scholarly journals Evidence of a calcium-ion-transport system in mitochondria isolated from flight muscle of the developing sheep blowfly Lucilia cuprina

1975 ◽  
Vol 146 (3) ◽  
pp. 601-608 ◽  
Author(s):  
F L Bygrave ◽  
A A Daday ◽  
F A Doy
Keyword(s):  
Transport System ◽  
Flight Muscle ◽  
Specific Binding ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Lucilia Cuprina ◽  
Transport Systems ◽  
Rat Liver Mitochondria ◽  
High Affinity ◽  
Energy Saturation

The EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-Ruthenium Red-quench technique (Reed & Bygrave, 1974a) was used to measure initial rates of Ca-2+ transport in mitochondria from flight muscle of the blowfly Lucilia cuprina. Evidence is provided for the existence in these mitochondria of a Ca-2+-transport system that has many features in common with that known to exist in rat liver mitochondria. These include requirement for energy, saturation at high concentrations of Ca-2+, a sigmoidal relation between initial rates of Ca-2+ transport and Ca-2+ concentration, a high affinity for free Ca-2+ (Km approx. 5 muM) and high affinity for the Ca-2+-transport inhibitoy, Ruthenium Red (approx. 0.03 nmol of carrier-specific binding-sites/mg of protein; Ki approx. 1.6 × 10- minus 8 M). Controlled respiration can be stimulated by Ca-2+ after a short lag-period provided the incubation medium contains KCl and not sucrose. The ability of Lucilia mitochondria to transport Ca-2+ critically depends on the stage of mitochondrial development; Ca-2+ transport is minimal in mitochondria from pharate adults, is maximal between 0 and 2h post-emergence and thereafter rapidly declines to reach less than 20% of the maximum value by about 2-3 days post-emergence. Respiration in mitochondria from newly emerged flies does not respond to added Ca-2+; that from 3-5-day-old flies is stimulated approx. 50%. Whereas very low concentrations of Ca-2+ inhibit ADP-stimulated respiration and oxidative phosphorylation in mitochondria from newly emerged flies (Ki approx. 60 ng-ions of Ca-2+/mg of protein); much higher concentrations (approx. 200 ng-ion/mg of protein) are needed to inhibit these processes in those from older flies. The potential of this system for studying the function and development of metabolite transport systems in mitochondria is discussed.

scholarly journals The inhibition of mitochondrial calcium transport by lanthanides and Ruthenium Red

1974 ◽  
Vol 140 (2) ◽  
pp. 143-155 ◽  
Author(s):  
Ken C. Reed ◽  
Fyfe L. Bygrave
Keyword(s):  
Binding Sites ◽  
Calcium Transport ◽  
Specific Binding ◽  
Protein A ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Calcium ◽  
Binding Studies ◽  
Specific Binding Sites

An EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-quench technique was developed for measuring initial rates of 45Ca2+ transport by rat liver mitochondria. This method was used in conjunction with studies of Ca2+-stimulated respiration to examine the mechanisms of inhibition of Ca2+ transport by the lanthanides and Ruthenium Red. Ruthenium Red inhibits Ca2+ transport non-competitively with Ki 3×10-8m; there are 0.08nmol of carrier-specific binding sites/mg of protein. The inhibition by La3+ is competitive (Ki=2×10-8m); the concentration of lanthanide-sensitive sites is less than 0.001nmol/mg of protein. A further difference between their modes of action is that lanthanide inhibition diminishes with time whereas that by Ruthenium Red does not. Binding studies showed that both classes of inhibitor bind to a relatively large number of external sites (probably identical with the ‘low-affinity’ Ca2+-binding sites). La3+ competes with Ruthenium Red for most of these sites, but a small fraction of the bound Ruthenium Red (less than 2nmol/mg of protein) is not displaced by La3+. The results are discussed briefly in relation to possible models for a Ca2+ carrier.

scholarly journals Oxidative phosphorylation. The specific binding of trimethyltin and triethyltin to rat liver mitochondria

1970 ◽  
Vol 118 (1) ◽  
pp. 171-179 ◽  
Author(s):  
W. N. Aldridge ◽  
B. W. Street
Keyword(s):  
Adipose Tissue ◽  
Oxidative Phosphorylation ◽  
Brown Adipose Tissue ◽  
Binding Site ◽  
Rat Liver ◽  
Specific Binding ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Affinity Constants ◽  
Brown Adipose

1. The binding of trimethyltin and triethyltin to rat liver mitochondria was determined and the results were analysed by the method of Scatchard (1949). 2. One binding site (site 1) has the correct characteristics for the site to which trimethyltin and triethyltin are attached when they inhibit oxidative phosphorylation. For each compound the concentration of site 1 is 0.8nmol/mg of protein and the ratios of their affinity constants are the same as the ratio of the concentrations inhibiting oxidative phosphorylation. 3. Binding site 1 is present in a fraction derived from mitochondria containing only 15% of the original protein. In this preparation ultrasonication rapidly destroyed site 1. 4. Dimethyltin and diethyltin do not prevent binding of triethyltin to rat liver mitochondria, whereas triethyl-lead does. 5. Trimethyltin and triethyltin bind to mitochondria from brown adipose tissue and the results indicate a binding site 1 similar to that in rat liver mitochondria. 6. The advantages and limitations of this approach to the study of inhibitors are discussed.

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

Proton gradient-dependent renal transport of glycine: evidence for vesicle studies

1990 ◽  
Vol 258 (2) ◽  
pp. F388-F396 ◽  
Author(s):  
H. Roigaard-Petersen ◽  
H. Jessen ◽  
S. Mollerup ◽  
K. E. Jorgensen ◽  
C. Jacobsen ◽  
...  
Keyword(s):  
Transport System ◽  
Membrane Vesicles ◽  
Proton Gradient ◽  
Transport Systems ◽  
Rabbit Kidney ◽  
Renal Transport ◽  
High Affinity ◽  
Luminal Membrane ◽  
Pars Recta ◽  
Pars Convoluta

The characteristics of renal transport of glycine by luminal membrane vesicles isolated from either proximal convoluted part (pars convoluta) or proximal straight part (pars recta) of rabbit proximal tubule were investigated. In vesicles from pars convoluta two transport systems have been characterized: a Na(+)-dependent system with intermediate affinity (half-saturation 3.64 mM) and a Na(+)-independent system that, in the presence of H+ gradient (extravesicular greater than intravesicular), can accelerate the transport of glycine into these vesicles. This is the first demonstration of H(+)-glycine cotransport across the luminal membrane of rabbit kidney proximal convoluted tubule. By contrast, in membrane vesicles from pars recta, transport of glycine was strictly dependent on Na+ and occurred via a dual transport system, namely a high-affinity (half-saturation 0.34 mM) and a low-affinity system (half-saturation 8.56 mM). The demonstration of competition between the H(+)-gradient dependent uptake of glycine, L-alanine, and L-proline, but insignificant inhibition with L-phenylalanine in vesicles from pars convoluta suggests that glycine, L-proline, and L-alanine probably share a common proton gradient-dependent transport system. In vesicles from pars recta, the Na(+)-dependent uptake of glycine was inhibited by low concentrations of L-alanine and L-phenylalanine, whereas addition of L-proline to the incubation medium did not significantly alter the uptake of glycine, suggesting that the Na(+)-dependent high-affinity system for glycine located in pars recta is shared with the high-affinity L-alanine and L-phenylalanine but not L-proline transport system.

scholarly journals Effect of micromolar concentrations of manganese ions on calcium-ion cycling in rat liver mitochondria

1983 ◽  
Vol 212 (3) ◽  
pp. 773-782 ◽  
Author(s):  
B P Hughes ◽  
J H Exton
Keyword(s):  
Rat Liver ◽  
Calcium Ion ◽  
Incubation Medium ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Rat Liver Mitochondria ◽  
Manganese Ions ◽  
Dose Dependent Decrease ◽  
Incubation Temperatures ◽  
Dose Dependent

The effects of micromolar concentrations of Mn2+ on the rat liver mitochondrial Ca2+ cycle were investigated. It was found that the addition of Mn2+ to mitochondria which were cycling 45Ca2+ led to a rapid dose dependent decrease in the concentration of extramitochondrial 45Ca2+ of about 1 nmol/mg of protein. The effect was complete within 30 s, was half maximal with 10 microM Mn2+ and was observed in the presence of 3 mM Mg2+ and 1 mM ATP. It occurred over a broad range of incubation temperatures, pH and mitochondrial Ca2+ loads. It was not observed when either Mg2+ or phosphate was absent from the incubation medium, or in the presence of Ruthenium Red. These findings indicate that micromolar concentrations of Mn2+ stimulate the uptake of Ca2+ by rat liver mitochondria, and provide evidence for an interaction between Mg2+ and Mn2+ in the control of mitochondrial Ca2+ cycling.

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)

Pathway for uncoupler-induced calcium efflux in rat liver mitochondria: inhibition by Ruthenium Red

Biochemistry ◽  
1984 ◽  
Vol 23 (8) ◽  
pp. 1645-1651 ◽  
Author(s):  
Paolo Bernardi ◽  
Venturina Paradisi ◽  
Tullio Pozzan ◽  
Giovanni Felice Azzone
Keyword(s):  
Rat Liver ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Rat Liver Mitochondria ◽  
Calcium Efflux
Sign in / Sign up

Export Citation Format

Share Document