Inactivation of human glutamate dehydrogenase by aluminum

Aluminum inactivated glutamate dehydrogenase (GDH) by a pseudo-first-order reaction at micromolar concentrations. A double-reciprocal plot gave a straight line with a k_inact of 2.7 min^-1 and indicated the presence of a binding step prior to inactivation. The inactivation was strictly pH dependent and a marked increase in sensitivity to aluminum was observed as the pH decreased. At a pH higher than 8.5, no inactivation was observed. The completely inactivated GDH contained 2 mol of aluminum per mole of enzyme subunit monomer. When preincubated with enzyme, several chelators such as citrate, NaF, N-(2-hydroxyethyl) ethylenediaminetriacetic acid or ethylenediaminetriacetic acid efficiently protected the enzyme against the aluminum inactivation. In a related experiment, only citrate and NaF released the aluminum from the completely inactivated aluminum-enzyme complex and fully recovered the enzyme activity. Ferritin, NADP⁺, or nerve growth factor did not show any effects on the recovery of the aluminum-inactivated GDH activity. The dissociation constant for the aluminum-enzyme complex was calculated to be 5.3 M. Although aluminum has been known to form a complex with nucleotides, no such effects were observed in the inactivation of GDH by aluminum as determined using GDHs mutated at the ADP-binding site, NAD⁺-binding site or GTP-binding site. Circular dichroism studies showed that the binding of aluminum to the enzyme induced a decrease in helices and sheets and an increase in random coil. Therefore, inactivation of GDH by aluminum is suggested to be due to the conformational change induced by aluminum binding. These results suggest a possibility that aluminum-induced alterations in enzymes of the glutamate system may be one of the causes of aluminum-induced neurotoxicity.Received 25 July 2003; received after revision 27 August 2003; accepted 15 September 2003     

Irreversible inactivation of yeast glucose-6-P dehydrogenase by penicillin G

Summary Yeast glucose-6-P dehydrogenase is irreversibly inactivated by penicillin G. Kinetic data show that 1 molecule of penicillin G reacts with each active unit when the enzyme is inactivated The rate of inactivation increases greatly with increasing pH. This irreversible inactivation by penicillin G is largely prevented by pyridoxal-P, a reversible inactivator of this enzyme. Prior treatment of penicillin G with penicillinase totally abolishes its ability to inactivate the enzyme.This work was supported by grant RR-8006 from the General Research Branch, Division of Research Resouces, NIH (USA).     

Role of Drosophila alkaline ceramidase (Dacer) in Drosophila development and longevity

Ceramidases catalyze the hydrolysis of ceramides to generate sphingosine (SPH) and fatty acids, and ceramide metabolism is implicated in various biological responses in Drosophila melanogaster. Here we report the cloning, biochemical characterization, and functional analysis of a Drosophila alkaline ceramidase (Dacer). Dacer, a membrane-bound protein of 284 amino acids, shares homology with yeast and mammalian alkaline ceramidases. Overexpression of Dacer in High Five insect cells increases ceramidase activity in the alkaline pH range, indicating that Dacer is a bona fide alkaline ceramidase. Dacer mRNA is highly expressed in the midgut and at the pupal stage. An inactivation of Dacer by insertional mutagenesis increases the levels of ceramides in both Drosophila pupae and adult flies. Dacer inactivation increases Drosophila pre-adult development time, lifespan, and anti-oxidative stress capacity. Collectively, these results suggest that Dacer plays an important role in the Drosophila development and longevity by controlling the metabolism of ceramides.     

Inactivation of yeast glucose-6-P dehydrogenase by aspirin

Summary Glucose-6-P dehydrogenase is irreversibly inactivated by treatment with Na salts of aspirin. Kinetic data show that 1 molecule of aspirin reacts with each active unit when the enzyme is inactivated. The rate of inactivation is enhanced with increasing pH but is reduced in the presence of glucose-6-P or NADP⁺. Na salicylate fails to inactivate the enzyme.This work was supported by grant RR-8006 from the General Research Branch, Division of Research Resources, NIH (USA).     

Immobilization of chicken liver fructose 1,6-bisphosphatase on CNBr-activated Sepharose

Summary Chicken liver fructose 1,6-bisphosphatase is readily immobilized on CNBr-activated Sepharose. The immobilization alters some enzymatic properties. They include broader pH activity curve, loss of activation by K⁺ or NH ₄ ⁺ , increased resistance to inactivation by trypsin, decreased sensitivity to AMP inhibition, and loss of cooperative interaction among AMP-binding sites. The immobilized enzyme retains about 38% or 19% of the specific activity of the native enzyme when the activity is measured in the absence or presence of K⁺, resepctively.This work was supported by grant RR-8006 from the General Research Branch, Division of Research Resources, NIH (USA).     

Effect of exogenous iron on the viability of pathogenicNaegleria fowleri in serum

Summary WhenNaegleria fowleri (Lee) was incubated in newborn calf and human serum an amebicidal effect was observed. Heat inactivation of both sera resulted in the recovery of viable amebae after incubation in these sera. Exogenous iron added to non-heat inactivated calf serum improved viability slightly but was without effect when added to human serum not heat inactivated. Exogenous iron greatly enhanced growth and/or viability in heat inactivated calf serum. Viability of amebae also was considerably enhanced in human serum which was heat inactivated when pH was lowered in conjunction with iron supplements.Appreciation is expressed to Dr Ronald R. Weik (Dept. Biochemistry, Louisiana State Univ., New Orleans, LA. 70119) and Dr David T. John (Dept. Microbiology, Medical College of Virginia, Richmond, VA. 23298) for providing theNaegleria fowleri (Lee) used in this investigation.     

Dependence of 6β-acetoxy-7α-hydroxyroyleanone block of Kv1.2 channels on C-type inactivation

Voltage-gated K⁺ (Kv) channels exhibit slow or C-type inactivation during continuous depolarization. A selective pharmacological agent targeting C-type inactivation is hitherto lacking. Here, we report that 6β-acetoxy-7α-hydroxyroyleanone (AHR), a diterpenoid compound isolated from Taiwania cryptomerioides, can selectively modify C-type inactivation of Kv1.2 channels. Extracellular, but not intracellular, AHR (50 μM) dramatically accelerated the slow decay of Kv currents and left-shifted the steady-state inactivation curve. AHR blocked Kv currents with an IC₅₀ of 17.7 μM. AHR did not affect the kinetics and voltage-dependence of Kv1.2 channel activation. Channel block by AHR was independent of intracellular K⁺ concentration. In addition, effect of AHR was much attenuated in a Kv1.2 V370G mutant defective in C-type inactivation. Therefore, block of Kv1.2 channels by AHR did not appear to involve direct occlusion of the outer pore but depended on C-type inactivation. AHR could thus be a probe targeting Kv channel C-type inactivation gate.     

Spermine protects protein kinase C from phospholipid-induced inactivation

Phosphatidylserine (PS), an activator of protein kinase C (PKC) in the assay of protein phosphorylation, inhibited this enzyme in a time-dependent manner following preincubation in the absence of Ca²⁺. The phospholipid-induced inactivation of kinase activity was dependent on the PS content and on the charge density of liposomes. This inactivation of PKC could be reduced, but not completely eliminated, by addition of Ca²⁺. In the present work the effect of a naturally occurring polyamine (spermine) on the PS-induced inactivation of PKC was investigated. The presence of spermine during preincubation without Ca²⁺ was effective in suppressing the PS-induced inactivation of PKC over the period (20 min) required for PS to inhibit the enzyme by 95%. PKC exists in two membrane-bound states: a reversible one which can be dissociated by Ca²⁺ chelators (membrane-associated form) and an irreversible one which is chelator-stable (membrane-inserted form). Gel filtration experiments on the PKC-PS complex formed in the presence of Ca²⁺ indicated that less insertion of enzyme into liposomes occurred in the presence of spermine and that the kinase activity of the reversibly membrane-associated PKC was protected from PS inactivation.     

Description of some simple tests allowing the distinction between bacteriocins sensu stricto and other antibacterial agents produced by bacteria]

In contrast with other antibacterial agents produced by bacteria, the bacteriocins of Gram -- bacteria (briefly: cins G--) are characterized by their primary lethal action, their inactivation by trypsin, their resistance to pH 2 (in the crude state) and insensitivity to DNase I after treatment with 7 M urea. Only 4 among 26 studied cins G + have the 4 above-cited properties and share most properties of cines G--.     

Thioglycolate de sodium et hormones hypothalamo-neurohypophysaires

Summary A kinetic study of hormonal inactivation in the presence of sodium thioglycolate was carried out using the rat uterus bioassay. In all cases we observed a total inactivation of the hormonal activities, whether the uterine horn is in the presence or not of magnesium. The hormones under consideration divide into 2 groups: the arginine-vasotocin group (rapidly inactivated), and the ichtyotocin and oxytocin group (more slowly inactivated). But, in all cases the rate of inactivation is linear.

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Avec la collaboration technique de Mr.G. Hoeltzel.     

Über die Bildung von Malondialdehyd bei der Bestrahlung von Glycerin

Summary A strong pH dependence of the radiation induced formation of malondialdehyde in 1% glycerol solution was observed. The following G-values were found: 0,202 at pH 2,35, 0,017 at pH 5,50 and 0,168 at pH 8,55.     

Immobilization of chicken liver fructose 1,6-bisphosphatase on CNBr-activated Sepharose

Chicken liver fructose 1,6-bisphosphatase is readily immobilized on CNBr-activated Sepharose. The immobilization alters some enzymatic properties. They include broader pH activity curve, loss of activation by K+ or NH+4, increased resistance to inactivation by trypsin, decreased sensitivity to AMP inhibition, and loss of cooperative interaction among AMP-binding sites. The immobilized enzyme retains about 38% or 19% of the specific activity of the native enzyme when the activity is measured in the absence or presence of K+, respectively.     

The role of hsp70 in protection and repair of luciferase activity in vivo; experimental data and mathematical modelling

The stably transfected rat cell line HR24 expressing high levels of the inducible human hsp70 and its parental cell line Rat-1 were used for in vivo studies to analyse the role of hsp70 during thermal protein denaturation and the subsequent renaturation. In order to monitor denaturation and renaturation of a cellular protein in vivo, both cell lines were transiently transfected with firefly luciferase (Luc). The continuous monitoring of Luc activity during and after heat stress allowed a detailed analysis of the inactivation and reactivation kinetics in cells grown in monolayers. The aim of these studies was to distinguish a protective effect of increased hsp70 levels during heat shock-induced protein inactivation from a stimulation of reactivation. In this paper we show that in cells that are stably transfected with hsp70, thermal Luc inactivation decreased, and subsequent reactivation yielded higher activity levels, compared with the parental cells. The difference in early inactivation kinetics observed in the two cell lines suggests an immediate effect of the presence of an extra amount of hsp70 on enzyme inactivation. Using different mathematical models, the heat-induced inactivation and reactivation kinetics was compared with simulations of denaturation and renaturation. It is concluded that the model in which it is assumed that hsp70 is able to interact with partially denatured proteins, which did not yet lose their enzymatic activity, most optimally explains the experimental observations. Received 2 December 1998; received after revision 19 February 1999; accepted 18 March 1999     

Ein Beitrag zur irreversiblen Temperaturinaktivierung der Gewebsatmung

Summary (1) Tissue slices of the liver and pieces of the skin of summer-frogs were incubated at 47.5°C for 30–150 min. Their respiration was measured during this incubation period and thereafter at a temperature of 37.5°C.(2) As a sign of an irreversible inactivation of the enzymes, the average respiration was inhibited. However, after certain periods of incubation at 47.5°C, over-shoot phenomena in the O₂-uptake at the temperature of 37.5°C were seen.     

Agglutininbildung durch verschiedene Proteinfraktionen vonSalmonella ballerup

Summary The author has examined the agglutinogenic property of nucleoprotein and its components, the nucleic acid and proteins fromS. ballerup. The nucleoprotein was obtained by the extraction of fresh bacteria desintegrated by means of glass powder at pH 8.4 and precipitated with acetic acid at pH 4.6, and its components by splitting the nucleoprotein with 0.5% sodium carbonate at pH 5.5. The protein component was separated from the nucleic acid by forming a chloroform-proteingel. The protein component was submitted to a further purification by dissolving at pH 7 and precipitating at pH 5.5. Rabbits to whom 4–8 mg of the substance were given in 10–12 doses were used for the immunization. The agglutinins were determined byHeidelberger andKabat's quantitative method with aS. ballerup suspension. The maximum quantity of agglutinin is shown in mg nitrogen per 1 cm³ of serum. In comparison with the results obtained by the immunization with whole germs, it follows that, by means of nitrogen compounds, the nucleoprotein has the most substantial agglutinogenic effect, and the nucleic acid the poorest one.     

Reversible inactivation of the nitrate reductase of rice plants

Summary Studies of the inactivation of the rice nitrate reductase showed that the nitrate-reducing moiety and not the diaphorase moiety was reversibly inactivated by NADH and cyanide. Ferricyanide could reverse the inactivation, and nitrate could protect the enzyme against inactivation. Although the general characteristics of the reversible inactivation of rice nitrate reductase appeared similar to those of the algal nitrate reductase, it was found that the rice enzyme was automatically reactivated when NADH and cyanide were removed. Attempts to isolate inactivated nitrate reductase from ammonium-treated tissue were unsuccessful.     

Voltage-gated sodium channel-associated proteins and alternative mechanisms of inactivation and block

Voltage-gated sodium channels mediate inward current of action potentials upon membrane depolarization of excitable cells. The initial transient sodium current is restricted to milliseconds through three distinct channel-inactivating and blocking mechanisms. All pore-forming alpha subunits of sodium channels possess structural elements mediating fast inactivation upon depolarization and recovery within milliseconds upon membrane repolarization. Accessory subunits modulate fast inactivation dynamics, but these proteins can also limit current by contributing distinct inactivation and blocking particles. A-type isoforms of fibroblast growth factor homologous factors (FHFs) bear a particle that induces long-term channel inactivation, while sodium channel subunit Navβ4 employs a blocking particle that rapidly dissociates upon membrane repolarization to generate resurgent current. Despite their different physiological functions, the FHF and Navβ4 particles have similarity in amino acid composition and mechanisms for docking within sodium channels. The three competing channel-inactivating and blocking processes functionally interact to regulate a neuron’s intrinsic excitability.     

Activity of two components of serum ribonuclease under conditions of physical exercise

Summary Serum ribonuclease activity before and after physical exercise in healthy persons was estimated. It is found that a work load of 6000 kgm/5 min increased ribonuclease activity measured at pH 8.5 and decreased the activity of the same enzyme measured at pH 7.0 in the presence of ZnSO₄. The observed changes were more pronounced in untrained than in trained persons.