the enzyme became progressively more oxidised during the period of its degradation in vivo. 5. By titrating crude broken cell suspensions with anti-isocitrate lyase antibody, preliminary evidence was obtained for transfer of the enzyme from the soluble fraction to an insoluble form as part of the process of disappearance.     

The enhancement of iron-dependent luminol peroxidation by 2,2′-Dipyridyl and nitrilotriacetate

The generation of radicals from luminol and H₂O₂, in the presence of iron and iron chelates was monitored by measuring the chemiluminescence produced by further oxidation of these radicals. 2,2′-Dipyridyl enhanced the production of chemiluminescence in the presence of FeSO₄, farritin and haemosiderin but not FeCI₃ or horseance of both FeSO₄ and FeCI₃ but not ferritin or haemosiderin. The enhancement of chemiluminescence by iron chelation may have analytical applications and the process by which these iron chelates are able to generate radicals from the nitrogenous base luminol may be similar to that responsible for their toxic effects on DNA.