Reactivation by glycerol and ethylene glycol of inactivatedδ-aminolevulinic acid synthetase ofRhodopseudomonas spheroides

Summary Reactivation effects by glycerol and ethylene glycol of inactivated ALA synthetase ofR. spheroides were observed. Accompanying the reactivation of the inactivated enzyme, K _m value for PLP decreased to levels similar to those of the freshly prepared enzyme.     

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).     

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.     

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).     

Functional consequences of treating turkey liver fructose-1,6-bisphosphatase with penicillin G.

Treatment of turkey liver fructose-1,6--bisphosphatase with penicillin G progressively inactivated the enzyme and desensitized the enzyme toward high substrate inhibition. The treatment also led to reduced sensitivity to AMP inhibition and the loss of cooperative interaction among AMP-binding sites. These altered properties were not reversed by dialysis, but were prevented when treatment with penicillin G was perfomed in the presence of substrate.     

Parameters influencing inactivation-dissociation/reactivation-association phenomena induced by variations of ionic strength of L (+)lactate: cytochrome c oxidoreductase (cytochrome b2) extract of the yeast Hansenula anomala

H-flavocytochrome b2, a tetramer enzyme, is inactivated, at low ionic strength and can be reactivated, increasing the ionic strength of the medium. The inactivation-reactivation process was structurally manifested by a dissociation-association phenomenon between subunits. It was clearly shown that the inactivation-dissociation process appeared independent of enzyme concentration whereas the reactivation-association phenomenon was enzyme concentration dependent. However, proteins protect H-flavocytochrome b2 from inactivation-dissociation, only when electrostatic interactions are possible between the two proteins: Horse heart cytochrome c was a good protector whereas serum albumin had no protector effect.     

Mangelnde Reaktivierung reduzierter RNase durch Zajdela-Hepatom-Ascites-Zellen

Summary Decreased reactivation of inactivated RNase by microsomes from a hepatoma as compared to normal liver was observed and is described in more detail. This hampered reactivation of enzymes in general might be responsible for other enzyme deletions in tumours.     

Structure/function analysis of a critical disulfide bond in the active site of l-xylulose reductase

l-Xylulose reductase (XR) is involved in water re-absorption and cellular osmoregulation. The crystal structure of human XR complemented with site-directed mutagenesis (Cys138Ala) indicated that the disulfide bond in the active site between Cys138 and Cys150 is unstable and may affect the reactivity of the enzyme. The effects of reducing agents on the activities of the wild-type and mutant enzymes indicated the reversibility of disulfide-bond formation, which resulted in three-fold decrease in catalytic efficiency. Furthermore, the addition of cysteine (>2 mM) inactivated human XR and was accompanied by a 10-fold decrease in catalytic efficiency. TOF-MS analysis of the inactivated enzyme showed the S-cysteinylation of Cys138 in the wild-type and Cys150 in the mutant enzymes. Thus, the action of human XR may be regulated by cellular redox conditions through reversible disulfide-bond formation and by S-cysteinylation. Received 25 January 2009; received after revision 12 February 2009; accepted 16 February 2009 H.-T. Zhao, S. Endo: These two authors contribute equally to this work.     

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     

Functional consequences of treating turkey liver fructose-1,6-bisphosphatase with penicillin G

Summary Treatment of turkey liver fructose-1,6-bisphosphatase with penicillin G progressively inactivated the enzyme and desensitized the enzyme toward high substrate inhibition. The treatment also led to reduced sensitivity to AMP inhibition and the loss of cooperative interaction among AMP-binding sites. These altered properties were not reversed by dialysis, but were prevented when treatment with penicillin G was performed in the presence of substrate.This work was in part supported by grant RR-8006 from the General Research Branch, Division of Research Resources, NIH (USA) and in part by Faculty Research Grant from Southern School of Pharmacy, Mercer University, Atlanta (Georgia, USA).     

Reactivation of yeast glucose-6-phosphate dehydrogenase denaturated by saturated fatty acids

Summary Activity of yeast glucose-6-phosphate dehydrogenase, inactivated by treatment with saturated fatty acids, can be partially restored by incubation in a medium of suitable ionic composition. The effectiveness of ions in the reactivation process is inversely related to their chaotropic properties. Time-dependence of reactivation extent suggests a 2-step mechanism of enzyme inactivation and the existence of an intermediate form that aggregates through a 2nd-order reaction, producing irreversibly inactive enzyme.This work was supported by a grant of the Italian Consiglio Nazionale delle Ricerche.     

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.     

Model of the interaction between trypsin and alpha 2-macroglobulin of porcine serum]

A comparative study of the dissociation into subunits of Porcine alpha2 M, either native or bound to trypsin (Tn), has been carried out in order to determine the modifications of the alpha2 M structure due to the formation of the Tn-alpha2 M complex. Analytical ultra-centrifugation at pH 3.5 shows that the dissociation is smaller when alpha 2 M is bound to trypsin. Electrophoresis in 4% polyacrylamide gels, in presence of 0.1% SDS, of alpha2 M and Tn-alpha2 M incubated in 1% SDS leads to the same conclusion; the enzyme must stabilize the quaternay structure of alpha2 M. In presence of SDS + beta-mercaptoethanol, only a molecular weight (M.W.) 200,000 band is revealed in electrophoresis pattern of native alpha2 M. In the case of reduced Tn-alpha2 M, some other bands of M.W. 100,000, 50,000, 30,000 appear. When trypsin is inactivated by TLCK 100,000 M.W. band is present, accompanied by the 200,000 M.W. band whose intensity is function of the alpha2 M concentration. The 100,000 M.W. band appears therefore characteristic of the formation of the complex which must imply a proteolytic cleavage in the middle of the 100,000 polypeptidic chain of alpha2 M. A model of the complex is proposed in which the enzyme forms a proteic bridge between the two halves of the alpha2 M molecule.     

Aktivierung einer alkalischen Phosphatase

Summary -Glycerophosphatase prepared from the intestinal mucosa of the calf was purified by fractionated precipitation with alcohol. A further concentration of the enzyme activity was attained by electrophoresis.The activity of the purified enzyme solution was reduced to of its original value when dialysed for 48 hours atp _H 4.5. Atp _H 6 and atp _H 10.5 only a less pronounced decrease of the activity occurred.By addition of heat-inactivated-glycerophosphatase to the enzyme solution which was partly inactivated by dialysis atp _H 4.5 the activity of the latter was increased by about 100%.     

Photodynamic studies on acid ribonuclease from pea cotyledons

Summary In crude extracts, pea cotyledon acid ribonuclease is not inactivated by photodynamic treatment, but after 150-fold purification it is markedly inactivated when illuminated in the presence of rose bengal at pH 7.1. Data suggests that histidine photo-oxidation reduces catalytic activity.     

Evaluation of oral vaccination against mouse-typhoid in a new animal model

Summary In a simple, new animal model the spread of mouse-typhoid within a mouse-colony was studied and oral vaccination against this disease was evaluated. Live vaccine was superior to inactivated vaccine.     

Einwirkung einiger Carbonylreagentien auf das Virus der Maul- und Klauenseuche (MKS)

Summary The virus of foot and mouth disease is inactivated by hydroxylamine, hydrazine and semicarbazide. Such inactivated virus does not differ serologically from intact virus, and represents an interesting antigen for purposes of immunization.

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Herrn Dr.G. A. Moosbrugger, Leiter des Eidg. Vakzine-Instituts in Basel, danken wir, dass er uns die Durchführung dieser Untersuchungen ermöglicht hat.     

Suppression of intrinsic B-cell function in Dengue-infected mice.

Mice infected with Dengue virus show a depressed immune response to lipopolysaccharide (LPS), a helper T-cell-independent antigen, when LPS was administered on day 0, 6 and 12 post infection. Mice injected with inactivated virus failed to show immunosuppression.     

The reactivation of human interferons by guanidine thiocyanate

The addition of 1.5 M guanidine thiocyanate (GuSCN) reactivates inactive human leukocyte interferon. The biological activity of inactivated human fibroblast interferon can be only partially recovered with GuSCN if additional (thermal) energy is supplied.     

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.