Nam Hea Lee

The role of sulfhydryl groups in oxidative phosphorylation and ion transport by rat liver mitochondria

Abstract The influence of the sulfhydryl reagent 5,5′-dithio-bis-(2-nitrobenzoic acid) (DNTB) on metabolism of liver mitochondria was studied under different experimental conditions. 1. 1. DTNB prevented the stimulation of respiration produced by ADP and inorganic phosphate in the presence of glutamate or succinate but had little effect on 2,4-dinitrophenol-stimulated respiration. 2. 2. The formation of ATP from ADP and phosphate was depressed by DTNB. The inhibition could be reversed by dithiothreitol. 3. 3. The increase in mitochondrial oxygen uptake produced by calcium and phosphate was depressed by DTNB. 4. 4. The uptake of calcium by mitochondria in the presence of ATP and glumate was only partially inhibited by DTNB. In contrast, the stimulatory effect of inorganic phosphate on calcium transport was completely prevented. 5. 5. In the presence of glutamate and no added ATP the uptake of calcium was associated with an entrance of phosphate. DTNB almost abolished the uptake of phosphate and inhibited calcium uptake by about 60%. The decrease in calcium uptake produced by DTNB was equal, mole for mole, to the decrease in phosphate uptake. 6. 6. DTNB completely prevented the extrusion of calcium exhibited by calcium-loaded mitochondria incubated in the presence of inorganic phosphate and a low concentration of magnesium. 7. 7. N-Ethylmaleimide had effects similar to DTNB but in addition severely inhibited 2,4-dinitrophenol-stimulated respiration with glutamate as substrate. 8. 8. It was concluded that a reactive site involving a sulfhydryl group is intimately involved in either the entrance of inorganic phosphate into the mitochondrion or in the formation of a phosphorylated intermediate essential for oxidative phosphorylation and ion transport.

Respiratory inhibition of isolated rat liver mitochondria by eugenol.

Abstract Eugenol inhibited mitochondrial respiration dose-dependently in vitro and uncoupled oxidative phosphorylation from electron transfer. This phenol derivative appeared to compete at higher concentrations with the known uncoupler of oxidative phosphorylation, 2,4-dinitrophenol. Isoeugenol was less active than eugenol, and homovanillic acid was the least active of the three compounds. The effect of eugenol on mitochondrial respiration was different according to the site of entry of electrons from substrates. Eugenol inhibited respiration at a lower concentration with glutamate rather than succinate as substrate. The suggestion was made that the effect of eugenol on a nicotinamide adenine dinucleotide (NAD)-linked substrate was one of electron chain inhibition, but beyond Co-enzyme Q, both NAD-linked and flavin-linked substrates uncoupled oxidative phosphorylation. The results suggest that at the mitochondrial level all three agents can mediate cell injury by acting as potent uncouplers of the respiratory assembly.

Calcium accumulation by chondrocyte mitochondria.

Chick epiphyseal plate mitochondria observed in vitro suggest that energy dependent Ca++ uptake was maximal in the presence of ATP and a respiratory substrate. However, nucleotides other than ATP had no effect on this type of cation uptake. The observation that Ca++ accumulation was sensitive to the presence of 2,4-DNP and a number of respiratory inhibitors suggested that the mechanism of cation accumulation was similar to that described in tissues that do not undergo biological mineralization. Non-energy supported Ca++ acumulation was studied in the presence of rotenone and antimycin A. Under these conditions, the amount of Ca++ bound by skeletal tissue mitochondria was greater than bound by mitochondria obtained from noncalcifying tissues. Following isopycnic centrifugation, the Ca++ loaded mitochondria banded at different sucrose densties but the Ca++ affinity of mitochondria at each density band was similar. Hence, no particular mitochondrial species seems to be responsible for cation transport.

Effects of Ca2+ on the respiratory activity of chondrocyte mitochondria☆

Abstract The first step in the process of biological mineralization is considered to be the intramitochondrial accumulation of Ca 2+ ions. To investigate this concept, mitochondria were isolated from cells of the calcifying epiphyseal plate and their Ca 2+ content was determined. In the presence of the inhibitor, 5,5′-dithiobis(2-nitrobenzoic acid), the Ca 2+ content ranged from 80 nmol/mg of protein in the proliferating-hypertrophic zone to over 300 nmol in calcifying cartilage and the zone of provisional mineralization. The existence of a Ca 2+ gradient suggests that with progressive mineralization there is an accompanying increase in intramitochondrial Ca 2+ levels. In all epiphyseal zones, mitochondrial Ca 2+ efflux occurred in the presence of uncoupling agents. The rapidity and extent of efflux suggested that mitochondrial Ca 2+ was in ionic form or was complexed to a low molecular weight anion. Finally, the respiratory activities of epiphyseal plate mitochondria were examined. The study showed that despite their Ca 2+ load, mitochondria from zones of early and late calcification exhibited normal respiratory characteristics. However, the addition of Ca 2+ did not elicit a respiratory jump. The refractivity of chondrocyte mitochondria to Ca 2+ -stimulated respiration indicated that these organelles have a unique ability to store Ca 2+ without incurring damage to their respiratory chain.

The effect of oxygen, phosphoenolpyruvate and pH on the release of calcium from chondrocyte mitochondria

Abstract Chondrocyte mitochondria were isolated from the epiphyseal growth plate of the chick and calcium release was measured at an oxygen tension corresponding to the pO 2 of the most hypoxic region of the plate. At this oxygen tension no significant release of calcium was observed. Since this result suggested that low oxygen tension had no direct effect on mitochondria calcium efflux, an indirect effect via glycolytic intermediates was explored. There was no release of calcium when mitochondria were incubated with 1,2-diphosphoglycerate, 3-phosphor glycerate, or lactate. However, in the absence of added ATP, phosphoenolpyruvate (PEP) caused inhibition of calcium uptake and release of more than 50% of endogeneous calcium; this effect was maximal in the presence of a NAD-linked substrate. Analysis of PEP levels in the growth plate showed that the highest quantity was present in the most hypoxic zone. As a low oxygen tension would stimulate glycolysis, the effects of pH on calcium transport were also studied. It was found that at a low pH (4.0–6.5) there was release of mitochondrial calcium. The findings from this investigation suggest that the hypoxic conditions at the calcification front stimulate glycolysis. This results in an increase in PEP, a decrease in ATP and a drop in cytosolic pH. In concert, these factors induce calcium release from chondrocyte mitochondria.

Effects of a disulfide (Ellman's reagent) and thiols on oxidative phosphorylation and ion transport by rat liver mitochondria

Abstract The disulfide, 5,5′-dithio- bis -2-nitrobenzoic acid (DTNB), inhibits oxidative phosphorylation and uptake of calcium and phosphate ions by rat liver mitochondria. Half-maximal inhibition of oxidative phosphorylation is obtained with an amount of inhibitor of approximately 5 nmoles/mg of protein. Complete inhibition of oxidative phosphorylation and uptake of phosphate as well as abolition of phosphate-linked calcium uptake results from preincubation of mitochondria with DTNB followed by removal of excess inhibitor by washing. DTNB-treated mitochondria can be restored to normal function by additional incubation with dithiothreitol (DTT) followed by removal of excess DTT by washing. Oxidative phosphorylation during state 3 respiration can be completely stopped by DTNB and restored by DTT. It is concluded that one or more highly reactive -SH groups are essential for oxidative phosphorylation and ion transport in mitochondria.

A method for isolating respiring mitochondria from epiphyseal cartilage

Abstract Enzymic and ultrasonic methods for isolating a respiring mitochondrial fraction from chick epiphyseal cartilage were evaluated. It was found that sonication was the method of choice. Utilizing the “Polytron,” a fraction with elevated cytochrome oxidase activity was obtained. The effects of ADP, DNP, and oligomycin on oxygen consumption indicated that mitochondria in this fraction were biochemically intact and were performing coupled oxidative phosphorylation.

Oxidative phosphorylation by chondrocyte mitochondria

A mitochondrial fraction was isolated from chick epiphyseal cartilage using a Polytron. The oxidative phosphorylating properties and principle dehydrogenase activities of these mitochondria were studied. The highest rate of oxidation was noted when succinate was used as a substrate. Lesser levels of respiration were recorded with isocitrate in the presence of NADP. Succinate, isocitrate, oxoglutarate, malate and glycerol-3-phosphate dehydrogenases were found in chondrocyte mitochondria. Of these, succinate and NADP-linked isocitrate dehydrogenases were the most active. The results indicate that chondrocyte mitochondria have the enzymic capability to perform oxidative phosphorylation. The presence of NADP-linked isocitric dehydrogenase supports the view that this enzyme may play a key role in citrate formation.

Ca2+ transport by chondrocyte mitochondria of the epiphyseal growth plate.

SummaryIn a study of the Ca2+ kinetics of mitochondria of chick epiphyseal chondrocytes, the rate of Ca2+ uptake was linear up to a medium Ca2+ concentration of 30 μm. The half maximal transport rate occurred at 34 μm Ca2+. The Ca2+ uptake rate, expressed as a function of time, was 35 nmoles/mg protein/min; the presence of Mg2+ had little effect on Ca2+ accumulation. While these kinetic parameters did not differ significantly from mitochondria of cells of nonmineralizing tissues, the respiratory characteristics of the chondrocyte organelles exhibited functional differences. Thus, up to 350 nmoles Ca2+/mg protein, chondrocyte mitochondria performed coupled oxidative phosphorylation. Calcium uptake was energy supported, while Ca2+ binding was low. Addition of respiratory inhibitors and uncouplers to these mitochondria resulted in a rapid loss of more than 80% of the total Ca2+. The Ca/Pi ratio of the extrudate was very similar to the ratio of these ions in cartilage septum fluid. In the most mineralized zones of the epiphyseal plate, there was little change in the state 4 respiratory rate, but nonspecific Ca2+ binding was elevated and a high percentage of the total Ca2+ was in a nonextrudable form. The results indicate that in cells preparing for mineralization, much of the total mitochondrial Ca2+ is in a form that can be transported to the calcification front. In cells close to the calcification front, nonextrudable Ca2+ may form calcium phosphate granules described by other investigators.

Effect of tetracycline on chondrocyte mitochondria—an explanation of tetracycline-induced defects of mineralized tissues☆

Abstract To investigate the mechanism of tetracycline (TC)-induced defects of mineralization, the effect of the antibiotic on Ca2+ transport by chick chondrocyte mitochondria was studied. Multiple injections of TC resulted in a profound drop in the intramitochondrial Ca2+ concentration and an increase in the serum Ca2+ level. The antibiotic caused a reduction in Ca2+ uptake and an increase in Ca2+ efflux from the mitochondria in vitro; in the presence of 1 mM TC, the effect on efflux was greater than uptake. The addition of ATP to TC-treated mitochondria did not prevent Ca2+ release. Using rat liver mitochondria, it was found that. while the antibiotic had an oligomycin-like effect on respiration, this inhibitory action could be prevented by preincubating the organelles with 10 mM Mg2+ The results of the studies both in vitro and in vivo suggest that TC interferes with mitochondrial Ca2+ translocation. It is concluded that critical levels of intramitochondrial Ca2+ are prerequisite for the normal mineralization process and that interference with Ca2+ accumulation leads to defective mineralization.