Deactivated State Research Articles

Nonradiative deactivation and triplet states in donor-aryl-acceptor compounds (dialkylaminobenzonitriles)

Intersystem crossing rates and triplet state properties of TICT (twisted intramolecular charge transfer) model compounds (dimethylaminobenzonitrile — DMABN — and its methyl substituted derivatives, 3-cyanoindoline, 3-cyanobenzquinuclidine — CBQ — and benzonitrile) were studied by various experimental and theoretical methods (emission spectroscopy, transient absorption techniques, EPR spectroscopy and semiempirical quantum chemical calculations) with the aim to obtain a quantitative deactivation pattern of this class of compounds in solvents of different polarity. The lowest triplet state, populated after excitation, is generally, a non-charge-transfer state, i.e. the 3A 1 of planar DMABN, irrespective of solvent polarity and compound flexibility, with the only exception of the rigidly perpendicular CBQ, which relaxes to the 3A 1 state of the benzonitrile moiety. In non-polar solvents intersystem crossing increases with increasing charge transfer character of the excited singlet; in ethanol, the intersystem crossing rate is about the same for all compounds studied, with the exception of benzonitrile, and in general significantly lower than in a non-polar environment. From the mechanistic point of view, intersystem crossing from 1TICT can be described as a thermal charge recombination process in the normal Marcus region. With increasing solvent polarity, the decreasing energy gap between these two states then entails a decreasing intersystem crossing rate and an increasing 1TICT lifetime, as experimentally observed. In hydroxylic solvents, internal conversion to the ground state is the second dominant deactivation pathway of 1TICT states; it shows a significant deuterium isotope effect.

CO oxidation over Au/ZrO 2 catalysts: Activity, deactivation behavior, and reaction mechanism

Gold catalysts supported on amorphous zirconia have been prepared by coprecipitation. These catalysts exhibit a high initial CO oxidation activity, which is due to a synergy between the zirconia and the supported gold particles. The activity decreases significantly over a time scale of ≈20 h. The deactivation behavior is only weakly influenced by pretreatments in O 2, CO, or CO 2. However, the long-time stability is found to depend strongly on the O 2/CO feed ratio. With stoichiometric reaction mixtures (O 2/CO = 0.5), the activity stays nearly constant over the time of observation. In the presence of excess oxygen (O 2/CO = 1 or 40, respectively), rapid deactivation is observed. From a careful monitoring of the carbon balance, and from thermal desorption/mass spectrometry experiments, a progressive covering of the surface by oxygenated carbon species is deduced. Diffuse reflectance FTIR experiments indicate the presence of carbonates, formate, and several types of differently bound CO molecules on the surface. Temperature-dependent experiments reveal that these species are involved in a series of interconnected equilibria, which are coupled to the relevant adsorption/de sorption processes. Oxidized and reduced states of the catalyst can be distinguished. The deactivated state in excess oxygen is characterized by a high concentration of surface formate. On the deactivated catalyst, oxydation of formate and of CO appears to be slow, possibly as a consequence of site blocking.

Ca(2+)-calmodulin-dependent protein kinases Ia and Ib from rat brain. II. Enzymatic characteristics and regulation of activities by phosphorylation and dephosphorylation.

In addition to physical properties (DeRemer, M. F., Saeli, R. J., and Edelman, A. M. (1992) J. Biol. Chem. 267, 13460-13465), enzymatic and regulatory characteristics indicate that calmodulin (CaM) kinase Ia and CaM kinase Ib are distinct entities. The Km values for ATP and site 1 peptide were similar between the two kinases, however, CaM kinase Ib is approximately 20-fold more sensitive to CaM than is CaM kinase Ia. The kinases also displayed differential sensitivities to divalent metal ions. For both kinases, site 1 peptide, synapsin I, and syntide-2 were highly preferred substrates relative to others tested. A 72-kDa protein from a heat-treated extract of rat pancreas was phosphorylated by CaM kinase Ib but not by CaM kinase Ia. CaM kinase Ia activity displayed a pronounced lag in its time course suggesting enzyme activation over time. Preincubation of CaM kinase Ia in the combined presence of Ca(2+)-CaM and MgATP led to a time-dependent increase in its site 1 peptide kinase activity of up to 15-fold. The extent of activation of CaM kinase Ia correlated with the extent of autophosphorylation. The enzyme retained full Ca(2+)-CaM dependence in the activated state which was rapidly reversible by treatment with protein phosphatase 2A catalytic subunit. Thus, the activation of CaM kinase Ia is a result of its Ca(2+)-CaM-dependent autophosphorylation. CaM kinase Ib was not activated by preincubation under autophosphorylating conditions yet lost approximately 90% of its activity toward either an exogenous substrate (site 1 peptide) or itself (autophosphorylation) after incubation with protein phosphatase 2A catalytic subunit. The deactivated state was not reversed by subsequent incubations under autophosphorylating conditions. Thus, CaM kinase Ib activity is dependent upon phosphorylation by a regulating kinase(s) which is resolved from CaM kinase Ib during purification of the latter.

Operative mortality with implantation of the automatic cardioverter-defibrillator

Operative mortality was studied in 939 consecutive patients undergoing initial implantation of an automatic implantable cardioverter-defibrillator (AICD ™) at 15 hospitals. Twenty-nine (3.1%) patients died during the first 30 days after surgery. Among patients who survived beyond the first 30 postoperative days, ejection fraction data were available in 219; compared with the mortality group, these survivors had a significantly higher ejection fraction (34 ± 15 vs 26 ± 10%, respectively, p < 0.001), despite similar age, sex, underlying heart disease, type of presenting arrhythmia and prevalence of concomitant surgery. The causes of perioperative death were sudden in 7 (24%), tachyarrhythmic/non-sudden in 5 (17%), cardiac nonarrhythmic in 9 (31%), and noncardiac in 8 (28%). Twenty-four (83%) of the deaths occurred before hospital discharge, and in all 9 instances of in-hospital sudden and tachyarrhythmic/nonsudden death, the initial recorded rhythm was sustained ventricular tachycardia or fibrillation; in 5 (56%) of these 9 patients the AICD had been in a deactivated state since implantation. Other possible contributory factors in the 12 sudden or tachyarrhythmic/nonsudden deaths included acute myocardial ischemia or infarction in 2 (17%), and “device proarrhythmia” in 3 (25%) that were AICD-related in 2 and secondary to an antitachycardia pacemaker in another; defibrillation threshold testing was not performed in 3 patients (1 of whom had terminal ventricular fibrillation). Thus, in this multicenter experience with thoracotomy requiring AICD implantation, operative (30-day) mortality was 3.1% and correlated inversely with left ventricular ejection fraction. Further studies are needed to explore the potential role of management practices in the occurrence of sudden or tachyarrhythmic/nonsudden death, which accounted for 41% of all postoperative fatalities in this AICD implantation series.

Differential in situ expansion and gene expression of CD4+ and CD8+ tumor‐infiltrating lymphocytes following adoptive immunotherapy in a murine tumor model system

In previous reports, we demonstrated that adoptively transferred T cells homed to the tumor site (among other sites) and that amplification of immune responses occurred in situ leading to the generation of cytotoxic CD8+ tumor-infiltrating lymphocytes (TIL) and macrophages. The present report extends these findings and shows that following adoptive immunotherapy (AIT) of mice bearing the immunogenic transplanted methylcholanthrene-induced rhabdomyosarcoma (MCA/76-9) there was a differential expansion of CD4+ and CD8+ TIL, the numbers peaking on days 6 and 8, respectively. At this time, CD8+ TIL accounted for the majority of Thy-1+ cells. Northern analyses of RNA extracted from positively selected (by panning) Thy-1+, CD8+ and CD4+ TIL isolated 8 days after AIT indicated the following: in five separate experiments, CD4+ cells expressed three- to sixfold more interleukin (IL)2 mRNA and six- to eightfold more IL6 mRNA than CD8+ cells, while CD8+ TIL expressed three- to sixfold more IL2 receptor (IL2R) mRNA and four- to sixfold more interferon-gamma mRNA than CD4+ cells. TIL cultured in 10% fetal bovine serum failed to release IL2 over a 24-h period, whereas both IL6 and interferon-gamma activities were demonstrable. The level of IL2R mRNA expression was reflected by a vigorous proliferative response of CD8+ TIL to exogenous recombinant IL2 and only a low response by CD4+ cells suggesting that most of the CD4+ TIL were in the resting stage. This was confirmed when it was shown that the incubation of panned CD4+ TIL with IL2 supplemented with irradiated spleen cells and "spent" 76-9 tumor culture supernatant (as a source of antigen) induced expansion of TIL resulting in a population consisting of greater than 90% CD4+ TIL. The overall data suggest that the relatively deactivated state of the CD4+ TIL at this particular time reflects the status of the rejection process in terms of the absence or low concentration of stimulating tumor-associated antigen.

Slow active/inactive transition of the mitochondrial NADH-ubiquinone reductase

NADH-ubiquinone reductase of bovine heart submitochondrial particles as prepared is unable to catalyze either the direct or reverse electron transfer from NADH to ubiquinone. The deactivated state of the enzyme in coupled particles was revealed as: (i) the absence of the rotenone-sensitive, Δ \\ ̃ gm H + - dependent succinate-ferricyanide reductase activity; (ii) a prominent lag in the aerobic succinate-supported, Δ \\ ̃ gm H + - dependent NAD + reduction; and (iii) a lag in the rotenone-sensitive NADH-ubiquinone reductase or NADH oxidase activities. Being inactive as NADH-ubiquinone reductase (direct or reverse), the enzyme is fully active as rotenone-insensitive NADH-ferricyanide reductase. The enzyme can be activated by preincubation with substrates (NADH or NADPH) only under the conditions where the turnover of the NADH-ubiquinone reductase reaction (but not in the NADH-ferricyanide reductase) occurs. Partial activation of the enzyme was observed when the particles were preincubated with rotenone. Neither NADH under the conditions when the ubiquinone pool was reduced nor succinate plus Δ \\ ̃ gm H + or dithionite were able to activate the enzyme. Once activated, the enzyme remains in the active state for quite a long time (more than 5 h at 0 °C). The deactivation rate is extremely temperature-dependent, being insensitive to NAD +, ferricyanide or succinate. A comparison of the enzyme activation/deactivation kinetics showed that the same mechanism is involved in the slow activation of the direct and reverse electron transfer from NADH to ubiquinone. Activated particles catalyze the aerobic Δ \\ ̃ gm H + - dependent succinate-supported reverse electron transfer in the absence of ATP at a rate comparable with that of NADH-ubiquinone reductase.

Interactions of oxaloacetate with Escherichia coli fumarate reductase

Fumarate reductase of Escherichia coli is converted to a deactivated state when tightly bound by oxaloacetate (OAA). Incubation of the inhibited enzyme with anions or reduction of the enzyme by substrate restores both the activity of the enzyme and its sensitivity to thiol reagents. In these respects the enzyme behaves like cardiac succinate dehydrogenase. Close to an order of magnitude difference was found to exist between the affinities of OAA for the oxidized ( K D ∼ 0.12 μM) and reduced ( K D ∼ 0.9 μM) forms of fumarate reductase. Redox titrations of deactivated fumarate reductase preparations have confirmed that reductive activation, as in cardiac succinate dehydrogenase ( B. A. C. Ackrell, E. B. Kearney, and D. Edmondson (1975) J. Biol. Chem. 250, 7114–7119 ), is the result of reduction of the covalently bound FAD moiety and not the non-heme iron clusters of the enzyme. However, the processes differed for the two enzymes; activation of fumarate reductase involved 2 e − and 1H +, consistent with reduction of the flavin to the anionic hydroquinone form, whereas the process requires 2 e − and 2H + in cardiac succinate dehydrogenase. The reason for the difference is not known. The redox potential of the FAD FADH 2 couple in FRD ( E m ∼ −55 mV) was also slightly more positive than that in cardiac succinate dehydrogenase (—90 mV).

Reversible deactivation of beta-lactamase by quinacillin. Extent of the conformational change in the isolated transitory complex.

Conditions have been established where the deactivation of the beta-lactamase from Staphylococcus aureus PC1 by the penicillin substrate, quinacillin, is close to complete but fully reversible. The temperature-dependence of the rate of re-activation indicated a half-life of about 170 min for the deactivated state at 0 degrees C. Measurement of the relative viscosity of mixtures of enzyme and quinacillin at 8.4 degrees C ruled out any significant difference in shape or solvation between the deactivated and the normal enzyme. C.d. measurements of the deactivated protein, separated from excess quinacillin, showed that the quinacillin side-chain chromophore was bound in an asymmetric environment. The ellipticity associated with the bound quinacillin chromophore decreased with the same first-order rate constant as that for reappearance of enzyme activity. These findings support the accumulation of a deactivated state that contains bound quinacillin or a derivative. Quinacillin caused a 3-fold increase in the rate of 3H exchange-out (at a rate that was low compared with that for the substantially unfolded or expanded protein). However, there was rapid exchange-out of about 50 3H atoms on addition of 1 M-urea to the deactivated enzyme, whereas the same concentration had no effect on the exchange-out of 3H from native enzyme. The interpretation that quinacillin increases the susceptibility of the native state to unfolding in the presence of urea is supported by the demonstration that SO4(2)- ions decreased the rate and extent of deactivation but had no effect on the rate of re-activation, as predicted from the observation that SO4(2)- ions, in competition with urea, stabilize the native state relative to the partially unfolded state H [Mitchinson & Pain (1985) J. Mol. Biol. 184, 331-342].

Cumene cracking on NaHY and NaH-ZSM—5 type zeolites as catalysts

Cumene cracking on NaHY zeolites with different degrees of cation exchange and on highly exchanged ZSM—5 type zeolites was investigated. In all cases the predominant reaction was cracking of cumene to benzene and propylene. The course of the side reactions was dependant on the type of zeolite, and for NaHY zeolites, on the degree of cation exchange and the state of deactivation of the catalyst. On NaHY zeolites saturated hydrocarbons, butanes, pentanes and hexanes appear as the products of secondary transformations of propylene occurring via formation and disproportionation of the oligomer. The other products of secondary reactions are toluene, ethylbenzene and n-propylbenzene. The amount of side products increases with the increasing acidity of zeolite thus indicating that side reactions need more strongly acid groups than the dealkylation of benzene. In the case of ZSM—5 type zeolite primary propylene turns partially into other olefins; butenes and pentenes. The differences in the behaviour of NaHY and ZSM—5 type zeolites are discussed and interpreted as the result of shape selectivity.

Reconstitution of quinone reduction and characterization of Escherichia coli fumarate reductase activity.

Resolution of the fumarate reductase complex (ABCD) of Escherichia coli into reconstitutively active enzyme (AB) and a detergent preparation containing peptides C and D resulted in loss of quinone reductase activity, but the phenazine methosulfate or fumarate reductase activity of the enzyme was unaffected. An essential role for peptides C and D in quinone reduction was confirmed by restoration of this activity on recombination of the respective preparations. Neither peptide C nor peptide D by itself proved capable of permitting quinone reduction and membrane binding by the enzyme when E. coli cells were transformed with plasmids coding for the enzyme and the particular peptides. Transformation of a plasmid coding for all subunits resulted in a 30-fold increase in membrane-bound complex, which exhibited, however, turnover numbers for succinate oxidation and fumarate reduction that were intermediate between the high values characteristic of chromosomally produced complex and the relatively low values found for the isolated complex. It is also shown that preparations of the isolated complex and membrane-bound form of the enzyme, as obtained from anaerobically grown cells, are in the deactivated state owing to the presence of tightly bound oxalacetate and thus must be activated prior to assay.

Secretion of toxic oxygen products by macrophages: regulatory cytokines and their effects on the oxidase.

We are attempting to identify cytokines that regulate macrophage secretion of reactive oxygen intermediates (ROI) and to analyse the biochemical basis of their effects. In both humans and mice, interferon-gamma (IFN-gamma) appears to be the chief factor secreted by clonally unselected lymphocytes that enhances macrophage oxidative metabolism and antiprotozoal activity. In vivo administration of recombinant IFN-gamma enhances the ROI secretory capacity of monocytes in humans, and the secretion of ROI and killing of protozoa by peritoneal macrophages in mice. A protein secreted by murine tumours and certain non-malignant cells exerts opposing effects. This macrophage deactivation factor (MDF) both blocks the induction of activation by IFN-gamma and reverses pre-existent activation. MDF action is non-toxic and selective, suppressing the secretion of ROI, killing of intracellular protozoa, and expression of Ia antigen, without inhibiting secretion of several other products, or synthesis of protein, ingestion of particles or adherence to culture vessels. The suppressive effect of MDF is reversed over several days after its removal. This reversal is hastened by IFN-gamma. Profound suppression of oxidative metabolism accompanies the differentiation of murine monocytes into Kupffer cells. The capacity of Kupffer cells to secrete ROI and kill intracellular protozoa remains deficient even after exposure to IFN-gamma. Thus, four states of macrophage activation can provisionally be discerned: the transition of mouse peritoneal macrophages from the non-activated to the activated state is accompanied by a ninefold increase in affinity of the superoxide-producing enzyme for NADPH, without a marked increase in cellular Vmax or content of cytochrome b559. The MDF-induced transition of mouse peritoneal macrophages from the activated to the deactivated state is accompanied by both an increase in Km and a decrease in apparent V max of the oxidase. There are no changes in the phorbol myristate acetate receptor number or affinity, glucose transport, NADPH levels, cytochrome b559 content, catalase (EC 1.11.1.6) GSH, GSH peroxidase (EC 1.11.1.9), GSH reductase (EC 1.6.4.2) or myeloperoxidase, consistent with the suppressed ROI secretory capacity and antiprotozoal activity of these cells. The Kupffer cell, whose non-responsiveness to IFN-gamma may mark it as inactivated, appears to lack detectable NADPH oxidase activity, despite the probable presence of cytochrome b559, and in this regard differs from both non-activated and deactivated macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

Properties of ATP tightly bound to catalytic sites of chloroplast ATP synthase.

Under steady state photophosphorylating conditions, each ATP synthase complex from spinach thylakoids contains, at a catalytic site, about one tightly bound ATP molecule that is rapidly labeled from medium 32Pi. The level of this bound [32P]ATP is markedly reduced upon de-energization of the spinach thylakoids. The reduction is biphasic, a rapid phase in which the [32P] ATP/synthase complex drops about 2-fold within 10 s, followed by a slow phase, kobs = 0.01/min. A decrease in the concentration of medium 32Pi to well below its apparent Km for photophosphorylation is required to decrease the amount of tightly bound ATP/synthase found just after de-energization and before the rapid phase of bound ATP disappearance. The [32P]ATP that remains bound after the rapid phase appears to be mostly at a catalytic site as demonstrated by a continued exchange of the oxygens of the bound ATP with water oxygens. This bound [32P]ATP does not exchange with medium Pi and is not removed by the presence of unlabeled ATP. The levels of tightly bound ADP and ATP arising from medium ADP were measured by a novel method based on use of [beta-32P]ADP. After photophosphorylation and within minutes after the rapid phase of bound ATP loss, the measured ratio of bound ADP to ATP was about 1.4 and the sum of bound ADP plus ATP was about 1/synthase. This ratio is smaller than that found about 1 h after de-energization. Hence, while ATP bound at catalytic sites disappears, bound ADP appears. The results suggest that during and after de-energization the bound ATP disappears from the catalytic site by hydrolysis to bound ADP and Pi with subsequent preferential release of Pi. These and related observations can be accommodated by the binding change mechanism for ATP synthase with participation of alternating catalytic sites and are consistent with a deactivated state arising from occupancy of one catalytic site on the synthase complex by an inhibitory ADP without presence of Pi.

Return of fear: The role of inhibition

In a previous study investigating the relationship between subjective, physiological and behavioural changes during treatment of specific phobias, a marked between-session increase was found in subjective fear after high, but not after low intensity phobic stimulation although the groups showed similar fear ratings and heart rates immediately after treatment (Grey et al, 1979). It was hypothesized that the high intensity stimulation led to ‘treatment fatigue’; a state of deactivation, the recovery from which led to a return of fear. In this study high intensity in vivo stimulation was used for the treatment of phobic subjects under conditions of distributed and massed practice. No differences emerged between treatment conditions. A small group of Ss with a conspicuously high heart rate (mean level 120 bpm) when rating zero fear, showed a significant increase in fear after one week's interval, although they had shown similar improvement to the Ss during treatment. Subjects in both treatment conditions showed lower heart rate at all post-treatment assessments than pre-treatment. The high initial heart rate Ss continued to have higher heart rate than other Ss throughout the experiment, although they showed a decrease after treatment. It was only these Ss with desynchronously high heart rate, i.e. high heart rate with zero fear ratings, who suffered a return of fear, whereas high heart rate during high fear did not predict either response to treatment or return of fear. A further finding was a lack of correlation between the behavioural and physiological indices of fear, namely, between distance and heart rate at high levels of fear.

Pig gastric and small-intestinal mucus glycoproteins: proposed role in polymeric structure for protein joined by disulphide bridges.

of the value before heating, but the rotation of the control enzyme decreased further, suggesting that cross-links were favouring the acquisition of native-like structure, whereas the unsubstituted protein was irreversibly denatured at 8OOC. Penicillinase activity was determined from the decrease in either A,,, (1 mM-benzylpeniciIlin) or (55pM-cephaloridine) in 1 mM-EDTA (disodium salt) and 39 rn~-KH,P0, /6 1 mMNa,HPO,, pH 7.0 at 2OOC. The benzylpenicillinase activity of the control enzyme was 201pmol/min per mg. The rate of cephaloridine hydrolysis after maximal deactivation was 0.053 pmol/min per mg. Treatment with dimethyl suberimidate for either 90, 150 or 1140min resulted in a decrease in benzylpenicillinase specific activity to 78% (s.D. = 2.0 (n = 3)l of that of the control enzyme. No deactivation by benzylpenicillin was observed with either the control or the treated enzyme. Both control and treated enzymes were deactivated by cephaloridine. However, the deactivated rate of cephaloridine hydrolysis with the treated enzyme was 45.5% [s.D. = 1.3 (n = 3)1 of the rate with the control enzyme. In summary, treatment with dimethyl suberimidate at pH 8.4 resulted in a product with almost normal activity with benzylpenicillin but with less than half of the normal deactivated activity with cephaloridine. The small effect on the activity with benzylpenicillin suggests that cross-linking did not directly involve the active site. We suggest that some of the (presumably heterogeneous) cross-links stabilize part of the protein conformation that is barely involved in any isomerization during the catalytic cycle with benzylpenicillin. The additional stability could contribute to lower activity in the deactivated state by an indirect effect on the active site. Alternatively, if the deactivated enzyme consists of an equilibrium mixture of active and inactive molecules, cross-linking could retard a conformational change that is necessary for the reactivation process but that is not necessary for deactivation. Our results contrast with the correlation between destruction of conformational 'flexibility' and protection against deactivation that has been reported for another penicillinase (Klemes 8c Citri, 1979). ,

Evidence for a reversible functional state of neutrophil chemotactic deactivation.

Neutrophils pretreated with a chemotactic factor become deactivated, that is, unresponsive to a subsequent chemotactic stimulus. A decay of surface esterase activity, an increased adhesiveness, an auto-oxidative damage of the cell structures and microtubule hyperpolymerization have been previously proposed as the cause of the chemotactic deactivation. To further understand the cellular mechanisms involved in this process, we have studied the locomotory behaviour of deactivated neutrophils in an experimental system allowing differentiation among the various types of migratory cellular response to chemotactic factors. Thus, we have found that deactivated neutrophils retain a normal capacity of random locomotion, but entirely lose their true chemotactic responsiveness. Diamide, which disrupts assembled microtubules in concanavalin A-treated cells, was able to completely restore the true chemotactic response of deactivated cells. These observations suggest that neutrophil deactivation is a reversible cellular event dependent on the functional state of the microtubular system.

Activation of Succinate Dehydrogenase by FMNH2 and by Photoreduction

Abstract Reports in the literature have indicated that the fumarate reductase activity of succinate dehydrogenase, as measured by the fumarate-FMNH2 assay, is not influenced by the state of activation of the enzyme. This conclusion was based on the observation that the activity in the fumarate reductase assay, measured at 15°, where significant activation by succinate or malonate is not observed, was the same whether the enzyme was fully activated or essentially in the deactivated state. It is shown in the present paper that under the conditions of the fumarate-FMNH2 assay full activation of succinate dehydrogenase occurs even at 15°. The activating agent responsible is the reduced flavin, which acts as electron donor in this assay. Flavin mononucleotide, reduced by light-EDTA, causes full activation, but the energy of activation for the process is 17 Cal per mole, a value much lower than that characterizing activation by substrates, competitive inhibitors, or reduced coenzyme Q10. Light irradiation of the purified soluble enzyme in the presence of EDTA but without added flavin also causes complete activation.

Regulation of Succinate Dehyrogenase in Higher Plants

The spectrophotometric phenazine methosulfate assay of succinate dehydrogenase was adapted to use with cauliflower (Brassica oleracea) and mung bean (Phaseolus aureus) mitochondria with suitable modifications to overcome the permeability barrier to the dye. Procedures in the literature for the isolation and sonic disruption of mitochondria from these sources were modified to assure maximal yield and stability of the enzyme. In tightly coupled mung bean mitochondria, as isolated, about half of the succinate dehydrogenase is in the deactivated state, and the enzyme is further extensively deactivated on sonication or freeze-thawing. In cauliflower mitochondria most of the enzyme is in the deactivated form, and little or no further deactivation occurs on sonication or freeze-thawing. Incubation of mitochondria from either source with succinate leads to full activation of the enzyme. The energy of activation for the conversion of the deactivated to the activated form in membranal preparations under the influence of substrate is about 30,000 cal/mole, essentially the same value as in animal tissues. Activation of the enzyme also occurs under the influence of a variety of other agents, among which the action of anions as activators is documented in the present paper. Activation is accompanied by the release of very tightly bound oxaloacetate. As in animal tissues, the enzyme appears to contain covalently bound flavin (histidyl 8alpha-FAD), and the turnover number is 19,400 moles of succinate oxidized/mole of histidyl flavin at pH 7.5, 38 C.

Tightly bound oxalacetate and the activation of succinate dehydrogenase

Soluble succinate dehydrogenase prepared from acetone powders of submitochondrial particles is almost entirely in the deactivated state and contains 0.5 mole of oxalacetate (OAA) per mole of histidyl flavin. OAA is dissociated by succinate, malonate, IDP, ITP, and high concentrations of anions at elevated temperatures, but not significantly in the cold, with concurrent activation of the enzyme; the high energy of activation observed for OAA release and for activation suggests that a conformation change in the protein is involved. On removal of OAA, a reversible activation-deactivation cycle dependent on the pH is demonstrable. Submitochondrial particles behave similarly but appear to contain 1 mole of tightly bound OAA per histidyl flavin in the deactivated state.

A new column system for isothermal gas chromatographic analysis of light gases (H2, O2, N2, CO, CH4, CO2, C2H4, C2H6 and C2H2) employing a column switch technique

A new column system for the isothermal analysis of H2, O2, N2, CO, CH4, CO2, C2H4, C2H6 and C2H2 is described. Higher hydrocarbons and water are backflushed from the system. The use of 13X molecular sieve in a relatively deactivated state shortens the elution times of the light components and improves peak shapes. Both factors enable quantitative analyses to be carried out satisfactorily at lower concentrations than would otherwise be possible. Analysis of all compounds listed can be carried out in eleven minutes. Sparingly activated 13X molecular sieve columns have proved to be very stable and rarely require reactivation. The method of column switching employed avoids the use of mechanical valves in the sample path and is suitable for automatic operation.