scholarly journals Altered release of carnitine palmitoyltransferase activity by digitonin from liver mitochondria of rats in different physiological states

1985 ◽  
Vol 230 (2) ◽  
pp. 389-394 ◽  
Author(s):  
V A Zammit ◽  
C G Corstorphine
Keyword(s):  
Liver Mitochondria ◽  
Diabetic Rats ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Intermembrane Space ◽  
Marker Enzyme ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Cpt I ◽  
Carnitine Palmitoyltransferase Activity

The release of carnitine palmitoyltransferase (CPT) activity from rat liver mitochondria by increasing concentrations of digitonin was studied for mitochondrial preparations from fed, 48 h-starved and diabetic animals. A bimodal release of activity was observed only for mitochondria isolated from starved and, to a lesser degree, from diabetic rats, and it appeared to result primarily from the enhanced release of approx. 40% and 60%, respectively, of the total CPT activity. This change in the pattern of release was specific to CPT among the marker enzymes studied. For all three types of mitochondria there was no substantial release of CPT concurrently with that of the marker enzyme for the soluble intermembrane space, adenylate kinase. These results illustrate that the bimodal pattern of release of CPT reported previously for mitochondria from starved rats [Bergstrom & Reitz (1980) Arch. Biochem. Biophys. 204, 71-79] is not an immutable consequence of the localization of CPT activity on either side of the mitochondrial inner membrane. Sequential loss of CPT I (i.e. the overt form) from the mitochondrial inner membrane did not affect the concentration of malonyl-CoA required to effect fractional inhibition of the CPT I that remained associated with the mitochondria. The results are discussed in relation to the possibility that altered enzyme-membrane interactions may account for some of the altered regulatory properties of CPT I in liver mitochondria of animals in different physiological states.

scholarly journals Role of carnitine palmitoyltransferase I in the regulation of hepatic ketogenesis during the onset and reversal of chronic diabetes

1988 ◽  
Vol 249 (2) ◽  
pp. 409-414 ◽  
Author(s):  
B D Grantham ◽  
V A Zammit
Keyword(s):  
Insulin Treatment ◽  
Regular Insulin ◽  
Liver Mitochondria ◽  
Diabetic Rats ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Kinetic Properties ◽  
Malonyl Coa ◽  
Time Courses ◽  
Cpt I

1. The kinetic properties of overt carnitine palmitoyltransferase (CPT I, EC 2.3.1.21) were studied in rat liver mitochondria isolated from untreated, diabetic and insulin-treated diabetic animals. A comparison was made of the time courses required for the changes in these properties of CPT I to occur and for the development of ketosis during the induction of chronic diabetes and its reversal by insulin treatment. 2. The development of hyperketonaemia over the first 5 days of insulin withdrawal from streptozotocin-treated rats was accompanied by parallel increases in the activity of CPT I and in the I0.5 (concentration required to produce 50% inhibition) of the enzyme for malonyl-CoA. 3. The rapid reversal of the ketotic state by treatment of chronically diabetic rats with 6 units of regular insulin was not accompanied by any change in the properties of CPT I over the first 4 h. Higher doses of insulin (15 units), delivered throughout a 4 h period, resulted in an increase in the affinity of CPT I for malonyl-CoA, but the sensitivity of the enzyme to the inhibitor was still significantly lower than in mitochondria from normal animals. 4. Conversely, when insulin treatment was continued over a 24 h period, full restoration of the sensitivity of the enzyme to malonyl-CoA was achieved. However, the activity of the enzyme was only decreased marginally. 5. These results are discussed in terms of the possibility that the major regulatory sites of the rate of hepatic oxidation may vary in different phases of the induction and reversal of chronic diabetes.

scholarly journals Hepatic mitochondrial inner membrane properties and carnitine palmitoyltransferase A and B. Effect of diabetes and starvation

1985 ◽  
Vol 232 (2) ◽  
pp. 445-450 ◽  
Author(s):  
L J Brady ◽  
L J Silverstein ◽  
C L Hoppel ◽  
P S Brady
Keyword(s):  
Membrane Fluidity ◽  
Diabetic Rats ◽  
Carnitine Palmitoyltransferase ◽  
Membrane Properties ◽  
Inner Membrane ◽  
Fed State ◽  
Treatment Groups ◽  
Mitochondrial Inner Membrane ◽  
Malonyl Coa ◽  
Carnitine Palmitoyltransferase Activity

Intact mitochondria and inverted submitochondrial vesicles were prepared from the liver of fed, starved (48 h) and streptozotocin-diabetic rats in order to characterize carnitine palmitoyltransferase kinetics and malonyl-CoA sensitivity in situ. In intact mitochondria, both starved and diabetic rats exhibited increased Vmax., increased Km for palmitoyl-CoA, and decreased sensitivity to malonyl-CoA inhibition. Inverted submitochondrial vesicles also showed increased Vmax. with starvation and diabetes, with no change in Km for either palmitoyl-CoA or carnitine. Inverted vesicles were uniformly less sensitive to malonyl-CoA regardless of treatment, and diabetes resulted in a further decrease in sensitivity. In part, differences in the response of carnitine palmitoyltransferase to starvation and diabetes may reside in differences in the membrane environment, as observed with Arrhenius plots, and the relation of enzyme activity and membrane fluidity. In all cases, whether rats were fed, starved or diabetic, and whether intact or inverted vesicles were examined, increasing membrane fluidity was associated with increasing activity. Malonyl-CoA was found to produce a decrease in intact mitochondrial membrane fluidity in the fed state, particularly at pH 7.0 or less. No effect was observed in intact mitochondria from starved or diabetic rats, or in inverted vesicles from any of the treatment groups. Through its effect on membrane fluidity, malonyl-CoA could regulate carnitine palmitoyltransferase activity on both surfaces of the inner membrane through an interaction with only the outer surface.

scholarly journals ‘Pore’ formation is not required for the hydroperoxide-induced Ca2+ release from rat liver mitochondria

1992 ◽  
Vol 285 (1) ◽  
pp. 65-69 ◽  
Author(s):  
J Schlegel ◽  
M Schweizer ◽  
C Richter
Keyword(s):  
Rat Liver ◽  
Pore Formation ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Specific Permeability ◽  
Specific Pathway

It has recently been suggested by several investigators that the hydroperoxide- and phosphate-induced Ca2+ release from mitochondria occurs through a non-specific ‘pore’ formed in the mitochondrial inner membrane. The aim of the present study was to investigate whether ‘pore’ formation actually is required for Ca2+ release. We find that the t-butyl hydroperoxide (tbh)-induced release is not accompanied by stimulation of sucrose entry into, K+ release from, and swelling of mitochondria provided re-uptake of the released Ca2+ (‘Ca2+ cycling’) is prevented. We conclude that (i) the tbh-induced Ca2+ release from rat liver mitochondria does not require ‘pore’ formation in the mitochondrial inner membrane, (ii) this release occurs via a specific pathway from intact mitochondria, and (iii) a non-specific permeability transition (‘pore’ formation) is likely to be secondary to Ca2+ cycling by mitochondria.

scholarly journals The uptake of silicic acid by rat liver mitochondria

1978 ◽  
Vol 172 (3) ◽  
pp. 557-568 ◽  
Author(s):  
R N Johnson ◽  
B E Volcani
Keyword(s):  
Silicic Acid ◽  
Diffusion Mechanism ◽  
Mitochondrial Protein ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Energy Status ◽  
Matrix Space ◽  
Inner Membrane ◽  
Simple Diffusion ◽  
Mitochondrial Inner Membrane

1. To gain insight into a putative role for mitochondria in silicon metabolism, mitochondrial uptake (by which it is meant the removal from the medium) of silicic acid [Si(OH)4] was studied under conditions minimizing SI(OH)4 polymerization. 2. Measurements of mitochondrial respiration and swelling indicated indirectly a significant uptake of Si(OH)4 as a weak acid, but this was not confirmed when 31Si(OH)4 was used as a tracer. 31Si(OH)4 occupied a mitochondrial volume similar to that of 3H2O and was relatively unaffected by mitochondrial energy status and by the pH gradient across the mitochondrial inner membrane. 3. Uptake was directly proportional to Si(OH)4 concentration in the range 0-3 mM. 4. The uptake consisted of two components: under all conditions examined, the greater quantity, amounting to 1-2nmol of Si(OH)4/mg of mitochondrial protein, was bound, a major portion of it external to the inner membrane, with the lesser quantity free within the matrix space. 5. Equilibration of 31Si(OH)4 between medium and matrix was a slow process, having a half-time of approx. 10 min at 22 degrees C. 6. Mersalyl and N-ethylmaleimide inhibited the uptake by preferentially lowering the amount of Si(OH)4 bound. Their action was somewhat variable, depending on the precise nature of the suspending medium, and suggesting that the bound material may represent polymerized forms of Si(OH)4. 7. It is concluded that Si(OH)4 may penetrate the mitochondrial inner membrane by a simple diffusion mechanism.

scholarly journals Cholesterol increase in mitochondria: its effect on inner-membrane functions, submitochondrial localization and ultrastructural morphology

1993 ◽  
Vol 289 (3) ◽  
pp. 703-708 ◽  
Author(s):  
S Echegoyen ◽  
E B Oliva ◽  
J Sepulveda ◽  
J C Díaz-Zagoya ◽  
M T Espinosa-García ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Atpase Activity ◽  
Succinate Dehydrogenase ◽  
Outer Membrane ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Inner Membrane ◽  
Ultrastructural Morphology ◽  
Mitochondrial Inner Membrane

The effect of cholesterol incorporation on some functions of the mitochondrial inner membrane and on the morphology of rat liver mitochondria was studied. Basal ATPase and succinate dehydrogenase activities remained unchanged after cholesterol was incorporated into the mitochondria; however, uncoupled ATPase activity was partially inhibited. The presence of several substrates and inhibitors did not change the amount of cholesterol incorporated, which was localized mostly in the outer membrane. Electron-microscope observations revealed the presence of vesicles between the outer and inner membranes; these vesicles increased in number with the amount of cholesterol incorporated. The data suggest that cholesterol induces the formation of vesicles from the outer membrane, and modifies the activity of stimulated ATPase.

scholarly journals Discrimination of distinct proteinases at the four structural levels of rat liver mitochondria

1986 ◽  
Vol 233 (1) ◽  
pp. 283-286 ◽  
Author(s):  
M C Duque-Magalhães ◽  
P Régnier
Keyword(s):  
Outer Membrane ◽  
Rat Liver ◽  
Degradation Products ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Intermembrane Space ◽  
Peptidase Activity ◽  
Inner Membrane ◽  
Mitochondrial Fractions ◽  
The Matrix

Rat liver mitochondrial fractions corresponding to four morphological structures (matrix, inner membrane, intermembrane space and outer membrane) contain proteinases that cleave casein components at different rates. Proteinases of the intermembrane space preferentially cleave kappa-casein, whereas the proteinases of the outer membrane, inner membrane and matrix fractions degrade alpha S1-casein more rapidly. Electrophoretic separation of the degradation products of alpha S1-casein and kappa-casein in polyacrylamide gels shows that different polypeptides are produced when the substrate is degraded by the matrix, by both membranes and by the intermembrane-space fraction. Some of the degradation products resulting from incubation of the caseins with the mitochondrial fractions are probably the result of digestion by contaminating lysosomal proteinase(s). The matrix has a high peptidase activity, since glucagon, a small peptide, is very rapidly degraded by this fraction. These observations strongly suggest that distinct proteinases, with different specificities, are associated respectively with the intermembrane space and with both membrane fractions.

scholarly journals Target size analysis by radiation inactivation of carnitine palmitoyltransferase activity and malonyl-CoA binding in outer membranes from rat liver mitochondria

1989 ◽  
Vol 263 (1) ◽  
pp. 89-95 ◽  
Author(s):  
V A Zammit ◽  
C G Corstorphine ◽  
M P Kolodziej
Keyword(s):  
Rat Liver ◽  
Molecular Size ◽  
Protein S ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Size Analysis ◽  
Rat Liver Mitochondria ◽  
Radiation Inactivation ◽  
Malonyl Coa ◽  
Cpt I

The functional molecular sizes of the protein(s) mediating the carnitine palmitoyltransferase I (CPT I) activity and the [14C]malonyl-CoA binding in purified outer-membrane preparations from rat liver mitochondria were determined by radiation-inactivation analysis. In all preparations tested the dose-dependent decay in [14C]malonyl-CoA binding was less steep than that for CPT I activity, suggesting that the protein involved in malonyl-CoA binding may be smaller than that catalysing the CPT I activity. The respective sizes computed from simultaneous analysis for molecular-size standards exposed under identical conditions were 60,000 and 83,000 DA for malonyl-CoA binding and CPT I activity respectively. In irradiated membranes the sensitivity of CPT activity to malonyl-CoA inhibition was increased, as judged by malonyl-CoA inhibition curves for the activity in control and in irradiated membranes that had received 20 Mrad radiation and in which CPT activity had decayed by 60%. Possible correlations between these data and other recent observations on the CPT system are discussed.

scholarly journals Carnitine palmitoyltransferase activities (EC 2.3.1.–) of rat liver mitochondria

1970 ◽  
Vol 119 (3) ◽  
pp. 547-552 ◽  
Author(s):  
D. W. Yates ◽  
P. B. Garland
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Type I ◽  
Type Ii ◽  
Inner Mitochondrial Membrane ◽  
Group Transfer ◽  
Carnitine Palmitoyltransferase Activity

1. A continuously recording and sensitive fluorimetric assay is described for carnitine palmitoyltransferase. This assay has been applied to whole or disintegrated mitochondria and to soluble protein fractions. 2. When rat liver mitochondria had been disintegrated by ultrasound, the specific activity of carnitine palmitoyltransferase was 15–20m-units/mg of protein. Only one-fifth of this activity was assayable (with added substrates) before mitochondrial disintegration. 3. It is concluded that there are two carnitine palmitoyltransferase activities in rat liver mitochondria, of which one (type I) is relatively superficial in location and catalyses an acyl-group transfer between added CoA and carnitine, whereas the other (type II) is less superficial and catalyses an acyl-group transfer in unbroken mitochondria between added carnitine and intramitochondrial CoA. The existence of two distinct carnitine palmitoyltransferases was predicted by Fritz & Yue (1963). 4. In unbroken mitochondria, type I transferase is accessible to the inhibitor 2-bromostearoyl-CoA whereas the type II transferase is inaccessible. 5. A major part of the total carnitine palmitoyltransferase activity of rat liver mitochondria is membrane-bound and of type II. 6. These observations, when considered in conjunction with the penetration of mitochondria by CoASH or carnitine, indicate that the type II transferase is attached to the inner mitochondrial membrane.

scholarly journals d-Lactate transport and metabolism in rat liver mitochondria

2002 ◽  
Vol 365 (2) ◽  
pp. 391-403 ◽  
Author(s):  
Lidia de BARI ◽  
Anna ATLANTE ◽  
Nicoletta GUARAGNELLA ◽  
Giovanni PRINCIPATO ◽  
Salvatore PASSARELLA
Keyword(s):  
Lactate Dehydrogenase ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Lactate Metabolism ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
The Matrix ◽  
Inside Out

In the present study we investigated whether isolated rat liver mitochondria can take up and metabolize d-lactate. We found the following: (1) externally added d-lactate causes oxygen uptake by mitochondria [P/O ratio (the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation) = 2] and membrane potential (Δψ) generation in processes that are rotenone-insensitive, but inhibited by antimycin A and cyanide, and proton release from coupled mitochondria inhibited by α-cyanocinnamate, but not by phenylsuccinate; (2) the activity of the putative flavoprotein (d-lactate dehydrogenase) was detected in inside-out submitochondrial particles, but not in mitochondria and mitoplasts, as it is localized in the matrix phase of the mitochondrial inner membrane; (3) three novel separate translocators exist to mediate d-lactate traffic across the mitochondrial inner membrane: the d-lactate/H+ symporter, which was investigated by measuring fluorimetrically the rate of endogenous flavin reduction, the d-lactate/oxoacid antiporter (which mediates both the d-lactate/pyruvate and d-lactate/oxaloacetate exchanges) and d-lactate/malate antiporter studied by monitoring photometrically the appearance of the d-lactate counteranions outside mitochondria. The d-lactate translocators, in the light of their different inhibition profiles separate from the monocarboxylate carrier, were found to differ from each other in the Vmax values and in the inhibition and pH profiles and were shown to regulate mitochondrial d-lactate metabolism in vitro. The d-lactate translocators and the d-lactate dehydrogenase could account for the removal of the toxic methylglyoxal from cytosol, as well as for d-lactate-dependent gluconeogenesis.

scholarly journals Biochemical effects of the hypoglycaemic compound diphenyleneiodonium. Catalysis of anion–hydroxyl ion exchange across the inner membrane of rat liver mitochondria and effects on oxygen uptake

1972 ◽  
Vol 129 (1) ◽  
pp. 39-54 ◽  
Author(s):  
P. C. Holland ◽  
H. S. A. Sherratt
Keyword(s):  
Rat Liver ◽  
Indirect Evidence ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Hydroxyl Ion ◽  
Respiration Of Mitochondria ◽  
Free Media ◽  
Stimulation Of

1. The hypoglycaemic compound diphenyleneiodonium causes rapid and extensive swelling of rat liver mitochondria suspended in 150mm-NH4Cl, and in 150mm-KCl in the presence of 2,4-dinitrophenol and valinomycin. This indicates that diphenyleneiodonium catalyses a compulsory exchange of OH-for Cl-across the mitochondrial inner membrane. Br-and SCN-were the only other anions found whose exchange for OH-is catalysed by diphenyleneiodonium. 2. Diphenyleneiodonium inhibited state 3 respiration of mitochondria and slightly stimulated state 4 respiration with succinate or glutamate as substrate in a standard Cl--containing medium. 3. Diphenyleneiodonium did not inhibit state 3 respiration significantly in two Cl--free media (based on glycerol 2-phosphate or sucrose) but caused some stimulation of state 4. 4. In Cl--containing medium diphenyleneiodonium only slightly inhibited the 2,4-dinitrophenol-stimulated adenosine triphosphatase and it had little effect in the absence of Cl-. 5. The inhibition of respiration in the presence of Cl-is dependent on the Cl-–OH-exchange. 2,4-Dichlorodiphenyleneiodonium is ten times as active as diphenyleneiodonium both in causing swelling of mitochondria suspended in 150mm-NH4Cl and in inhibiting state 3 respiration in Cl--containing medium. Indirect evidence suggests that the Cl-–OH-exchange impairs the rate of uptake of substrate anions. 6. It is proposed that stimulation of state 4 respiration in the absence of Cl-depends, at least in part, on an electrogenic uptake of diphenyleneiodonium cations. 7. Tripropyl-lead acetate, methylmercuric iodide and nine substituted diphenyleneiodonium derivatives also catalyse Cl-–OH-exchange across the mitochondrial membrane. 8. Diphenyleneiodonium is compared with the trialkyltin compounds, which are also known to mediate Cl-–OH-exchange and which have in addition strong oligomycin-like effects on respiration. It is concluded that diphenyleneiodonium is specific for catalysing anion–OH-exchange and will be a useful reagent for investigating membrane-dependent systems.

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