scholarly journals NN′-dicyclohexylcarbodi-imide-sensitivity of bovine heart mitochondrial NADH: ubiquinone oxidoreductase. Inhibition of activity and binding to subunits

1988 ◽  
Vol 249 (2) ◽  
pp. 339-344 ◽  
Author(s):  
P T Vuokila ◽  
I E Hassinen
Keyword(s):  
Time Course ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Dependent Manner ◽  
Submitochondrial Particles ◽  
Concentration Dependent Manner ◽  
Oxidoreductase Activity ◽  
Ubiquinone Oxidoreductase ◽  
Nadh Ubiquinone Oxidoreductase ◽  
Electron And Proton Transport ◽  
Molecular Masses

Dicyclohexylcarbodi-imide (DCCD) inhibition of NADH: ubiquinone oxidoreductase was studied in submitochondrial particles and in the isolated form, together with the binding of the reagent to the enzyme. DCCD inhibited the isolated enzyme in a time- and concentration-dependent manner. Over the concentration range studied, a maximum inhibition of 85% was attained within 60 min. The time course for the binding of DCCD to the enzyme was similar to that of activity inhibition. The NADH:ubiquinone oxidoreductase activity of the submitochondrial particles was also sensitive to DCCD, and the locus of binding of the inhibitor was studied by subsequent resolution of the enzyme into subunit polypeptides. Only two subunits (molecular masses 13.7 and 21.5 kDa) were labelled by [14C]DCCD, whereas, when the enzyme in its isolated form was treated with [14C]DCCD, six subunits (13.7, 16.1, 21.5, 39, 43 and 53 kDa) were labelled. Comparison with the subunit labelling of F1F0-ATPase and ubiquinol:cytochrome c oxidoreductase indicated that the labelling pattern of NADH:ubiquinone oxidoreductase, and enzyme complex with a multitude of subunits, is unique and not due to contamination by other inner-membrane proteins. The correlation between the electron- and proton-transport functions and the DCCD-binding components remains to be established.

Properties of neuroprotective cell-permeant Ca2+ chelators: effects on [Ca2+]i and glutamate neurotoxicity in vitro

1994 ◽  
Vol 72 (4) ◽  
pp. 1973-1992 ◽  
Author(s):  
M. Tymianski ◽  
M. P. Charlton ◽  
P. L. Carlen ◽  
C. H. Tator
Keyword(s):  
Time Course ◽  
Dependent Manner ◽  
Protective Effects ◽  
Tetraacetic Acid ◽  
Acetoxymethyl Ester ◽  
Neuroprotective Effects ◽  
Concentration Dependent Manner ◽  
Fura 2 ◽  
Glutamate Neurotoxicity

1. Cell-permeant Ca2+ chelators such as 1,2-bis-(2-amino-phenoxy)ethane- N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) protect neurons against excitotoxic and ischemic neuronal injury in vitro and in vivo. Here we provide the first steps toward characterizing the mechanisms by which these agents produce their neuroprotective effects. 2. Cultured mouse spinal neurons were simultaneously loaded with the Ca2+ indicator fura-2 and with one of three permeant chelators derived from the fast Ca2+ buffer BAPTA, or with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester (EGTA-AM). Adding these chelators did not interfere with the fluorescence spectrum of fura-2 and had no effect on baseline [Ca2+]i. 3. The neurons were challenged with 250 microM L-glutamate for 50 min, producing a marked transient [Ca2+]i increase followed by a decay of [Ca2+]i to a lower “plateau.” About 80% of control neurons succumbed to this excitotoxic insult. Neurons that survived adjusted their plateau [Ca2+]i to lower levels than those that succumbed. 4. Neurons that were pretreated with permeant Ca2+ chelators became more resistant to these neurotoxic challenges. 5. We examined whether this reduction in glutamate neurotoxicity could be related to the given buffer's known Ca2+ affinity (Kd), its Ca2+ binding kinetics, and its ability to attenuate glutamate-induced [Ca2+]i increases. 6. Pretreatment of neurons with BAPTA analogues having Kds ranging from 100 to 3,600 microM 1) attenuated the amplitude and 2) lengthened the time constant describing the rise and decay of the glutamate-evoked [Ca2+]i transient. The magnitude of these effects paralleled the affinity of the chelator for Ca2+. 7. BAPTA-AM and its analogues dramatically attenuated the early neurotoxicity of glutamate, reducing cell deaths by up to 80%. However, in contrast with the graded effects of chelators having different Ca2+ affinities on Ca2+ transients, all BAPTA analogues were equally protective. These protective effects did not relate to the chelators' Ca2+ affinity within a Kd range of 100 nM (for BAPTA) to 3,600 nM (for 5,5'-dibromo BAPTA). 8. BAPTA-AM protected neurons in a concentration-dependent manner with 50% protection obtained with 10 microM, a concentration having no effect on the [Ca2+]i transient amplitude. 9. EGTA, a slow Ca2+ buffer with a similar Ca2+ affinity to BAPTA produced the same effects as BAPTA on [Ca2+]i transient kinetics. However, it was far less protective than BAPTA. 10. The time course of early glutamate neurotoxicity was altered by the BAPTA analogues, but not EGTA. BAPTA analogues caused a small increase in cell deaths in the first minutes of each experiment, followed by relative sparing from further neurodegeneration. 11. The ability of low Ca2+ affinity chelators such as 5,5'-dibromo BAPTA to protect neurons without markedly attenuating measured [Ca2+]i increases conflicts with the hypothesis that global elevations in [Ca2+]i are responsible for triggering neurotoxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of KCa-channel activity by cyclic ADP-ribose and ADP-ribose in coronary arterial smooth muscle

1998 ◽  
Vol 275 (3) ◽  
pp. H1002-H1010 ◽  
Author(s):  
Pin-Lan Li ◽  
Ai-Ping Zou ◽  
William B. Campbell
Keyword(s):  
Smooth Muscle ◽  
Time Course ◽  
Control Level ◽  
Internal Surface ◽  
Threshold Concentration ◽  
Dependent Manner ◽  
Channel Activity ◽  
Arterial Smooth Muscle ◽  
Concentration Dependent Manner ◽  
Cyclic Adp Ribose

The enzymatic pathway responsible for the production and metabolism of cyclic ADP-ribose (cADP-R) in small bovine coronary arteries was characterized, and the role of cADP-R and ADP-ribose (ADP-R) in the regulation of the activity of large-conductance Ca2+-activated K+(KCa) channels was determined in vascular smooth muscle cells (SMC) prepared from these vessels. We found that cADP-R and ADP-R were produced when the coronary arterial homogenates were incubated with 1 mM β-NAD. The time course of the enzyme reactions showed that the maximal conversion rate (1.37 ± 0.03 nmol ⋅ min−1 ⋅ mg protein−1) of β-NAD to cADP-R was reached after 3 min of incubation. As incubation time was prolonged, the production of ADP-R was increased to a maximal rate of 3.66 ± 0.03 nmol ⋅ min−1 ⋅ mg protein−1, whereas cADP-R production decreased. Incubation of the homogenate with cADP-R produced a time-dependent increase in the synthesis of ADP-R. Comparison of coronary arterial microsomes with cytosols shows that the production of both cADP-R and ADP-R in microsomes was significantly greater. In excised inside-out membrane patches of single coronary SMC, the KCa channels were activated when β-NAD, the precursor for both cADP-R and ADP-R, was applied to the internal surface. This effect of β-NAD may be associated with the production of ADP-R, because the KCa-channel activity was increased by ADP-R in a concentration-dependent manner. The open-state probability of the KCa channels increased from a control level of 0.08 ± 0.03 to 0.17 ± 0.05 even at the lowest ADP-R concentration (0.1 μM) studied. However, cADP-R reduced the KCa-channel activity, and the threshold concentration of cADP-R that decreased the average channel activity of the KCa channels was 1 μM. These results provide evidence that cADP-R is produced and metabolized in the coronary arterial smooth muscle and that a cADP-R/ADP-R pathway participates in the control of the KCa-channel activity in vascular SMC.

Inhibition of Maize (Zea mays L.) Root Plasma Membrane-Bound Redox Activities by Coumarins

1994 ◽  
Vol 49 (7-8) ◽  
pp. 447-452 ◽  
Author(s):  
Sabine Lüthje ◽  
José A. Gonzaléz-Reyes ◽  
Placido Navas ◽  
Olaf Döring ◽  
Michael Böttger
Keyword(s):  
Zea Mays ◽  
Plasma Membrane ◽  
Zea Mays L ◽  
Membrane Vesicles ◽  
Dependent Manner ◽  
Plasma Membrane Vesicles ◽  
Two Phase ◽  
Concentration Dependent Manner ◽  
Oxidoreductase Activity ◽  
Membrane Bound

Modulation of plasma membrane-bound NADH:hexacyanoferrate III oxidoreductase activities by dicumarol and warfarin was investigated with plasma membrane vesicles of Zea mays L. (cv. Sil Anjou 18) roots, prepared by aqueous two phase partitioning. Vesicles were about 65% right-side out orientated as demonstrated by enzyme latency of vanadate sensitive ATPase activity. Dicumarol or warfarin, respectively, inhibited NADH:hexacyanoferrate III oxidoreductase activity in a concentration-dependent manner and inhibition could be reversed partially by addition of quinones

Effects of local anesthetics on Na+ channels containing the equine hyperkalemic periodic paralysis mutation

1998 ◽  
Vol 275 (2) ◽  
pp. C389-C400 ◽  
Author(s):  
Rajan L. Sah ◽  
Robert G. Tsushima ◽  
Peter H. Backx
Keyword(s):  
Local Anesthetics ◽  
Time Course ◽  
Slow Component ◽  
Periodic Paralysis ◽  
Dependent Manner ◽  
Wild Type ◽  
Hyperkalemic Periodic Paralysis ◽  
Concentration Dependent Manner ◽  
Drug Concentrations ◽  
Recovery From Inactivation

We examined the ability of local anesthetics to correct altered inactivation properties of rat skeletal muscle Na+channels containing the equine hyperkalemic periodic paralysis (eqHPP) mutation when expressed in Xenopusoocytes. Increased time constants of current decay in eqHPP channels compared with wild-type channels were restored by 1 mM benzocaine but were not altered by lidocaine or mexiletine. Inactivation curves, which were determined by measuring the dependence of the relative peak current amplitude after depolarization to −10 mV on conditioning prepulse voltages, could be shifted in eqHPP channels back toward that observed for wild-type (WT) channels using selected concentrations of benzocaine, lidocaine, and mexiletine. Recovery from inactivation at −80 mV (50-ms conditioning pulse) in eqHPP channels followed a monoexponential time course and was markedly accelerated compared with wild-type channels (τWT= 10.8 ± 0.9 ms; τeqHPP= 2.9 ± 0.4 ms). Benzocaine slowed the time course of recovery (τeqHPP,ben = 9.6 ± 0.4 ms at 1 mM) in a concentration-dependent manner. In contrast, the recovery from inactivation with lidocaine and mexiletine had a fast component (τfast,lid = 3.2 ± 0.2 ms; τfast,mex = 3.1 ± 0.2 ms), which was identical to the recovery in eqHPP channels without drug, and a slow component (τslow,lid = 1,688 ± 180 ms; τslow,mex = 2,323 ± 328 ms). The time constant of the slow component of the recovery from inactivation was independent of the drug concentration, whereas the fraction of current recovering slowly depended on drug concentrations and conditioning pulse durations. Our results show that local anesthetics are generally incapable of fully restoring normal WT behavior in inactivation-deficient eqHPP channels.

Propofol Inhibits Cardiac L-Type Calcium Current in Guinea Pig Ventricular Myocytes

1996 ◽  
Vol 84 (3) ◽  
pp. 626-635 ◽  
Author(s):  
Ching-Yue Yang ◽  
Chih-Shung Wong ◽  
Chuan-Cheng Yu ◽  
Hsiang-Ning Luk ◽  
Cheng-I Lin
Keyword(s):  
Steady State ◽  
Guinea Pig ◽  
Time Course ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Hill Coefficient ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Inactivation Curve ◽  
Whole Cell

Background Propofol may exert negative inotropic and chronotropic actions in the heart. Single-channel studies show that propofol affects the kinetics of opening and closing of cardiac L-type calcium channels (ICa(L)) without altering channel conductance. The aim of this study was to investigate the mechanisms of depressant effects of propofol on cardiac whole-cell ICa(L). Methods Single ventricular myocytes were freshly dissciated from guinea pig hearts using enzymatic isolation. One-suction electrode voltage-clamp technique (whole-cell mode) was used. LCa(L) was separated from other contaminated ionic currents. Propofol was applied in the commercial 10% Intralipid emulsion formula (Zeneca, UK). Results In isolated cardiomyocytes, propofol significantly inhibited whole-cell ICa(L) in a concentration-dependent manner (K D = 52.0 microM; Hill coefficient = 1.3). The solvent (Intralipid) did not affect ICa(L). Propofol decreased ICa(L) at all potentials tested along the voltage axis and reduced the slope conductance. The threshold potential for activation and the peak potential of the current-voltage relationship were not changed by propofol. The steady-state activation curves overlapped in the absence and the presence of 56 microM propofol. In contrast, the steady-state inactivation curve was shifted in the hyperpolarizing direction. The time course of the recovery from inactivation was delayed by 56 microM propofol. The blocking action on ICa(L) of propofol shows marked resting block and use-dependent block. Propofol caused more pronounced inhibition at a higher stimulation frequency. The effect of propofol on the inactivation process was even more clear on ICa(L). Conclusions The authors conclude tha propofol, at supratherapeutic concentrations, inhibits cardiac ICa(L). This inhibition is mainly due to a shift of inactivation curve and a reduction in slope conductance.

scholarly journals Preservation of NADH ubiquinone-oxidoreductase activity by Src kinase-mediated phosphorylation of NDUFB10

2012 ◽  
Vol 1817 (5) ◽  
pp. 718-725 ◽  
Author(s):  
Etienne Hebert-Chatelain ◽  
Caroline Jose ◽  
Nicolas Gutierrez Cortes ◽  
Jean-William Dupuy ◽  
Christophe Rocher ◽  
...  
Keyword(s):  
Src Kinase ◽  
Oxidoreductase Activity ◽  
Ubiquinone Oxidoreductase ◽  
Nadh Ubiquinone Oxidoreductase

scholarly journals Effects of proteolytic digestion by chymotrypsin on the structure and catalytic properties of reduced nicotinamide–adenine dinucleotide–ubiquinone oxidoreductase from bovine heart mitochondria

1977 ◽  
Vol 165 (2) ◽  
pp. 295-301 ◽  
Author(s):  
Susan E. Crowder ◽  
C. Ian Ragan
Keyword(s):  
Complex I ◽  
Time Course ◽  
Nadh Dehydrogenase ◽  
Sodium Dodecyl ◽  
Complex Iii ◽  
Type Ii ◽  
Oxidoreductase Activity ◽  
Ubiquinone Oxidoreductase ◽  
Nadh Cytochrome ◽  
Reduced Nicotinamide Adenine Dinucleotide

1. Incubation of NADH–ubiquinone oxidoreductase (Complex I) with chymotrypsin caused loss of rotenone-sensitive ubiquinone-1 reduction and an increase in rotenone-insensitive ubiquinone reduction. 2. Within the same time-course, NADH–K3Fe(CN)6 oxidoreductase activity was unaffected. 3. Mixing of chymotrypsin-treated Complex I with Complex III did not give rise to NADH–cytochrome c oxidoreductase activity. 4. Gel electrophoresis in the presence of sodium dodecyl sulphate revealed selective degradation of several constituent polypeptides by chymotrypsin. 5. With higher chymotrypsin concentrations and longer incubation times, a decrease in NADH–K3Fe(CN)6 oxidoreductase was observed. The kinetics of this decrease correlated with solubilization of the low-molecular-weight type-II NADH dehydrogenase (subunit mol.wts. 53000 and 27000) and with degradation of a polypeptide of mol.wt. 30000. 6. Phospholipid-depleted Complex I was more rapidly degraded by chymotrypsin. Specifically, a subunit of mol.wt. 75000, resistant to chymotrypsin in untreated Complex I, was degraded in phospholipid-depleted Complex I. In addition, the 30000-mol.wt. polypeptide was also more rapidly digested, correlating with an increased rate of transformation to type II NADH dehydrogenase.

scholarly journals The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Evidence for stoicheiometric association

1978 ◽  
Vol 174 (3) ◽  
pp. 783-790 ◽  
Author(s):  
C I Ragan ◽  
C Heron
Keyword(s):  
Cytochrome C ◽  
Complex I ◽  
Structural Features ◽  
Complex Iii ◽  
Molar Ratio ◽  
Oxidoreductase Activity ◽  
Ubiquinone Oxidoreductase ◽  
Nadh Ubiquinone Oxidoreductase ◽  
Cytochrome C Reduction ◽  
Nadh Cytochrome

1. The NADH-ubiquinone oxidoreductase complex (Complex I) and the ubiquinol-cytochrome c oxidoreductase complex (Complex III) combine in a 1:1 molar ratio to give NADH-cytochrome c oxidoreductase (Complex I-Complex III). 2. Experiments on the inhibition of the NADH-cytochrome c oxidoreductase activity of mixtures of Complexes I and III by rotenone and antimycin indicate that electron transfer between a unit of Complex I-Complex III and extra molecules of Complexes I or III does not contribute to the overall rate of cytochrome c reduction. 3. The reduction by NADH of the cytochrome b of mixtures of Complexes I and III is biphasic. The extents of the fast and slow phases of reduction are determined by the proportion of the total Complex III specifically associated with Complex I. 4. Activation-energy measurements suggest that the structural features of the Complex I-Complex III unit promote oxidoreduction of endogenous ubiquinone-10.

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