scholarly journals The effects of diphenyleneiodonium on mitochondrial reactions. Relation of binding of diphenylene[125I]iodonium to mitochondria to the extent of inhibition of oxygen uptake

1976 ◽  
Vol 158 (2) ◽  
pp. 307-315 ◽  
Author(s):  
S J Gatley ◽  
H S A Sherratt
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Nadh Dehydrogenase ◽  
Liver Mitochondria ◽  
Nadh Oxidation ◽  
Affinity Constant ◽  
Rat Liver Mitochondria ◽  
High Affinity ◽  
Limiting Stage ◽  
Rate Limiting

1. Several ring-substituted derivatives of diphenyleneiodonium catalyse the exchange of Cl- and OH- ions across the inner membrane of rat liver mitochondria. They also inhibit state 3 and state 3u oxidations of glutamate plus malate in the presence of Cl- more than in its absence. Most have activities similar to diphenyleneiodonium, although 2,4-dichlorodiphenyleneiodonium is up to 50 times more active. 2. Diphenyleneiodonium inhibits soluble rat liver NADH dehydrogenase and NADH oxidation by rat liver sub-mitochondrial particles directly; 2,4-dichlorodiphenyleneiodonium is only about twice as inhibitory. 3. Liver mitochondria contain two classes of binding sites for diphenylene[125I]iodonium, namely high-affinity sites with an affinity constant of 3 X 10(5) M-1 (1–2 nmol/mg of protein), and low-affinity sites with an affinity constant of 1.3 X 10(3) M-1 (80 nmol/mg of protein). Both sites occur in hepatocytes with a relative enrichment of the low-affinity site. Nadh dehydrogenase preparations only apparently contain high-affinity binding sites. Only low-affinity sites occur in erythrocytes. 4. 2,4-Dichlorodiphenyleneiodonium competes with diphenylene[125I]iodonium for both low- and high-affinity sites, whereas tri-n-propyltin only competes for the low-affinity sites. 5. The high-affinity sites are apparently associated with NADH dehydrogenase and the low-affinity sites probably represent electrostatic binding of diphenylene[125I]iodonium to phospholipids. The high-affinity site does not appear to be associated with a rate-limiting stage of NADH oxidation.

scholarly journals Re-evaluation of the interaction of malonyl-CoA with the rat liver mitochondrial carnitine palmitoyltransferase system by using purified outer membranes

1990 ◽  
Vol 267 (1) ◽  
pp. 85-90 ◽  
Author(s):  
M P Kolodziej ◽  
V A Zammit
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Mitochondria ◽  
Subsequent Treatment ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Outer Membranes ◽  
High Affinity ◽  
Malonyl Coa ◽  
Order Of Magnitude

1. The interaction of malonyl-CoA with the outer carnitine palmitoyltransferase (CPT) system of rat liver mitochondria was re-evaluated by using preparations of highly purified outer membranes, in the light of observations that other subcellular structures that normally contaminate crude mitochondrial preparations also contain malonyl-CoA-sensitive CPT activity. 2. In outer-membrane preparations, which were purified about 200-fold with respect to the inner-membrane-matrix fraction, malonyl-CoA binding was largely accounted for by a single high-affinity component (KD = 0.03 microM), in contrast with the dual site (low- and high-affinity) previously found with intact mitochondria. 3. There was no evidence that the decreased sensitivity of CPT to malonyl-CoA inhibition observed in outer membranes obtained from 48 h-starved rats (compared with those from fed animals) was due to a decreased ratio of malonyl-CoA binding to CPT catalytic moieties. Thus CPT specific activity and maximal high-affinity [14C]malonyl-CoA binding (expressed per mg of protein) were increased 2.2- and 2.0-fold respectively in outer membranes from 48 h-starved rats. 4. Palmitoyl-CoA at a concentration that was saturating for CPT activity (5 microM) decreased the affinity of malonyl-CoA binding by an order of magnitude, but did not alter the maximal binding of [14C]malonyl-CoA. 5. Preincubation of membranes with either tetradecylglycidyl-CoA or 2-bromopalmitoyl-CoA plus carnitine resulted in marked (greater than 80%) inhibition of high-affinity binding, concurrently with greater than 95% inhibition of CPT activity. These treatments also unmasked an effect of subsequent treatment with palmitoyl-CoA to increase low-affinity [14C]malonyl-CoA binding. 6. These data are discussed in relation to the possible mechanism of interaction between the malonyl-CoA-binding site and the active site of the enzyme.

Characterization of the binding of 3,3′-di-iodo-l-thyronine to rat liver mitochondria

1997 ◽  
Vol 154 (1) ◽  
pp. 119-124
Author(s):  
A Lombardi ◽  
M Moreno ◽  
C Horst ◽  
F Goglia ◽  
A Lanni
Keyword(s):  
Rat Liver ◽  
Binding Capacity ◽  
Specific Binding ◽  
Local Environment ◽  
Liver Mitochondria ◽  
Ion Concentration ◽  
Affinity Constant ◽  
Rat Liver Mitochondria ◽  
Inner Mitochondrial Membrane ◽  
Thyroid State

Abstract The binding of labelled 3,3′-di-iodo-l-thyronine (3,3′-T2) to isolated rat liver mitochondria has been characterized. Specific binding could be detected only in the inner mitochondrial membrane, not in other mitochondrial subfractions. The composition of the incubation medium influenced the binding capacity, the best combination of high specific binding and low non-specific binding being observed in phosphate buffer, pH 6·4. The specific binding of 3,3′-T2 to mitochondria requires low ionic strength: concentrations of K+ and Na+ higher than 10 mmol/l and 0·1 mmol/l respectively resulted in a decreased binding capacity. The optimal calcium ion concentration was in the range 0·01–1·0 mmol/l. Varying magnesium ion, over the range of concentrations used (0·1–100 mmol/l), had no effect. Both ADP and ATP, at over 1 mmol/l, resulted in an inhibition of the specific binding. Incubation with protease resulted in a decrease in specific binding and an increase in non-specific binding, thus indicating the proteic nature of the binding sites. In addition to the above factors in the local environment the thyroid state of the animal might influence the 3,3′-T2-binding capacity. In fact, the thyroid state of the animal seemed not to have an influence on the affinity constant, but it did affect binding capacity. Journal of Endocrinology (1997) 154, 119–124

scholarly journals The effect of butacaine on adenine nucleotide binding and translocation in rat liver mitochondria

1975 ◽  
Vol 148 (3) ◽  
pp. 527-531 ◽  
Author(s):  
D R Fayle ◽  
G J Barritt ◽  
F L Bygrave
Keyword(s):  
Rat Liver ◽  
Local Anaesthetic ◽  
Binding Sites ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Nucleotide Binding ◽  
Local Anaesthetics ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Inner Membrane

The effect of the local anaesthetic, butacaine, on adenine nucleotide binding and translocation in rat liver mitochondria partially depleted of their adenine nucleotide content was investigated. The range of butacaine concentrations that inhibit adenine nucleotide translocation and the extent of the inhibition are similar to the values obtained for native mitochondria. Butacaine does not alter either the total number of atractyloside-sensitive binding sites of depleted mitochondria, or the affinity of these sites for ADP or ATP under conditions where a partial inhibition of the rate of adenine nucleotide translocation is observed. The data are consistent with an effect of butacaine on the process by which adenine nucleotides are transported across the mitochondrial inner membrane rather than on the binding of adenine nucleotides to sites on the adenine nucleotide carrier. The results are briefly discussed in relation to the use of local anaesthetics in investigations of the mechanism of adenine nucleotide translocation.

The inhibition of mammalian mitochondrial NADU oxidation by chloramphenicol and its isomers and analogues

1968 ◽  
Vol 46 (9) ◽  
pp. 1003-1008 ◽  
Author(s):  
K. B. Freeman ◽  
D. Haldar
Keyword(s):  
Cytochrome B ◽  
Respiratory Chain ◽  
Rat Liver ◽  
Nadh Dehydrogenase ◽  
Coenzyme Q ◽  
Liver Mitochondria ◽  
Heart Mitochondria ◽  
Rat Liver Mitochondria ◽  
Beef Heart ◽  
Beef Heart Mitochondria

Chloramphenicol and its isomers and analogues have been found to inhibit the oxidation of NADH, but not that of succinate, by beef heart mitochondria. They must therefore inhibit the NADH dehydrogenase segment of the respiratory chain. Chloramphenicol gave 50% inhibition at a concentration of 1 mM. The methylthio analogue of chloramphenicol inhibited NADH – coenzyme Q6 reductase but not NADH–ferricyanide reductase. Spectrophotometric observations suggest that these inhibitors act between NADH and flavin in coupled rat liver mitochondria and between flavin and cytochrome b in uncoupled beef heart mitochondria.

scholarly journals Time-dependence of inhibition of carnitine palmitoyltransferase I by malonyl-CoA in mitochondria isolated from livers of fed or starved rats. Evidence for transition of the enzyme between states of low and high affinity for malonyl-CoA

1984 ◽  
Vol 218 (2) ◽  
pp. 379-386 ◽  
Author(s):  
V A Zammit
Keyword(s):  
Rat Liver ◽  
Initial Rate ◽  
Liver Mitochondria ◽  
Time Dependent ◽  
Rat Liver Mitochondria ◽  
High Affinity ◽  
Malonyl Coa ◽  
Rate Of Increase ◽  
Cpt I ◽  
Zero Time

The degree of inhibition of CPT I (carnitine palmitoyltransferase, EC 2.3.1.21) in isolated rat liver mitochondria by malonyl-CoA was studied by measuring the activity of the enzyme over a short period (15s) after exposure of the mitochondria to malonyl-CoA for different lengths of time. Inhibition of CPT I by malonyl-CoA was markedly time-dependent, and the increase occurred at the same rate in the presence or absence of palmitoyl-CoA (80 microM), and in the presence of carnitine, such that the time-course of acylcarnitine formation deviated markedly from linearity when CPT I activity was measured in the presence of malonyl-CoA over several minutes. The initial rate of increase in degree of inhibition with time was independent of malonyl-CoA concentration. CPT I in mitochondria from 48 h-starved rats had a lower degree of inhibition by malonyl-CoA at zero time, but was equally capable of being sensitized to malonyl-CoA, as judged by an initial rate of increase of inhibition identical with that of the enzyme in mitochondria from fed rats. Double-reciprocal plots for the degree of inhibition produced by different malonyl-CoA concentrations at zero time for the enzyme in mitochondria from fed or starved animals indicated that the enzyme in the latter mitochondria was predominantly in a state with low affinity for malonyl-CoA (concentration required to give 50% inhibition, I0.5 congruent to 10 microM), whereas that in mitochondria from fed rats displayed two distinct sets of affinities: low (congruent to 10 microM) and high (less than 0.3 microM). Plots for mitochondria after incubation for 0.5 or 1 min with malonyl-CoA indicated that the increased sensitivity observed with time was due to a gradual increase in the high-affinity state in both types of mitochondria. These results suggest that the sensitivity of CPT I in rat liver mitochondria in vitro had two components: (i) an instantaneous sensitivity inherent to the enzyme which depends on the nutritional state of the animal from which the mitochondria are isolated, and (ii) a slow, malonyl-CoA-induced, time-dependent increase in sensitivity. It is suggested that the rate of malonyl-CoA-induced sensitization of the enzyme to malonyl-CoA inhibition is limited by a slow first-order process, which occurs after the primary event of interaction of malonyl-CoA with the mitochondria.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Studies on the nature of the high-affinity trialkyltin binding site of rat liver mitochondria

1982 ◽  
Vol 202 (1) ◽  
pp. 163-169 ◽  
Author(s):  
A P Dawson ◽  
B G Farrow ◽  
M J Selwyn
Keyword(s):  
Binding Site ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Affinity Binding ◽  
Mitochondrial Atpase ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Triphenyltin Chloride

1. The proteolipid fraction isolated from rat liver mitochondria pretreated with [3H]triphenyltin chloride is enriched in triphenyltin compared with the original mitochondria. 2. Part of this [3H]triphenyltin is eluted with a protein of Mr 5000-6000 on Sephadex LH20 chromatography. 2. Mössbauer spectra of the proteolipid fraction treated with 119Sn-enriched triethyltin chloride show a doublet which corresponds closely with that assigned previously [Farrow & Dawson (1978) Eur. J. Biochem. 86. 85-95] to the absorption of triethyltin bound to the high-affinity binding site of the mitochondrial ATPase.

scholarly journals Biochemical heterogeneity of rat liver mitochondria separated by rate zonal centrifugation

1972 ◽  
Vol 129 (1) ◽  
pp. 209-218 ◽  
Author(s):  
M. A. Wilson ◽  
J. Cascarano
Keyword(s):  
Cytochrome C ◽  
Succinate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Cytochrome B ◽  
Rat Liver ◽  
Nadh Dehydrogenase ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Osmotic Gradient ◽  
Glycerophosphate Dehydrogenase

1. Rat liver mitochondria were separated on the basis of their sedimentation coefficients in an iso-osmotic gradient of Ficoll–sucrose by rate zonal centrifugation. The fractions (33, each of 40ml) were collected in order of decreasing density. Fractions were analysed by spectral analysis to determine any differences in the concentrations of the cytochromes and by enzyme analyses to ascertain any differences in the activities of NADH dehydrogenase, succinate dehydrogenase and α-glycerophosphate dehydrogenase. 2. When plotted as% of the highest specific concentration, the contents of cytochrome a+a3 and cytochrome c+c1 were constant in all fractions but cytochrome b was only 65% of its maximal concentration in fraction 7 and increased with subsequent fractions. As a result, the cytochrome b/cytochrome a+a3 ratio almost doubled between fractions 7 and 25 whereas the cytochrome c+c1/cytochrome a+a3 ratio was unchanged. 3. Expression of the dehydrogenase activities as% of highest specific activity showed the following for fractions 6–26: NADH dehydrogenase activity remained fairly constant in all fractions; succinate dehydrogenase activity was 62% in fraction 6 and increased steadily to its maximum in fraction 18 and then decreased; the activity of α-glycerophosphate dehydrogenase was only 53% in fraction 6 and increased slowly to its peak in fractions 22 and 24. 4. These differences did not result from damaged or fragmented mitochondria or from microsomal contamination. 5. These results demonstrate that isolated liver mitochondria are biochemically heterogeneous. The importance of using a system for separating biochemically different mitochondria in studies of mitochondrial biogenesis is discussed.

Dual coenzyme activities of high-Km aldehyde dehydrogenase from rat liver mitochondria

1990 ◽  
Vol 68 (4) ◽  
pp. 751-757 ◽  
Author(s):  
C. Stan Tsai ◽  
D. J. Senior
Keyword(s):  
Steady State ◽  
Rat Liver ◽  
Aldehyde Dehydrogenase ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Binary Complex ◽  
Stopped Flow ◽  
Conformation Change ◽  
Rate Limiting ◽  
Flow Burst

Various kinetic approaches were carried out to investigate kinetic attributes for the dual coenzyme activities of mitochondrial aldehyde dehydrogenase from rat liver. The enzyme catalyses NAD+- and NADP+-dependent oxidations of ethanal by an ordered bi-bi mechanism with NAD(P)+ as the first reactant bound and NAD(P)H as the last product released. The two coenzymes presumably interact with the kinetically identical site. NAD+ forms the dynamic binary complex with the enzyme, while the enzyme-NAD(P)H complex formation is associated with conformation change(s). A stopped-flow burst of NAD(P)H formation, followed by a slower steady-state turnover, suggests that either the deacylation or the release of NAD(P)H is rate limiting. Although NADP+ is reduced by a faster burst rate, NAD+ is slightly favored as the coenzyme by virtue of its marginally faster turnover rate.Key words: aldehyde dehydrogenase, coenzyme preference.

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