Respiratory chain of rat heart mitochondria during heart preservation in formaldehyde

It has been shown by the method of low-temperature ESR-spectroscopy that partial blocking with formaldehyde of electron intake into the respiratory chain promotes the maintenance of a definite level of oxidation in the respiratory chain carriers and preservation of its nativity.     

Preservation of native properties of mitochondria in rat liver homogenate

A protocol is developed for preparation of concentrated rat liver homogenate preserving assemblies of mitochondria in isotonic KCl under 0 and 15 degrees C. Assemblies preserve ability for self-organization during storage in homogenate. All key energy functions of mitochondria can be investigated in such a homogenate. Oxidative phosphorylation and membrane potential are stable for 5-7 h and can be still observed on the next day. Substrate-level phosphorylation is better pronounced for mitochondria in KCl than in sucrose medium while Ca2+ capacity is greater and lipid peroxidation is much lower. Sucrose addition impairs these functions. The rate of phosphorylating respiration is lower in large assemblies and higher in small. Transition from large to small assemblies corresponds to the transition from quiescent state of animal to adrenaline induced active state. The proposed method is particularly convenient for clinical investigations with small bioptates.     

Population dynamics of mitochondria II. Turnover and ageing of rat liver mitochondria

Membrane-bound multi-protein complexes in mitochondria are provisionally classified into four categories based on possible mechanism of their assembly and degradation. These mechanisms may be investigated by the use of pulse-labeled radioactive markers which are not re-utilizable. Age dependent assembly is defined as that mechanism by which one or more of the pulse-labeled subunits are assembled into a complex, only while this complex is assembled. If the labeled sub-units can be taken up by the complex randomly during its life-span, then the mechanism is called age-independent assembly. Age-dependent degradation was defined as that mechanism by which the labeled subunits are decomposed, only when the complex is being degraded as an entity. If the labeled subunits are decomposed randomly, the mechanism is called age-independent degradation. Four categories are made by combining each of the assembly and degradation mechanisms. A differential equation was obtained to describe the fate of labeled sub-units that follow the age-dependent assembly and age-dependent degradation. Also derived was an equation for the age-independent assembly and age-dependent degradation. The other two categories which involve the age-independent degradation after age-dependent or age-independent assembly are described by single exponential kinetics. Practical application of the equations is illustrated with the use of experimental data on mitochondrial turnover found in the literature which suggests that the pulse-labeled proteins in rat liver mitochondria may follow the age-dependent assembly and degradation. The present attempts to introduce the concept of ageing into multi-protein complexes in mitochondria are the extensions of the steady state theory of mutation by Eyring & Stover (1970).     

Oxidative phosphorylation by pyridoxine-deficient rat brain mitochondria

Abstract— Oxygen uptake, ADP/O ratios and respiratory control ratios (RCR) were studied by the oxygen electrode technique in mitochondria prepared from adult and neonatal brains from normal and pyridoxine-deficient rats. The mitochondria from neonatal brain exhibited decreased rates of substrate oxidation, ADP/O ratios and respiratory control ratios in comparison to those obtained with mitochondria from the respective adult brains. The cytochrome contents of the neonatal brains were also less than those of the adults. Within the neonatal or adult groups, there were no differences in any of the parameters tested between the normal and pyridoxine-deficient rats.     

Restoration of some energy linked processes lost during the ageing of rat liver mitochondria

Some energy linked processes partially lost during the ageing of rat liver mitochondria, such as respiratory control index, ADP:O ratio and the Ca⁺⁺ and K⁺ uptake were restored to approximately the original level prior to ageing by addition of dithioerythritol to aged mitochondria.It is suggested that the maintenance of mitochondrial energy linked processes may depend upon the integrity of specific pairs of vicinal thiol groups of the membrane.     

Fumarate permeation in rat heart mitochondria

The possible fumarate translocation in rat heart mitochondria is examined. This substrate, which is claimed to be a non permeant ion in rat liver mitochondria appears to cross the mitochondrial membrane in cardiac mitochondria. This conclusion was proposed on the basis of experimental results which show swelling by rat heart mitochondria in ammonium fumarate, uptake by mitochondria of fumarate, Pi efflux from the matrix induced by fumarate and appearance of malate in the reaction mixture which follows the addition of fumarate to the mitochondria and depends on the fumarase activity. The existence of a carrier unknown so far as well as a possible physiological role of this transport is proposed.     

Studies of adenosine incorporation in Langendorff rat heart and rat heart mitochondria

The acid-insoluble product isolated from well-oxygenated Langendorff rat heart after perfusion with [¹⁴C]adenosine was purified by phenol extraction and subjected to specific phosphorolysis by pure polynucleotide phosphorylase. TLC analysis of the reaction mixture showed that ADP was the only radioactive product, proving that the original substance was a polyribonucleotide. Studies of the time course of labelling and of the distribution of the acid-insoluble product between the mitochondrial and nuclear fractions showed that both are labelled even after 1 min at 25 °C, but at short times and low temperature more radioactivity is found in the mitochondria. The kinetics of adenosine incorporation resemble those expected for the labelling of hnRNA and mRNA. Isolated, respiring mitochondria incorporate adenosine and adenine nucleotides into acid insoluble form by a process dependent on oxidative phosphorylation and the adenine nucleotide translocase that is specific for adenine derivatives. The results are discussed in terms of the hypothesis that the polyribonucleotide might be a storage form of adenine nucleotides: it is concluded that the bulk of the labelled product is unlikely to play a major role in energy metabolism.     

Uncoupled oxidation in rat heart mitochondria

Isolated heart mitochondria possessing a high phosphorylation efficiency with pyruvate and malate as substrates oxidize NADH and ascorbate unassociated with ADP phosphorylation. This uncoupled pathway is expressed partially when succinate or NAD-linked substrates are oxidized. The uncoupled oxidation is likely to be the result of the presence of a mitochondrial population with the high-permeable inner membrane in intact tissues. The nature and origin of a uncoupled respiratory system and its role in the thermoproduction of endotherms are discussed.     

Synthesis of proteins in heart mitochondria during postnatal development of the rat

Proteins of inner mitochondrial membranes of the albino rat myocardium during postnatal development of 1, 3 and 6 months old animals were electrophoretically separated in 10% polyacrylamide gel. The rate of 14C-amino acids incorporation into examined proteins was determined in vitro. Specific radioactivity of the total mitochondrial fraction decreased in the course of the postnatal development. That of outer membranes remained unchanged, though it sharply increased in inner membranes of mature animals as compared with animals aged one month. Levels of radioactive precursor incorporation in separate protein fractions of inner membranes of the myocardium mitochondria were estimated.     

Oxidative properties of swollen rat liver mitochondria.

Rat liver mitochondria undergo extensive swelling when they are incubated in hypotonic sucrose medium containing 5 mm P_i. After 30 min of swelling at 25 °C, a three- to fourfold increase in volume has occurred, accompanied by gross disorganization of the matrix as observed by electron microscopy. Succinate-supported respiration was unchanged, but the respiration of NAD-linked substrates was reduced and there was a complete and irreversible loss of phosphorylation in both cases. β-Hydroxybutyrate-supported respiration was regained completely on addition of NAD to the swollen mitochondria. α-Ketoglutarate- and malate + pyruvate-supported respiration was only partially restored by the addition of NAD. This inhibition of respiration in swollen mitochondria may be due to a disorganization of a putative complex of Krebs cycle enzymes on the inner surface of the inner membrane.     

Oxidative phosphorylation in rat spinal cord mitochondria in tetanus]

The mitochondria isolated from the spinal cord of rats with local tetanus utilized oxygen and inorganic phosphate more intensive than the mitochondria of normal animals. Ratios of P/O were similar. In experiments in vitro purified tetanus toxin in doses 5 X 10(2) = 1 X 10(5) MLD (for mice) did not effect on oxidation of intact mitochondria.     

Oxidative stress in mitochondria

In mitochondria, oxidative phosphorylation and enzymatic oxidation of biogenic amines by monoamine oxidase produce reactive oxygen and nitrogen species, which are proposed to cause neuronal cell death in neurodegenerative disorders, including Parkinson’s and Alzheimer’s disease. In these disorders, mitochondrial dysfunction, increased oxidative stress, and accumulation of oxidation-modified proteins are involved in cell death in definite neurons. The interactions among these factors were studied by use of a peroxynitrite-generating agent, N-morpholino sydnonimine (SIN-1) and an inhibitor of complex I, rotenone, in human dopaminergic SH-SY5Y cells. In control cells, peroxynitrite nitrated proteins, especially the subunits of mitochondrial complex I, as 3-nitrotyrosine, suggesting that neurons are exposed to constant oxidative stress even under physiological conditions. SIN-1 and an inhibitor of proteasome, carbobenzoxy-l-isoleucyl-γ-t-butyl-l-analyl-l-leucinal (PSI), increased markedly the levels of nitrated proteins with concomitant induction of apoptosis in the cells. Rotenone induced mitochondrial dysfunction and accumulation and aggregation of proteins modified with acrolein, an aldehyde product of lipid peroxidation in the cells. At the same time, the activity of the 20S β-subunit of proteasome was reduced significantly, which degrades oxidative-modified protein. The mechanism was proved to be the result of the modification of the 20S β-subunit with acrolein and to the binding of other acrolein-modified proteins to the 20S β-subunit. Increased oxidative stress caused by SIN-1 treatment induced a decline in the mitochondrial membrane potential, ΔΨm, and activated mitochondrial apoptotic signaling and induced cell death in SH-SY5Y cells. As another pathway, p38 mitogen-activated protein (MAP) kinase and exracellular signal-regulated kinase (ERK) mediated apoptosis induced by SIN-1. On the other hand, a series of neuroprotective propargylamine derivatives, including rasagiline [N-propargyl-1(R)aminoindan]and (−)deprenyl, intervened in the activation of apoptotic cascade by reactive oxygen species-reactive nitrogen species in mitochondria through stabilization of the membrane potential, ΔΨm. In addition, rasagiline induced antiapoptotic Bcl-2 and glial cell line-derived neurotrophic factor (GDNF) in SH-SY5Y cells, which was mediated by the ERK-nuclear factor (NF)-κB pathway. These results are discussed in relation to the interaction of oxidative stress and mitochondria in the regulation of neuronal death and survival in neurodegenerative diseases.