Milligram Of Mitochondrial Protein Research Articles

Exposure to marine benthic dinoflagellate toxins may lead to mitochondrial dysfunction

Even though marine dinoflagellates are important primary producers, many toxic species may alter the natural equilibrium of aquatic ecosystems and even generate human intoxication incidents, as they are the major causative agents of harmful algal blooms. In order to deepen the knowledge regarding benthic dinoflagellate adverse effects, the present study aims to clarify the influence of Gambierdiscus excentricus strain UNR-08, Ostreopsis cf. ovata strain UNR-03 and Prorocentrum lima strain UNR-01 crude extracts on rat mitochondrial energetic function and permeability transition pore (mPTP) induction. Our results, expressed in number of dinoflagellate cell toxic compounds tested in a milligram of mitochondrial protein, revealed that 934 cells mg prot−1 of G. excentricus, and 7143 cells mg prot−1 of both O. cf. ovata and P. lima negatively affect mitochondrial function, including by decreasing ATP synthesis-related membrane potential variations. Moreover, considerably much lower concentrations of dinoflagellate extracts (117 cells mg prot−1 of G. excentricus, 1429 cells mg prot−1 of O. cf. ovata and 714 cells mg prot−1 of P. lima) produced mPTP-induced swelling in Ca2+-loaded isolated mitochondria. The present study clearly demonstrates the toxicity of G. excentricus, O. cf. ovata and P. lima extracts at the mitochondrial level, which may lead to mitochondrial failure and consequent cell toxicity, and that G. excentricus always provide much more severe effects than O. cf. ovata and P. lima.

Mitochondrial energetics of benthic and pelagic Antarctic teleosts

Antarctic fauna are highly adapted to the frigid waters of the Southern Ocean. This study describes the in vitro temperature sensitivity of oxygen consumption rates measured in liver mitochondria from the pelagic notothenioid Pleuragramma antarcticum between 5 and 35 °C. Oxygen fluxes were measured after the addition of millimolar levels of pyruvate, malate, succinate and glutamate (state II, LEAK) and saturating levels of ADP [state III, oxidative phosphorylation (OXPHOS)]. State III respiration significantly decreased above 18.7 °C. A comparison of the oxidative capacities among P. antarcticum and other notothenioids showed significant differences in state III respiration, where benthic species exhibited about 50 % lower rates than P. antarcticum. In addition, state III respiration rates normalized per milligram of mitochondrial protein of P. antarcticum were up to eight times higher than state III rates reported in the literature for other notothenioids. The comparatively high respiration rates measured in this study may be explained by our approach, which engaged both complexes I and II under conditions of oxidative phosphorylation. State III rates of independently activated complexes I and II were found to range from 42 to 100 % of rates obtained when both complexes were activated simultaneously in the same species. The remarkable tolerance of P. antarcticum OXPHOS toward warmer temperatures was unexpected for an Antarctic stenotherm and may indicate that thermal sensitivity of their mitochondria is not the driving force behind their stenothermy.

ANESTHETIC PRECONDITIONING CONFERS ACUTE CARDIOPROTECTION VIA UP-REGULATION OF MANGANESE SUPEROXIDE DISMUTASE AND PRESERVATION OF MITOCHONDRIAL RESPIRATORY ENZYME ACTIVITY

The cellular and molecular mechanisms that underlie cardioprotection against I/R by anesthetic-induced preconditioning (APC) require further elucidation. Using isoflurane as a representative anesthetic, we evaluated the hypothesis that APC induces myocardial protection against I/R by attenuation of excessive reactive oxygen species and restoration of mitochondrial bioenergetics through postischemic up-regulation of manganese superoxide dismutase (MnSOD) expression and preservation of respiratory enzyme activity. Pentobarbital anesthetized open-chest Sprague-Dawley rats were subject to 30-min left coronary artery occlusion, followed by 120-min reperfusion. Before ischemia, rats were randomly assigned to receive 0.9% saline, two cycles of brief coronary artery occlusion and reperfusion, or a 30-min exposure to 1.0 minimum alveolar concentration isoflurane in the absence or presence of a specific mitochondrial adenosine triphosphate-sensitive potassium (KATP) channel blocker, 5-hydroxydecanoate; a membrane-permeable superoxide scavenger, 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl; or a NOS inhibitor, N(G)-nitro-L-arginine methyl ester. Isoflurane exposure induced an initial increase in myocardial superoxide (O2-), but not NO level. It also significantly decreased infarct size and restored mitochondrial respiratory enzyme activity or ATP production in I/R rat hearts, along with suppression of the O2- surge at reperfusion and increase in MnSOD expression or enzyme activity. These protective effects were abrogated by 5-hydroxydecanoate or 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl, but not by N(G)-nitro-L-arginine methyl ester pretreatment. These results suggest that opening of mitochondrial KATP channel, followed by O2- signaling, induces postischemic augmentation of MnSOD and preservation of mitochondrial respiratory enzyme activities, leading to attenuated cardiac O2- surge and restored ATP production during reperfusion, and underlie APC-induced cardioprotection.

Physiological basis of temperature-dependent biogeography: trade-offs in muscle design and performance in polar ectotherms

Polar, especially Antarctic, oceans host ectothermic fish and invertebrates characterized by low-to-moderate levels of motor activity; maximum performance is reduced compared with that in warmer habitats. The present review attempts to identify the trade-offs involved in adaptation to cold in the light of progress in the physiology of thermal tolerance. Recent evidence suggests that oxygen limitations and a decrease in aerobic scope are the first indications of tolerance limits at both low and high temperature extremes. The cold-induced reduction in aerobic capacity is compensated for at the cellular level by elevated mitochondrial densities, accompanied by molecular and membrane adjustments for the maintenance of muscle function. Particularly in the muscle of pelagic Antarctic fish, among notothenioids, the mitochondrial volume densities are among the highest known for vertebrates and are associated with cold compensation of aerobic metabolic pathways, a reduction in anaerobic scope, rapid recovery from exhaustive exercise and enhanced lipid stores as well as a preference for lipid catabolism characterized by high energy efficiency at high levels of ambient oxygen supply. Significant anaerobic capacity is still found at the very low end of the activity spectrum, e.g. among benthic eelpout (Zoarcideae). In contrast to the cold-adapted eurytherms of the Arctic, polar (especially Antarctic) stenotherms minimize standard metabolic rate and, as a precondition, the aerobic capacity per milligram of mitochondrial protein, thereby minimizing oxygen demand. Cost reductions are supported by the downregulation of the cost and flexibility of acid-base regulation. At maintained factorial scopes, the reduction in standard metabolic rate will cause net aerobic scope to be lower than in temperate species. Loss of contractile myofilaments and, thereby, force results from space constraints due to excessive mitochondrial proliferation. On a continuum between low and moderately high levels of muscular activity, polar fish have developed characteristics of aerobic metabolism equivalent to those of high-performance swimmers in warmer waters. However, they only reach low performance levels despite taking aerobic design to an extreme.

Identification and Properties of a Novel Intracellular (Mitochondrial) ATP-sensitive Potassium Channel in Brain

Protection of heart against ischemia-reperfusion injury by ischemic preconditioning and K(ATP) channel openers is known to involve the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). Brain is also protected by ischemic preconditioning and K(ATP) channel openers, and it has been suggested that mitoK(ATP) may also play a key role in brain protection. However, it is not known whether mitoK(ATP) exists in brain mitochondria, and, if so, whether its properties are similar to or different from those of heart mitoK(ATP). We report partial purification and reconstitution of a new mitoK(ATP) from rat brain mitochondria. We measured K(+) flux in proteoliposomes and found that brain mitoK(ATP) is regulated by the same ligands as those that regulate mitoK(ATP) from heart and liver. We also examined the effects of opening and closing mitoK(ATP) on brain mitochondrial respiration, and we estimated the amount of mitoK(ATP) by means of green fluorescence probe BODIPY-FL-glyburide labeling of the sulfonylurea receptor of mitoK(ATP) from brain and liver. Three independent methods indicate that brain mitochondria contain six to seven times more mitoK(ATP) per milligram of mitochondrial protein than liver or heart.

Chronic ethanol consumption causes alterations in the structural integrity of mitochondrial DNA in aged rats.

Chronic ethanol consumption adversely affects the respiratory activity of rat liver mitochondria. It causes increased cellular production of oxygen radical species and selectively decreases mitochondrial glutathione (GSH) levels. Here we show, using Southern hybridization techniques on total rat genomic DNA, that long-term (11-13 months) ethanol feeding, using the Lieber-DeCarli diet, results in a 36% (P <.05; n = 4) decrease in hepatic mitochondrial DNA (mtDNA) levels when compared with paired controls. UV quantitation of mtDNA isolated from hepatic mitochondria showed that chronic ethanol intake (11-13 months) causes a 44% (P <.01; n = 6) decrease in the amount of mtDNA per milligram of mitochondrial protein. No significant decline in mtDNA levels was seen in ethanol-fed animals maintained on the diet for 1 to 5 months. Ethanol feeding caused a 42% (P <.01; n = 4) and a 132% (P <.05; n = 3) increase in 8-hydroxydeoxyguanosine (8-OHdG) formation in mtDNA in animals maintained on the diet for 3 to 6 months and 10 to 11 months, respectively. In addition, agarose gel electrophoresis revealed a 49% increase (P <.05; n = 3) in mtDNA single-strand breaks (SSB) in animals fed ethanol for more than 1 year. These findings suggest that chronic ethanol consumption causes enhanced oxidative damage to mtDNA in older animals along with increased strand breakage, and that this results in its selective removal/degradation by mtDNA repair enzymes.

Are there distinct subcellular populations of mitochondria in rainbow trout red muscle?

Ultrastructural analysis typically shows vertebrate striated muscles to possess mitochondria residing primarily in two locations. One population is interlaced throughout the myofibrils and another occurs directly beneath the cell membrane. The two populations of mitochondria can be separated and studied in vitro. Subsarcolemmal mitochondria (SSmt) are released by mechanical shearing of the tissue, whereas protease treatment is required to release the intermyofibrillar population (IMFmt). These methods were applied to rainbow trout (Oncorhynchus mykiss) red muscle to investigate the possible existence of distinct populations in this tissue. The two populations were very similar in mitochondrial DNA content (mtDNA mg-1 mitochondrial protein) and enzymatically (activities of carnitine palmitoyl transferase, &bgr ;-hydroxyacyl CoA dehydrogenase, complex I, citrate synthase, cytochrome c oxidase expressed per milligram of mitochondrial protein). Respiration rates were the same for pyruvate and succinate, but IMFmt oxidized palmitoyl carnitine 26 % faster than SSmt (P<0.05). Apart from these minor differences in fatty acyl carnitine oxidation rates, no differences in biochemical or genetic properties were detected between populations. The lack of distinct subcellular populations in fish, in contrast to the situation in mammalian striated muscle, probably relates to the high mitochondrial volume density in fish red muscle.

The Proton Permeability of the Inner Membrane of Liver Mitochondria from Ectothermic and Endothermic Vertebrates and from Obese Rats: Correlations with Standard Metabolic Rate and Phospholipid Fatty Acid Composition

We measured the proton leak across the inner membrane of liver mitochondria isolated from six different vertebrate species and from obese and control Zucker rats. Proton leak at 37°C was similar in rat and pigeon, and in obese and control Zucker rats. Compared to rat, it was lower in cane toad, shingleback lizard, and the Madeiran lizard Lacerta dugessi. Proton leak at 20°C was similar in xenopus toad and higher in rainbow trout, compared to rat. In general, proton permeability and substrate oxidation activity were greater in liver mitochondria from endotherms than those from ectotherms. Analysis of this and previous data showed that proton leak per milligram of mitochondrial protein correlated with standard metabolic rate, and proton leak per milligram of inner membrane phospholipid correlated with 11 phospholipid fatty acid compositional parameters, including unsaturation index.

Diagnosis of Complex I Deficiency in Patients with Lactic Acidemia Using Skin Fibroblast Cultures

The requirement for a rapid and easy method of preparing mitochondrial fractions from cultured skin fibroblasts led us to compare the results obtained from such a preparation with the more traditional methods of cellular fractionation. Values for NADH–cytochromecreductase (rotenone sensitive) were compared for a series of three controls and nine patients with complex I (NADH–coenzyme Q reductase deficiency). Values obtained for deficient cell lines varied from 19 to 64% of the control values for the long mitochondrial preparation method and from 34 to 70% of control for the rapid preparation. Mean values were statistically significantly different from the lowest control cell line (P< 0.01) in all cases. The specific activity on the basis of activity per milligram of mitochondrial protein and of activity per unit of citrate synthase activity was lower in the rapid preparation of mitochondria by some 41%, indicating a lesser degree of mitochondrial purification. However, the overall result showed that this type of rapid preparation, which uses four 9-cm petri dishes of cultured cells, can be used to diagnose mitochondrial complex I deficiency. This method will find general use in the measurement of either mitochondrial enzymes of low specific activity or mitochondrial enzymes whose measurement is made difficult by contaminating nonmitochondrial enzymes.

Reduced antioxidative capacity in liver mitochondria from bile duct ligated rats.

Lipid peroxidation and antioxidative mechanisms were investigated in liver mitochondria from bile duct ligated rats (BDL rats) and correlated with the activity of enzyme complexes of the electron transport chain. In comparison to pair-fed control rats, BDL rats had increased concentrations of thiobarbituric acid reacting substances (TBARS) per gram of liver and per milligram of mitochondrial protein 3, 7, 14, and 28 days after surgery. The hepatic glutathione (GSH) content was decreased in BDL rats 28 days after surgery when expressed per gram of liver but equal between BDL and control rats when expressed per liver. The mitochondrial GSH content was decreased in BDL rats by 20% to 33% from day 7 after surgery. The concentrations of ubiquinone-9 and ubiquinone-10, substances involved in electron transport and efficient antioxidants, were both decreased in BDL rats 14 and 28 days after surgery per gram of liver and per milligram of mitochondrial protein. When expressed per liver, ubiquinone-9 was decreased in BDL rats from day 7 after surgery. In comparison with controls, the decrease in total mitochondrial ubiquinone content in BDL rats averaged 52% 14 days and 38% 28 days after surgery. The activity of the succinate:ferricytochrome c oxidoreductase (complexes II and III of the electron transport chain) was decreased in BDL rats at days 7, 14, and 28 after surgery, and the activity of the ferrocytochrome c:oxygen oxidoreductase (complex IV) was reduced at 14 and 28 days after surgery. The mitochondrial concentration of TBARS showed a negative and the concentrations of GSH and ubiquinone a positive correlation with the activity of the succinate:ferricytochrome c oxidoreductase.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced antioxidative capacity in liver mitochondria from bile duct ligated rats

Lipid peroxidation and antioxidative mechanisms were investigated in liver mitochondria from bile duct ligated rats (BDL rats) and correlated with the activity of enzyme complexes of the electron transport chain. In comparison to pair-fed control rats, BDL rats had increased concentrations of thiobarbituric acid reacting substances (TBARS) per gram of liver and per milligram of mitochondrial protein 3, 7, 14, and 28 days after surgery. The hepatic glutathione (GSH) content was decreased in BDL rats 28 days after surgery when expressed per gram of liver but equal between BDL and control rats when expressed per liver. The mitochondrial GSH content was decreased in BDL rats by 20% to 33% from day 7 after surgery. The concentrations of ubiquinone-9 and ubiquinone-10, substances involved in electron transport and efficient antioxidants, were both decreased in BDL rats 14 and 28 days after surgery per gram of liver and per milligram of mitochondrial protein. When expressed per liver, ubiquinone-9 was decreased in BDL rats from day 7 after surgery. In comparison with controls, the decrease in total mitochondrial ubiquinone content in BDL rats averaged 52% 14 days and 38% 28 days after surgery. The activity of the succinate:ferricytochrome c oxidoreductase (complexes II and III of the electron transport chain) was decreased in BDL rats at days 7, 14, and 28 after surgery, and the activity of the ferrocytochrome c:oxygen oxidoreductase (complex IV) was reduced at 14 and 28 days after surgery. The mitochondrial concentration of TBARS showed a negative and the concentrations of GSH and ubiquinone a positive correlation with the activity of the succinate:ferricytochrome c oxidoreductase. We conclude that the mitochondrial concentration of antioxidants such as GSH and ubiquinone decreases in BDL rats, whereas the concentration of lipid peroxidation products increases. Hepatic mitochondrial dysfunction in BDL rats may at least partially result from oxidative damage to mitochondrial lipids and/or proteins.

Mitochondrial respiration scavenges extramitochondrial superoxide anion via a nonenzymatic mechanism.

We determined that mitochondrial respiration reduced cytosolic oxidant stress in vivo and scavenged extramitochondrial superoxide anion (O2-.) in vitro. First, Saccharomyces cerevisiae deficient in both the cytosolic antioxidant cupro-zinc superoxide dismutase (Cu,Zn-SOD) and electron transport (Rho0 state) grew poorly (P < 0.05) in 21% O2 compared with parent yeast and yeast deficient only in electron transport or Cu,Zn-SOD, whereas anaerobic growth was the same (P > 0.05) in all yeast. Second, isolated yeast and mammalian mitochondria scavenged extramitochondrial O2-. generated by xanthine/xanthine oxidase. Yeast mitochondria scavenged 42% more (P < 0.05) extramitochondrial O2-. during pyruvate/malate-induced respiration than in the resting state. Addition of either antimycin (respiratory chain inhibitor) or FCCP (respiratory chain uncoupler) prevented increased O2-. scavenging. Mitochondria isolated from yeast deficient in the mitochondrial manganous superoxide dismutase (Mn-SOD) increased (P < 0.05) O2-. scavenging 56% during respiration. This apparent SOD activity, expressed in units of SOD activity per milligram of mitochondrial protein, was the same (9 +/- 0.6 vs. 10 +/- 1.0; P = 0.43) as the O2-. scavenging of mitochondria with Mn-SOD, suggesting that respiration-dependent mitochondrial O2-. scavenging was nonenzymatic. Finally, isolated rat liver and lung mitochondria also increased (P < 0.05) O2-. scavenging during respiration. We speculate that respiring mitochondria, via the protonmotive pump, present a polarized, proton-rich surface that enhances nonenzymatic dismutation of extramitochondrial O2-. and that this is a previously unrecognized function of mitochondrial respiration with potential physiological ramifications.

Thermogenic Capacity of Brown Adipose Tissue Is Reduced in Rats Fed a High Protein, Carbohydrate-Free Diet

The functional state of interscapular brown adipose tissue (IBAT) was examined in rats fed for 20–30 d a high protein, carbohydrate-free diet [70% (wt/wt) protein, 8% fat] or a balanced diet (66% carbohydrate, 17% protein, 8% fat). In rats fed the high protein diet, body weight did not differ from that of control rats, but relative IBAT weight (grams per 100 g body wt) and lipid concentration (per gram of tissue) were 37% and 14% lower, respectively. In vivo rates of lipogenesis in IBAT, epididymal and retroperitoneal adipose tissue of rats fed the high protein diet were 20, 30 and 40%, respectively, of control values. Mitochondrial protein and cytochrome oxidase activity per total IBAT were significantly lower in rats fed the high protein diet than in controls; GDP binding was lower even when expressed per total tissue or per milligram of mitochondrial protein. The increase of IBAT temperature following norepinephrine infusion was significantly smaller than in controls. It is suggested that the decrease in IBAT capacity in the rats fed the high protein diet was due, at least in part, to a sustained reduction of sympathetic activity.

Mitochondrial metabolism of cardiac and skeletal muscles from a fast (Katsuwonus pelamis) and a slow (Cyprinus carpio) fish

Tuna cardiac (atrium, compact and spongy ventricle) and skeletal muscle (red and white) were compared with carp tissues to determine the importance of mitochondrial differences in supporting the high aerobic capacities in tuna. Mitochondria isolated from red muscle of both species oxidized each of the physiological fuels at similar rates per milligram of mitochondrial protein, when differences in assay temperature are considered. The highest rate of oxygen consumption by ventricle mitochondria was 2 times greater in tuna than carp. The maximal oxidation rates were 3–4 times higher in ventricle than red muscle in both species. Tuna tissues had as much as 30–80% more mitochondrial protein per gram of tissue than carp. Morphometrically this was manifested as extremely densely packed mitochondrial cristae, rather than increased mitochondrial volume densities. In general, higher aerobic capacities of tuna ventricle and red muscle are primarily attributable to greater tissue mass and, to a lesser extent, differences in the nature or quantity of mitochondria per gram of tissue. Unlike ventricle and red muscle, tissues with relatively low mitochondrial contents in carp (white muscle, atrium) demonstrated several-fold higher mitochondrial contents in tuna. Enzyme analyses of tissue and isolated mitochondria suggest a greater dependence of tuna tissues on fatty acids as fuels. Activities of carnitine palmitoyl transferase (CPT) per milligram of protein were 2–2.5 times higher in tuna red muscle and ventricle mitochondria than in carp mitochondria from the same tissues. Whole tissue activity ratios of hexokinase/CPT, which indicate the relative importance of glucose and fatty acid metabolism, were 5 times higher in carp spongy ventricle and 12 times higher in carp compact ventricle. These data suggest that muscle aerobic capacity can be increased at several levels: tissue mass, mitochondrial volume density, cristae surface density, and mitochondrial specific activity. Large differences observed between carp and tuna muscles are due to cumulative effects of several of these factors.

Adrenalectomy increases brown adipose tissue metabolism in ob/ob mice housed at 35 degrees C.

Adrenalectomy arrests the development of obesity in ob/ob mice fed a high-starch diet and housed at a normal room temperature (20-25 degrees C) partly by stimulating the low thermogenic activity of brown adipose tissue (BAT). The present study was undertaken to determine if adrenalectomy would also lower energy retention and stimulate BAT metabolism in ob/ob mice housed in a warm environment (35 degrees C) where BAT thermoregulatory heat production is not needed. Adrenalectomy prevented hyperphagia and hyperinsulinemia and lowered the efficiency of energy retention in ob/ob mice housed at 35 degrees C, which is comparable to results obtained at 20-25 degrees C. Sympathetic nervous system stimulation of BAT (interscapular and subscapular depots) assessed by norepinephrine turnover was increased in adrenalectomized ob/ob mice. Thermogenic activity of BAT in adrenalectomized ob/ob mice (as assessed by GDP binding to isolated BAT mitochondria, GDP-inhibitable acetate-induced BAT mitochondrial swelling, and Mg2(/)-activated GDP binding to BAT mitochondria) was not elevated when results were expressed per milligram of mitochondrial protein but was elevated approximately 65% when expressed per interscapular and subscapular depots because adrenalectomy increased BAT mitochondrial mass. Adrenalectomy lowers the efficiency of energy retention and stimulates BAT metabolism even when ob/ob mice are housed in a warm environment.

Tissue specificity of the mitochondrial forms of malic enzyme in herring tissues

Abstract 1. 1. The activity of malic enzyme per wet weight or per milligram of mitochondrial protein in herring tissues showed significant tissue specificity. 2. 2. The specific activity of herring malic enzyme per mg of mitochondria from testes was 30 times greater than that of malic enzyme found in mitochondria of ovaries. 3. 3. The NAD(P)-dependent mitochondrial malic enzyme was present in all tissues tested and testis mitochondria contained the highest activity of this molecular form. 4. 4. Herring skeletal muscle mitochondria contained nearly identical activities of both molecular forms, the NADP- and NAD(P)-dependent malic enzyme, respectively. 5. 5. The results extend and support the proposal that mitochondria of some fish may contain two molecular forms of malic enzyme and have a unique intramitochondrial pahtway for malate metabolism.

Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites.

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.

Biological significance of enhanced mitochondrial membrane potential in regenerating liver

Liver mitochondrial membrane potential was assessed during regeneration following partial hepatectomy in rabbits. Absorbance change of safranine O per milligram of mitochondrial protein was used to evaluate mitochondrial membrane potential. Absorbance change was calibrated to the membrane potential in millivolts produced by valinomycin-induced potassium diffusion potential. At 24 hr after hepatectomy, absorbance change of safranine O per milligram of mitochondrial protein increased from 15.0 +/- 2.2 X 10(-3) to 37.4 +/- 3.3 X 10(-3) per mg (p less than 0.005). This represents a mitochondrial membrane potential increase from 77.0 +/- 4.6 to 124.4 +/- 6.7 mV. Phosphorylative activity increased from 59.9 +/- 5.0 to 106.1 +/- 7.4 nmoles ATP synthesized per mg per min (p less than 0.005). The enhancement of phosphorylative activity was closely linked to the elevation in liver mitochondrial membrane potential (r = 0.77, p less than 0.005). We suggest that elevation of mitochondrial membrane potential, coupled with enhanced oxidative and phosphorylative activities, plays an important role in the regeneration process following hepatectomy.

Calcium flux and endogenous calcium content in isolated mammalian growth-plate chondrocytes, hyaline-cartilage chondrocytes, and hepatocytes.

The role of chondrocyte mitochondria in endochondral ossification has been the subject of intensive investigation and controversy. The purpose of this study was to quantitate the endogenous calcium content and the maximum capacity for calcium accumulation and release in isolated mammalian growth-plate chondrocytes and hyaline-cartilage chondrocytes. The results indicated that the mitochondria of the isolated growth-plate and hyaline-cartilage chondrocytes possess a greater endogenous calcium content, a greater capacity for calcium accumulation, and a larger labile Ca+2 pool than do the mitochondria of hepatocytes. Growth-plate and hyaline-cartilage mitochondria had an endogenous calcium content of 908 and 142 nanomoles of Ca+2 per milligram of mitochondrial protein. The growth-plate mitochondria had a maximum calcium capacity of 5249 nanomoles of Ca+2 per milligram of mitochondrial protein. In comparison, the mitochondria of hepatocytes had a much smaller endogenous-calcium content and a smaller maximum Ca+2 capacity: twenty-one and 3262 nanomoles of Ca+2 per milligram of mitochondrial protein, respectively. The mitochondrial labile-calcium pool in both growth-plate and hyaline-cartilage chondrocytes was twofold greater than that in the mitochondria of hepatocytes. Chondrocyte mitochondria released approximately 2400 nanomoles of Ca+2 per milligram of mitochondrial protein, whereas hepatocyte mitochondria released 1200 nanomoles of Ca+2 per milligram. These results suggest that the chondrocyte mitochondria are specialized for calcium transport and are important in the calcification of the extracellular matrix of the growth plate.

Characteristics of Glutamate Dehydrogenase in Mitochondria Prepared from Corn Shoots

The amination of alpha-ketoglutarate (alpha-KG) by NADH-glutamate dehydrogenase (GDH) obtained from Sephadex G-75 treated crude extracts from shoots of 5-day-old seedlings was stimulated by the addition of Ca(2+). The NADH-GDH purified 161-fold with ammonium sulfate, DEAE-Toyopearl, and Sephadex G-200 was also activated by Ca(2+) in the presence of 160 micromolar NADH. However, with 10 micromolar NADH, Ca(2+) had no effect on the NADH-GDH activity. The deamination reaction (NAD-GDH) was not influenced by the addition of Ca(2+).About 25% of the NADH-GDH activity was solubilized from purified mitochondria after a simple osmotic shock treatment, whereas the remaining 75% of the activity was associated with the mitochondrial membrane fraction. When the lysed mitochondria, mitochondrial matrix, or mitochondrial membrane fraction was used as the source of NADH-GDH, Ca(2+) had little effect on its activity. The mitochondrial fraction contained about 155 nanomoles Ca per milligram of mitochondrial protein, suggesting that the NADH-GDH in the mitochondria is already in an activated form with regard Ca(2+). In a simulated in vitro system using concentrations of 6.4 millimolar NAD, 0.21 millimolar NADH, 5 millimolar alpha-KG, and 5 millimolar glutamate thought to occur in the mitochondria, together with 1 millimolar Ca(2+), 10 and 50 millimolar NH(4) (+), and purified enzyme, the equilibrium of GDH was in the direction of glutamate formation.