Impaired substrate utilization in mitochondria from strain 129 dystrophic mice

Mitochondria from skeletal muscle, heart and liver of strain 129/ReJ-dy dystrophic mice and their littermate controls were characterized with respect to their respiratory and phosphorylating activities. Skeletal muscle mitochondria from dystrophic mice showed significantly lower state 3 respiratory rates than controls with both pyruvate + malate and succinate as substrates (P < 0.01). ADP/O and Ca²⁺/O ratios were found to be normal. A decreased rate of NADH oxidation (0.01 <P < 0.05) by sonicated mitochondrial suspensions from dystrophic mice was also seen. High respiratory rates with ascorbate + phenazine methosulfate as substrates indicated that cytochrome oxidase was not rate limiting in the oxidation of either pyruvate + malate or succinate. Skeletal muscle mitochondria from dystrophic mice showed no deficiency in any of the cytochromes or coenzyme Q. Mg²⁺-stimulated ATPase activity was higher in dystrophic muscle mitochondria than in controls, but basal and oligomycin-insensitive activities were virtually identical to those of controls. A significant reduction in the intramitochondrial NAD⁺ content (0.01 <P < 0.02) was seen in dystrophic skeletal muscle as compared to controls. Heart mitochondria from dystrophic mice showed similar, though less extensive abnormalities while liver mitochondria were essentially normal. We concluded from these results that skeletal muscle mitochondria from strain 129 dystrophic mice possess impairments in substrate utilization which may result from (1) an abnormality in the transfer of electrons on the substrate side of coenzyme Q in the case of succinate oxidation; (2) a defect on the path of electron flow from NADH to cytochrome c, and (3) a deficiency of NAD⁺ in the case of NAD⁺-linked substrates.     

Stimulation of monoacylglycerophosphate formation by Z protein

Z protein has been purified from 110,000 × g rat liver supernatant using Sephadex G-100 and DEAE Sephadex. Z protein obtained in this manner was superior to albumin in stimulating the esterification of sn-glycerol-3-phosphate in the presence of palmityl-CoA and rat liver microsomes. These observations constitute direct evidence for the possible role of Z protein in fatty acid metabolism.     

Cellular energy metabolism during fetal development. IV. Fatty acid activation, acyl transfer and fatty acid oxidation during development of the chick and rat

We have investigated developmental profiles of ATP-dependent palmityl-CoA synthetase, acetyl-CoA synthetase, palmitylcarnitine transferase, and fatty acid oxidation in heart and liver of developing chicks and rats. Palmityl-CoA synthetase activity of rat liver and heart homogenates increased 6- to 10-fold during the first postnatal week. Chick embryo heart activity peaked between 13 and 16 days of development. The activity of embryonic chick livers was bimodal with highest activity seen at 7 and 16 days of development. Posthatching values were approximately 50–75% of the peak embryonic levels. Acetyl-CoA synthetase activity of rat liver and heart homogenates was low but also showed developmental increases following birth. Acetyl-CoA synthetase activity of chick embryonic hearts was greatest at 16 days of development. Palmitylcarnitine transferase activity of rat liver and heart homogenates showed a striking increase during the first week of life. Chick heart activity was similar to that observed for palmityl-CoA synthetase with a peak between 13 and 16 days of embryonic development. Coincident with the postnatal rise in fatty acid activation and palmitylcarnitine transferase activity in developing rats, the oxidation of palmityl-CoA plus carnitine and of palmitylcarnitine increased from barely measurable levels at birth to adult levels by 30 days of age. The increases that we observe probably relate to changes in the specific activity of the enzymes as well as to an increase in the absolute number of mitochondria during development.     

Regulation of long chain fatty acid activation in heart muscle.

Regulation of fatty acid activation was studied in whole tissue homogenates of rat heart. The palmityl-CoA synthestase activity was proportional to the fatty acid to albumin ratio in the incubation medium with maximal activity occurring at a molar ratio of about 5. Fatty acyl-CoA synthetase activity was inhibited by products of the reaction (AMP, pyrophosphate, and palmityl-CoA). The apparent Ki for palmityl-CoA inhibition was 5 muM and this inhibition could be relieved by CoA-SH or albumin. The Km for CoA-SH in the absence of palmityl-CoA was 7 muM and was increased to 24 muM by addition of 8 muM palmityl-CoA. Cytosolic and mitochondrial levels of CoA-SH and carnitine were estimated in whole tissue homogenates of heart and liver. From 90 to 100% of whole tissue CoA was recovered in the mitochondrial fraction of heart muscle and it was estimated that the cytosolic concentration of free CoA-SH probably never exceeds its Km value for fatty acid activation in this tissue. Therefore, the rate of fatty acid activation would be expected to depend on the availability of CoA-SH in the cytosolic space. By adjusting the concentration of CoA-SH in the cytosol to the rate of acetyl-CoA oxidation, carnitineacetyl-CoA transferase may function in cardiac muscle to couple the rate of fatty acid activation in the cytosolic compartment to acetyl-CoA oxidation in the mitochondria. Approximately 30% of whole tissue CoA-SH was located in the cytosolic space in liver. Heart muscle has about twice as much carnitine as liver but in both tissues 100% of whole tissue carintine was located in the cytosolic space. The ratio of carnitine to CoA-SH in the cytosolic space was estimated to be about 100 in heart and 17 in liver. This high ratio in cardiac muscle may function to channel fatty acids toward oxidation rather than toward synthesis of complex lipids.     

Relative importance of acetate, acetoacetate and D-beta-OH-butyrate in the transport of acetyl CoA from the mitochondria to the cytoplasm for fatty acid synthesis in mice.

Mice were injected intravenously with either 3-¹⁴C acetoacetate, 3-¹⁴C D-β-OH-butyrate or 1-¹⁴C acetate and the radioactivity of the fatty acids was measured. In liver, the values obtained for acetoacetate and β-OH-butyrate were identical and slightly higher than those for acetate. In carcass and adipose tissue, the values obtained for the β-OH-butyrate were lower than for the other two. In particle-free supernatant of liver and adipose tissue, almost no radioactivity was obtained from β-OH-butyrate, and only the acetoacetate and the acetate were used efficiently (in vitro studies). The incorporation of acetate and acetoacetate by adipose tissue supernatant is higher than that of citrate by liver supernatant.The cytoplasmic acetoacetyl CoA synthetase and acetyl CoA synthetase activity was found to be higher in adipose tissue than in the liver. β-OH-butyryl CoA synthetase was found to be much less active than the other two synthetases. Acetoacetyl CoA thiolase is very active in the mitochondria and supernatant of adipose tissue.Our results show that, in mice adipose tissue in particular, where the citrate-cleavage enzyme is not very active, acetyl CoA is probably transformed into acetoacetate so that it can leave the mitochondria to participate in cytoplasmic fatty acid synthesis.     

The binding of long chain fatty acid CoA to Z, a cytoplasmic protein present in liver and other tissues of the rat

A cytoplasmic protein (Z protein) has been shown to have a high affinity for the CoA derivatives of long chain fatty acids. Palmityl-CoA was reversibly bound to Z by a single class of high affinity binding sites with an apparent Kd ≈ 2.4 × 10^?7 moles at 4°C. Bromopalmitin, an inhibitor of fatty acid metabolism, as well as fasting, reduced the binding of palmityl-CoA to Z, while chlorphenoxy-isobutyrate enhanced the binding. Z protein in other tissues also showed a high affinity for palmityl-CoA.     

Multiple acid phosphatases in avian pectoral muscle--the postmicrosomal supernatant acid phosphatase is elevated in avian dystrophic muscle

There are at least three forms of acid phosphatase in avian pectoralis muscle differing in molecular weight, subcellular location, and response to various substrates and inhibitors. These enzymes are separated by differential sedimentation into postmicrosomal supernatant, lysosomal, and microsomal activities with apparent molecular weights in Triton X-100 of 68,000, 198,000, and 365,000, respectively. All of the enzymes show acid pH optima (pH approximately 5), but the postmicrosomal supernatant form is distinctly different from the other two forms in its resistance to most common phosphatase inhibitors and in its reduced activity against several organic phosphates. Quantitation of these three forms of acid phosphatase in normal and dystrophic avian pectoralis muscle shows that the postmicrosomal supernatant form is significantly elevated in dystrophic muscle; at 33 days ex ovo, 84% of the increased acid phosphatase activity in dystrophic muscle can be attributed to the postmicrosomal supernatant form. The microsomal form is only slightly elevated; the level of the lysosomal form is not altered.     

Decreased mitochondrial creatine kinase activity in dystrophic chicken breast muscle alters creatine-linked respiratory coupling

Dystrophic chicken breast muscle mitochondria contain significantly less mitochondrial creatine kinase than normal breast muscle mitochondria. Breast muscle mitochondria from normal 16- to 40-day-old chickens contain approximately 80 units of mitochondrial creatine kinase per unit of succinate:INT (p-iodonitrotetrazolium violet) reductase, a mitochondrial marker, while dystrophic chicken breast muscle mitochondria contain 36-44 units. Normal chicken heart muscle mitochondria contain about 10% of the mitochondrial creatine kinase per unit of succinate:INT reductase as normal breast muscle mitochondria. The levels in heart muscle mitochondria from dystrophic chickens are not affected significantly. Evidence is presented which shows that the reduced level of mitochondrial creatine kinase in dystrophic breast muscle mitochondria is responsible for an altered creatine linked respiration. First, both normal and dystrophic breast muscle mitochondria respire with the same state 3 and state 4 respiration. Second, the post-ADP state 4 rate of respiration of normal breast muscle mitochondria in the presence of 20 mM creatine continues at the state 3 rate. However, the state 4 rate of dystrophic breast muscle mitochondria and mitochondria from other muscle types with a low level of mitochondrial creatine kinase, such as heart muscle and 5-day-old chicken breast muscle, is slower than the state 3 rate. Third, dystrophic breast mitochondria synthesize ATP at the same rate as normal breast muscle mitochondria but rates of creatine phosphate synthesis in 20-50 mM Pi are reduced significantly. Finally, increasing concentrations of Pi displace mitochondrial creatine kinase from mitoplasts of normal and dystrophic breast muscle mitochondria with the same apparent KD, indicating that the outer surface of the inner mitochondrial membrane and the mitochondrial creatine kinase from dystrophic muscle are not altered.     

Palmityl-CoA and palmitylcarnitine interactions in mitochondrial fatty acid elongation

We have investigated interactions of palmityl-CoA and l-palmitylcarnitine as substrates for mitochondrial fatty acid elongation. l-Palmitylcarnitine is a more effective substrate primer for fatty acid elongation by intact mitochondria than is palmityl-CoA. Exogenous l-carnitine inhibited l-palmitylcarnitine-supported mitochondrial fatty acid elongation by both sonically disrupted and intact heart mitochondria, probably by shifting the equilibrium between palmitylcarnitine and palmityl-CoA toward palmitylcarnitine, thus removing palmityl-CoA from the reaction. d-Carnitine was without effect. d-Palmitylcarnitine inhibition of palmitylcarnitine transferase activity decreased palmitylcarnitine-stimulated mitochondrial fatty acid elongation but increased palmityl-CoA supported fatty acid elongation, presumably by increasing the effective concentration of palmityl-CoA in the assay medium. The data indicate that, although l-palmitylcarnitine is an effective substrate primer for mitochondrial fatty acid elongation, palmityl-CoA rather than palmitylcarnitine is the immediate precursor for fatty acid chain elongation.     

Inhibitory effect of acetazolamide on the activity of acetyl CoA carboxylase of mouse liver.

The effect of different concentrations of acetazolamide on the activities of acetyl CoA carboxylase and fatty acid synthetase was studied. Acetazolamide inhibits the activity of 100, 00 × g supernatant acetyl CoA carboxylase and that of this purified enzyme even in the presence of high concentrating of bicarbonate. It is without action on fatty acid synthetasse.     

Two complementary radiometric methods for the measurement of 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (SAICAR synthetase)

Two complementary methods have been devised for measuring the activity of 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (SAICAR synthetase, EC 6.3.2.6), a critical enzyme in the pathway of purine biosynthesis. In the first method, l-[4.¹⁴C]aspartic acid is condensed with 5-amino-4-imidazolecarboxylic acid ribonucleotide (AICOR) via the action of SAICAR synthetase. Unreacted l-[4-¹⁴C]aspartic acid is measured by scintillation spectrometry. In the second method, the reverse reaction of SAICAR synthetase is measured; radiactive 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide (SAICAR) is synthetized enzymatically, using a partial purified preparation of SAICAR synthetase from chicken liver. To the purified [¹⁴C]SAICAR is added: sodium arsenate, Tris-HCl buffer containing ADPMgCl₂ or buffer alone, and to initiate the reaction, a 12 000 × g supernatant or other suitable source of enzyme. As a consequence of the arsenolytic cleavage of [¹⁴C]SAICAR, l-[4-¹⁴C]aspartic acid is generated in stoichiometric amounts. The fourth carbon of this amino acid is then detached by selective enzymatic decarboxylation, trapped in 40% KOH and quantitated by scintillation spectrometry. The assays, performed as prescribed, are facile and notably sensitive; using them, the specific activity of SAICAR synthetase has been measured in acetone powders of the livers of representative members of the Vertebrata, and also in the principal viscera of the mouse. Of the livers examined, pigeon liver was the richest source of the investigated enzyme.     

Testicular 3 alpha-hydroxysteroid oxidoreductase activity in the developing male rat.

In the testes, 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone, DHT) is converted to 5α-androstane-3α,17β-diol (3α-diol) by the enzyme 3α-hydroxysteroid oxidoreductase (3α-HSO). This steroid has been shown to possess biological activity in the male rat. The secretion of 3α-diol is much greater in the prepubertal animal than in the adult. This study is designed to quantitate the activity of 3α-HSO in the cytosol fraction of testes from male rats throughout sexual development. Following homogenizatlon of whole testes, the 105,000 × g supernatant or cytosol fraction was incubated with ³H DHT and varying concentrations of unlabelled DHT in the presence of 0.25μm NADPH. The incubation was carried out at 34°C for 10 min at a pH of 7.4. The K_m of 3α-HSO in testicular cytosol was calculated to be 1.25μM. The specific activity of testicular cytosol 3α-HSO, expressed as pmoles of 3α-diol converted from DHT per min per mg testicular cytosol protein, was high in young rats from 10 to 22 days of age, and was followed by a decline between day 22 and 37, with activity remaining low throughout adulthood. Total testicular cytosol activity of 3α-HSO, expressed as nmoles of 3α-diol converted from DHT per min per pair of testes, gradually increased from day 10 to day 60 and remained high in the adult rat. In the post-pubertal period, a possible lack of available substrate, DHT, or possible endogenous testicular regulatory mechanisms acting on 3α-HSO activity might account for the actual decrease in 3α-diol concentration in the blood and testes of mature rats.     

Organ and intracellular localization of short-chain acyl-CoA synthetases in rat and guinea-pig.

1. Homogenates of rat epididymal fat pad, heart, kidney, lactating mammary gland, liver, skeletal muscle and small intestinal mucosa have been partitioned into a particulate and supernatant fraction. With reliable marker enzymes for the mitochondrial matrix and the cytosol: propionyl-CoA carboxylase and pyruvate kinase, the distributions of the acyl-CoA synthetase activities measured at 1 and 10 mM C2, C3 and C4 over mitochondria and cytosol have been calculated. From these values an estimate was made of the K0.5 of the fatty acids. 2. A distinct fatty acid-activating enzyme was assumed to be present in one of the compartments when that fatty acid was activated with a K0.5 less than or equal to 1.5 mM in an amount of greater than 13% of the total cellular activity. Adipose tissue, gut, liver and mammary gland, all organs of a high lipogenetic capacity, contained a cytosolic acetyl-CoA synthetase. At 1 mM acetate 60, 31, 77 and 83% of the total cellular activities in these organs were cytosolic in nature, with activities of 0.021, 0.32, 0.37 and 1.16 mumol C2 activated per min per g wet weight, respectively. 3. Mitochondrial acetyl-CoA and butyryl-CoA synthetases were found in adipose tissue, gut, heart, kidney, mammary gland and muscle. They were absent in liver. Adipose tissue and liver contained a mitochondrial propionyl-CoA synthetase with activities at 1 mM C3 of 0.014 and 1.50 mumol C3 activated per min per g wet weight, respectively. 4. At 1 mM, C2 was activated with decreasing rates by kidney, heart, mammary gland and gut (7.6-1.0 mumol C2 activated per min per g wet weight). C3 (1 mM) activation was about equal (1.6-1.9 mumol C3 activated per min per g wet weight) in liver, kidney and heart. C4 (1 mM) was activated with decreasing rates by heart, liver, kidney and gut (4.0-0.5 mumol C4 activated per min per g wet weight) in the order given. 5. The influence of the isolation method and the diet on fatty acid activation in small intestinal mucosal scrapings have been studied. To demonstrate the existence of cytosolic acetyl-CoA synthetase in fed animals a pre-treatment of everted intestine by low amplitude vibration has been found essential. Also C16 activation was highly (95%) decreased in a non-pre-vibrated preparation. 24 h starvation lowered cytosolic C2 and total C16 activation by 90 and 80%, respectively. Refeeding of starved rats with a balanced fat-free diet, and not with sucrose only, gave the same cytosolic C2 and total C16 activation as normally fed rats. 6. In guienea-pig heart, kidney, liver and muscle about the same partitions have been found as in the respective rat organs. The acetate activation in liver was factor 6 lower. Acetate and butyrate activation in guinea-pig muscle was much higher (6 and 37 times, respectively).     

A comparative study of superoxide dismutase activity in polymorphonuclear leukocytes, monocytes, and alveolar macrophages of the guinea pig.

Superoxide dismutase, an enzyme which catalyzes the dismutation of superoxide radical formed during the univalent reduction of oxygen, was quantitated by observing the inhibition of cytochrome C reduction in three cell fractions in guinea pig peritoneal PMNs and monocytes and compared to alveolar macrophages. No differences were found in the 16,000 × g pellets containing mitochondria, membranes, and granules and representing 96% of total SOD activity in PMNs and monocytes but only 48% total SOD activity in alveolar macrophages. The 100,000 × g microsomal pellet of alveolar macrophages contained 8% of total SOD activity and two-five times more activity than the respective fractions from monocytes and PMNs. However, there was 70 times more SOD in the 100,000 × g supernatant from alveolar macrophages containing 44% of total enzyme activity than in the same fraction of PMNs and monocytes containing less than 2% total SOD activity. SOD activity is mainly located in the 16,000 × g particulate fraction of PMN and monocytes but more equally distributed between the particulate fractions and cytosol of alveolar macrophages.     

Conversion of diglyceride to triglyceride by rat liver microsomes: a requirement for the 105,000 x g supernatant

This paper describes a requirement for the 105,000 × g supernatant of rat liver for the synthesis of triglyceride from diglyceride and palmityl coenzyme A by rat liver microsomes. ATP and magnesium chloride are also required. The incorporation of both [1-¹⁴C]-palmityl coenzyme A and [1-¹⁴C]-diolein into triglyceride has been observed. The 105,000 × g supernatant has no enzymatic activity for this reaction when incubated in the absence of microsomes. The supernatant contains a soluble, essential protein which is nondialyzable, heat sensitive, and destroyed by trypsin. Net synthesis of triglyceride has been demonstrated by chemical analysis.     

Oxidation of palmityl-CoA to CO2 by normal and atherosclerotic aortic mitochondria

Oxidation of [1-¹⁴C] palmityl-CoA by rabbit aortic mitochondria was assayed by the incorporation of radioactivity into CO₂. Oxidation of palmityl-CoA was stimulated by 15 μM malate, 0.1% albumin, 1.0 mM ADP, and 0.1 mM L-carnitine to the extent of 42, 12, 36, and 240% respectively. Oxidation by atherosclerotic mitochondria was reduced 70–80% as compared to normal mitochondria. The extent of stimulation of oxidation in the presence of carnitine was approximately the same for both the atherosclerotic and normal mitochondria, suggesting that a membrane barrier toward palmityl-CoA persists after the development of atherosclerosis.     

Defects in Mitochondrial ATP Synthesis in Dystrophin-Deficient Mdx Skeletal Muscles May Be Caused by Complex I Insufficiency

Duchenne Muscular Dystrophy is a chronic, progressive and ultimately fatal skeletal muscle wasting disease characterised by sarcolemmal fragility and intracellular Ca²⁺ dysregulation secondary to the absence of dystrophin. Mounting literature also suggests that the dysfunction of key energy systems within the muscle may contribute to pathological muscle wasting by reducing ATP availability to Ca²⁺ regulation and fibre regeneration. No study to date has biochemically quantified and contrasted mitochondrial ATP production capacity by dystrophic mitochondria isolated from their pathophysiological environment such to determine whether mitochondria are indeed capable of meeting this heightened cellular ATP demand, or examined the effects of an increasing extramitochondrial Ca²⁺ environment. Using isolated mitochondria from the diaphragm and tibialis anterior of 12 week-old dystrophin-deficient mdx and healthy control mice (C57BL10/ScSn) we have demonstrated severely depressed Complex I-mediated mitochondrial ATP production rate in mdx mitochondria that occurs irrespective of the macronutrient-derivative substrate combination fed into the Kreb’s cycle, and, which is partially, but significantly, ameliorated by inhibition of Complex I with rotenone and stimulation of Complex II-mediated ATP-production with succinate. There was no difference in the MAPR response of mdx mitochondria to increasing extramitochondrial Ca²⁺ load in comparison to controls, and 400 nM extramitochondrial Ca²⁺ was generally shown to be inhibitory to MAPR in both groups. Our data suggests that DMD pathology is exacerbated by a Complex I deficiency, which may contribute in part to the severe reductions in ATP production previously observed in dystrophic skeletal muscle.     

Argininosuccinate synthetase activity in cultured human lymphocytes

The activity of argininosuccinate synthetase (E.C. 6.3.4.5), a urea cycle enzyme, was measured in cultured human lymphocytes using a new radioactive assay. Control cells had a maximum specific activity of 15.7±8.7 nmoles per hour per milligram of protein and an apparent K _m for citrulline of 2 × 10^–4 m, whereas cells derived from a patient with citrullinemia had no detectable activity. A nutritional variant, selected out of the citrullinemic lymphocyte population by ability to grow in citrulline, had a maximum specific activity of 10.7±3.8 nmoles/hr/mg and an apparent K _m for citrulline of 2 × 10^–2 m. These measurements confirm the observation that citrullinemia is associated with a defect in argininosuccinate synthetase activity and provide further evidence that citrullinemia is expressed in cultured lymphocytes. The emergence of a nutritional variant with a partial defect in argininosuccinate synthetase enzyme suggests that this citrullinemic patient has a heterogeneous population of cells, some totally defective and others only partially defective in argininosuccinate synthetase. The new activity assay is described in detail.This research was supported by a National Institutes of Health Training Grant (5-TO1-GM-0071) and NIH Program Project Grant (2-PO1-GM-15419).     

Adenine nucleotide translocation in yeast mitochondria. Effect of inhibitors of mitochondrial biogenesis on the ADP translocase

1. Optimal test conditions for adenine nucleotide translocation in Candida utilis mitochondria are a standard medium, consisting of 0.63 M mannitol, 2 mM EDTA (or ethylene glycol tetraacetic acid, EGTA), 10 mM morpholinopropane sulfonic acid (pH 6.8), and a temperature of 0 °C.

2. Adenine nucleotide translocation in C. utilis mitochondria is an exchange-diffusion process. The whole pool of internal adenine nucleotides is exchangeable, ADP being the most readily exchangeable nucleotide. The rate of mitochondrial ADP exchange, but not the K_m value, depends on growth conditions. At 0 °C, the rate is about 3 to 4 nmoles ADP/min per mg protein for mitochondria obtained from yeast grown in the presence of 1.5% glucose; it rises to 11.5 nmoles when glucose is replaced by 3% ethanol in the growth medium. The K_m value for ADP is 2 μM. The Q₁₀ is about 2 between 0 and 20 °C. Among other exchangeable adenine nucleotides are ATP, dADP and the methylene and the hypophosphate analogues of ADP. Unlike mammalian mitochondria, C. utilis mitochondria are able to transport external UDP by a carboxyatractyloside-sensitive process.

3. Under conditions of oxidative phosphorylation (phosphate and substrate present in an aerated medium), added ADP is exchanged with internal ATP. A higher ATP/ADP ratio was found in the extramitochondrial space than in the intramito-chondrial space. The difference between the calculated phosphate potentials in the two spaces was 0.9–1.7 kcal/mole.

4. Atractyloside, carboxyatractyloside, bongkrekic acid and palmityl-CoA inhibit mitochondrial adenine nucleotide translocation in C. utilis as they do in mammalian mitochondria, but 2 to 4 times less efficiently. The inhibition due to atractyloside or palmityl-CoA is competitive with respect to ADP whereas that due to bongkrekic acid and carboxyatractyloside is non-competitive. Carboxyatractyloside and atractyloside inhibitions are additive. The apparent K_d for the binding of [³⁵S]-carboxyatractyloside and [¹⁴C]bongkrekic acid is 10–15 nM and the concentration of sites 0.4–0.6 nmole/mg protein in both cases. [³⁵S]Carboxyatractyloside binding is competitively displaced by atractyloside and vice versa.

5. Binding of [¹⁴C]ADP has been carried out with mitochondria depleted of their endogenous adenine nucleotides by incubation with phosphate and Mg²⁺ at 20 °C. The amount of bound [¹⁴C]ADP which is atractyloside removable is 0.08–0.16 nmole/mg protein.

6. The rate of ADP transport is quite different in mitochondria isolated from C. utilis, according to whether it is grown on glucose, or on ethanol or in the presence of chloramphenicol; for instance, it decreases by 10 times when 3% ethanol in the growth medium is replaced by 10% glucose and by 5 times when chloramphenicol is added to the medium. These variations are accompanied by parallel variations in cytochrome aa₃. The number of atractyloside-sensitive ADP binding sites is not modified by the above conditions of culture, nor the number of [³⁵S]carboxyatractyloside binding sites. The affinity for ADP is apparently not significantly modified, nor the size of the endogenous adenine nucleotide pool. In contrast to glucose repression or chloramphenicol inhibition, semi-anaerobiosis in C. utilis lowers significantly the mitochondrial binding capacity for carboxyatractyloside. Strict anaerobiosis in S. cerevisiae results in a practical loss of the cytochrome oxidase activity, and also of the carboxyatractyloside and ADP binding capacity. Transition from anaerobiosis to aerobiosis restores the cytochrome oxidase activity and the ADP and carboxyatractyloside binding capacities.     

Increase in placental 15-hydroxy-prostaglandin dehydrogenase in the first half of human pregnancy

NAD-dependent 15-hydroxy-prostaglandin dehydrogenase (PGDH) activity was measured in homogenates of 25 human placentae obtained between 7 and 17 weeks of gestation. PGDH activity, expressed in nanomoles PGF_2α metabolized per min, ranged from 0.2 to 5.4 nmoles per mg placental protein and from 1.5 to 80 nmoles per g wet weight. PGDH activity per mg protein and per g weight increased significantly in function of gestational age (p<0.001). Between 7–8 weeks' gestation and 15–16 weeks mean values increased tenfold from 0.4 to 3.0 nmoles per mg protein and from 2.7 to 36.6 nmoles per g wet weight. Per unit of weight these early placentae contained less PGDH activity than term controls, but this related mainly to their high water content. Per mg placental protein PGDH activities already equalled values found at term before the end of the first trimester. The data indicate that the development of terminal villi and the migration of trophoblast into the maternal spiral arteries is associated with a substantial increase in the placental capacity for prostaglandin metabolism.