Rate Of Malate Oxidation Research Articles

Combined Action of Melatonin and Water Deficiency on Growth and MDA Content of Hypocotyls and Roots of Lupine and Respiration of Mitochondria Isolated from These Organs

Melatonin is known to improve the tolerance of plants to adverse abiotic factors (AAF), including dehydration. Nevertheless, details of the action of this phytohormone are not sufficiently comprehended. In the current study, the effects of melatonin on the growth, water status, and content of malonic dialdehyde were evaluated in etiolated seedlings of narrow-leaved lupine (Lupinus angustifolius L.) subjected to dehydration. The respiration of mitochondria isolated from different organs of these plants was also analyzed. Here, the 4-day-old seedlings have been deprived of water for 1 day. This entailed the increased water deficit up to 22% in the roots and 6% in the hypocotyls. Such desiccation of the seedlings retarded the growth of their hypocotyls but increased that of the roots by 13% in comparison with the undesiccated control. Both root and hypocotyl tissues accumulated MDA under these conditions. The dehydration of the seedlings negatively affected the respiration of the mitochondria isolated from these organs. The rate of oxidation of the substrates, especially malate, was diminished at the metabolic State 3 mainly due to an inhibition of the cytochrome respiration pathway (CP). For example, upon oxidation of malate, the activity of CP decreased 1.8 times in the mitochondria from hypocotyls and four to five times in those from roots. Pretreatment of the seedlings with 0.1 µM melatonin prevented the dehydration-induced rise in the MDA level in the hypocotyls but, by contrast, stimulated this parameter by 27% in the roots. Under drought conditions combined with exogenous melatonin, the growth of hypocotyls was close to that in the undesiccated control, while the root growth was lower than that in the control. In the seedlings normally supplied with water, treatment with melatonin did not significantly influence the respiration of the mitochondria prepared from the hypocotyls or roots. However, under water shortage, melatonin completely blocked the inhibition of a substrate oxidation in the hypocotyl mitochondria, chiefly because of sustaining the CP activity. As a result, exogenous melatonin increased the rate of malate oxidation by 87% and succinate oxidation by 26% in metabolic state 3 in the mitochondria from the hypocotyls. Application of this hormone to the plants only partially reversed the dehydration-induced inhibition of the oxidative phosphorilation in the root mitochondria. In this case, the oxidation of respiratory substrates, especially succinate, and activity of CP were still inhibited. Possible causes of the differences between the hypocotyl and root cells of the lupine seedling in their responses to melatonin and water deficit, namely, an extent of oxidative stress and functions of mitochondria, are discussed.

Ultrastructure and metabolic activity of pea mitochondria under clinorotation

Experimental data on the mitochondrial ultrastructure and tissue respiration in root apex as well as metabolic activity of the organelles isolated from pea seedling roots after 5-day of clinorotation are presented. It was shown that mitochondrial condensation in the distal elongation zone correlated with an increased rate of oxygen uptake on 7%. We also observed increase in rate of malate oxidation and respiratory control ratio increased simultaneously with a decreased in efficiency of oxidative phosphorylation. Such character of mitochondrial rearrangements in simulated microgravity is assumed to be a consequence of adaptation to these conditions.

Malate metabolism in Hoya carnosa mitochondria and its role in photosynthesis during CAM phase III

This study investigated the respiratory properties and the role of the mitochondria isolated from one phosphoenolpyruvate carboxykinase (PCK)-CAM plant, Hoya carnosa, in malate metabolism during CAM phase III. The mitochondria showed high malate dehydrogenase (mMDH) and aspartate amino transferase (mAST), and a significant amount of malic enzyme (mME) activities. H. carnosa readily oxidized malate via mME and mMDH in the presence of some cofactors such as thiamine pyrophosphate (TPP), coenzyme A (CoA) or NAD(+). A high respiration rate of malate oxidation was observed at pH 7.2 with NAD(+) and glutamate (Glu). Providing AST and Glu simultaneously into the respiratory medium strongly increased the rates of malate oxidation, and this oxidation was gradually inhibited by an inhibitor of alpha-ketoglutarate (alpha-KG) carrier, pyridoxal-5'-phosphate (PLP). The mitochondria readily oxidized aspartate (Asp) or alpha-KG individually with low rates, while they oxidized Asp and alpha-KG simultaneously with high rates, and this simultaneous oxidation was also inhibited by PLP. By measuring the capacity of the mitochondrial shuttle, it was found that the OAA produced via mMDH seemed not to be transported outside the mitochondria, but mAST interconverted OAA and Glu to Asp and alpha-KG, respectively, and exported them out via a malate-aspartate (malate-Asp) shuttle. The data in this research suggest that during phase III of PCK-CAM, H. carnosa mitochondria oxidized malate via both mME and the mMDH systems depending on metabolic requirements. However, malate metabolism by the mMDH system did not operate via a malate-OAA shuttle similarly to Ananas comosus mitochondria, but it operated via a malate-Asp shuttle similarly to Kalanchoë daigremontiana mitochondria.

Oxidations of various substrates and effects of the inhibitors on purified mitochondria isolated from Kalanchoë pinnata

KalanchoĂŤ pinnata mitochondria readily oxidized succinate, malate, NADH, and NADPH at high rates and coupling. The highest respiration rates usually were observed in the presence of succinate. The high rate of malate oxidation was observed at pH 6.8 with thiamine pyrophosphate where both malic enzyme (ME) and pyruvate dehydrogenase were activated. In CAM phase III of K. pinnata mitochondria, both ME and malate dehydrogenase (MDH) simultaneously contributed to metabolism of malate. However, ME played a main function: malate was oxidized via ME to produce pyruvate and CO2 rather than via MDH to produce oxalacetate (OAA). Cooperative oxidation of two or three substrates was accompanied with the dramatic increase in the total respiration rates. Our results showed that the alternative (Alt) pathway was more active in malate oxidation at pH 6.8 with CoA and NAD+ where ME operated and was stimulated, indicating that both ME and Alt pathway were related to malate decarboxylation during the light. In K. pinnata mitochondria, NADH and NADPH oxidations were more sensitive with KCN than that with succinate and malate oxidations, suggesting that these oxidations were engaged to cytochrome pathway rather than to Alt pathway and these capacities would be desirable to supply enough energy for cytosol pyruvate orthophosphate dikinase activity.

Respiratory properties and malate metabolism in Percoll-purified mitochondria isolated from pineapple, Ananas comosus (L.) Merr. cv. smooth cayenne.

An investigation was made of the respiratory properties and the role of the mitochondria isolated from one phosphoenolpyruvate carboxykinase (PCK)-CAM plant Ananas comosus (pineapple) in malate metabolism during CAM phase III. Pineapple mitochondria showed very high malate dehydrogenase (MDH), and low malic enzyme (ME) and glutamate-oxaloacetate transaminase (GOT) activities. The mitochondria readily oxidized succinate and NADH with high rates and coupling, while they only oxidized NADPH in the presence of Ca(2+). Pineapple mitochondria oxidized malate with low rates under most assay conditions, despite increasing malate concentrations, optimizing pH, providing cofactors such as coenzyme A, thiamine pyrophosphate, and NAD(+), and supplying individually external glutamate or GOT. However, providing glutamate and GOT simultaneously strongly increased the rates of malate oxidation. The OAA easily permeated the mitochondrial membranes to import into or export out of pineapple mitochondria during malate oxidation, but the mitochondria did not consume external Asp or alpha-KG. These results suggest that OAA played a significant role in the mitochondrial malate metabolism of pineapple, in which malate was mainly oxidized by active mMDH to produce OAA which could be exported outside the mitochondria via a malate-OAA shuttle. Cytosolic GOT then consumed OAA by transamination in the presence of glutamate, leading to a large increase in respiration rates. The malate-OAA shuttle might operate as a supporting system for decarboxylation in phase III of PCK-CAM pineapple. This shuttle system may be important in pineapple to provide a source of energy and substrate OAA for cytosolic PCK activity during the day when cytosolic OAA and ATP was limited for the overall decarboxylation process.

Developmental Changes in the NAD Content in Sugar-Beet Root Mitochondria and Their Effect on the Oxidative Activity of These Organelles

The NAD content was determined in mitochondria isolated from sugar-beet roots at various stages of plant development. A high NAD content (7.6 ± 0.9 nmol/mg mitochondrial protein) was observed in the mitochondria of actively growing roots of 80–95-day-old plants, but it decreased ca. twofold by the end of the first year of plant development, before the roots were harvested for storage. The mitochondria isolated from roots stored at low temperature for two to three months and those after five to eight days of regrowth during the second year of plant development manifested an even lower NAD content (2.2 ± 0.4 and 2.0 ± 0.5 nmol/mg protein, respectively). A drastic decrease in the NAD content in mitochondria from stored roots did not result from the impairment of the inner membrane of these organelles and was evidently regulatory in its nature. The effect of developmental changes in the intramitochondrial NAD content on the malate oxidation pattern was studied. In the mitochondria of stored roots, the low NAD content limited the rate of malate oxidation in state 3, because the addition to the reaction mixture of exogenous NAD, which can be transported to the mitochondrial matrix, promoted malate oxidation by 30–50%. Rotenone inhibited malate oxidation in the stored-root mitochondria by more than 70%; in this case, the rate of rotenone-resistant malate oxidation in these organelles increased by several times in the presence of exogenous NAD. In the mitochondria of the growing root, exo-genous NAD did not affect the rate of malate oxidation, and rotenone inhibited it only by 25–35%. The analysis of the data obtained here and the published evidence suggests the existence of a universal mechanism of respiration control and the regulation of the functional activity of plant mitochondria. This mechanism acts through a change in the NAD content in the organelle matrix. This NAD can be used in the course of plant development, e.g., during the transition of sugar-beet-root cells in the dormant state, when the respiration rate must decline.

Complex I impairment, respiratory compensations, and photosynthetic decrease in nuclear and mitochondrial male sterile mutants of Nicotiana sylvestris.

We have previously shown that in Nicotiana sylvestris cytoplasmic male-sterile (CMS) mutants where the mtDNA lacks the nad7 gene coding for a subunit of respiratory Complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3), glycine (Gly) oxidation was lower than in the wild type and insensitive to rotenone, suggesting Complex I dysfunction. In contrast, the oxidation rate of exogenous NADH and the capacity of the cyanide-resistant respiration (AOX) were enhanced. Here we report that, in contrast to Gly, the rate of malate oxidation was not affected, but proceeded totally in a rotenone-insensitive pathway, strongly suggesting that survival of CMS plants depends on the activation of internal and external alternative NAD(P) H dehydrogenases and that Gly decarboxylase activity depends on Complex I functioning. A similar defect in Complex I activity and Gly oxidation was found in the NMS1 nuclear mutant, defective in the processing of the nad4 transcript, but alternative NAD(P) H dehydrogenases were less activated. In CMS and NMS1, the fraction of the AOX pathway was increased, as compared to wild type, associated with higher amounts of aox transcripts, AOX protein, and plant resistance to cyanide. Non-phosphorylating respiratory enzymes maintained normal in vivo respiration levels in both mutants, but photosynthesis was decreased, in correlation with lower leaf conductance, emphasizing mitochondrial control on photosynthesis.

Purification and physical and kinetic characterization of an NAD+ -dependent malate dehydrogenase from leaves of pineapple (Ananas comosus )

An NAD + -dependent malate dehydrogenase (MDH, EC 1.1.1.37) was purified and characterized from leaves of pineapple (Ananas comosus), a plant with Crassulacean acid metabolism (CAM). The purified enzyme had a subunit molecular mass of 39.5 kDa. Its activity showed a maximum at pH 6.8-7.0 and decreased sharply towards pH 8.0. This activity profile coincided with a change in the aggregation state, as determined by gel filtration on high-performance liquid chromatography from a dimer at pH 7.0 to a tetramer at pH 8.0. This isozyme is one of at least 5 MDH in pineapple leaves distinguishable by non-denaturing isoelectric focusing and displayed an isoelectric point of 5.8. The ratio of oxaloacetate reduction versus malate oxidation rates varied between 431 and 52 at pH 6.8 and 7.5, respectively. Antibodies raised against the purified pineapple leaf MDH immunodecorated a single 39.5-kDa polypeptide in denatured crude leaf extracts, but did not cross-react with extracts from purified pineapple mitochondria possessing high MDH activity. The purified MDH was recognized by monoclonal antibodies raised against the cytosolic MDH from Echinococcus granulosus. These and other distinctive traits, such as its isoelectric point and its subunit mass, suggest that the purified isozyme is the cytosolic MDH. Its properties are consistent with an implied function in the night acidification typical of CAM plants, although it is less clear if it also has a role in the daytime decarboxylation of malate.

Effects of short-term drought and rewatering on the activity of mitochondrial enzymes and the oxidative capacity of leaf mitochondria from a CAM plant, Aptenia cordifolia

Summary Changes in the oxidative properties of isolated leaf mitochondria and in the activities of several mitochondrial enzymes, involved in CAM, were examined in response to a short-term drought (10 d) and to rewatering in the CAM plant Aptenia cordifolia . Integrity of the isolated organelles was always high (>90%) and further purification did not increase activity or intactness. To evaluate the influence of mitochondrial activities on CAM, variations in malic and citric acid accumulation, and in diurnal gas exchange were concomitantly followed in a controlled environment chamber study. While daytime CO 2 uptake ceased within 2 days due to stomatal closure and nocturnal CO 2 uptake gradually diminished, malic and citric acid accumulation increased slighdy during drought. The latter could not be due to enhanced mitochondrial oxidative activity, because its relative measure, the Cytochrome c (Cyt c ) oxidase activity, decreased reversibly during drought. In addition, complete malate oxidation may occur to only a minor extent. Malate oxidation activity largely depended on malic enzyme (ME) activity, which was high in A. cordifolia if compared with other CAM species. Drought induced reduction of glycine oxidation activity may result in a larger relative capacity for malate oxidation because competition for NAD would decrease and changes of in vivo malate oxidation rates could then meet the additional demand for oxidative capacity during drought. Mitochondrial sensitivity to cyanide was always high, while that to rotenone was low, in particular when malate was used as a substrate. Increased net citric acid accumulation in response to drought may result from the larger inhibition of the citrate degrading system relative to citrate synthesis, although the capacity for citric acid oxidation and the citrate synthetase (CS) activity declined during drought. Changes in mitochondrial oxidative properties and enzyme activities, observed during drought, were reversible upon rewatering.

Adenylate control of respiration in plants: the contribution of rotenone‐insensitive electron transport to ADP‐limited oxygen consumption by soybean mitochondria

The aim was to test the hypothesis that rotenone‐insensive electron transport (bypass of complex I) may underlie rapid state 4 (ADP‐limited) mitochondrial respiration. A comparison of mitochondria from soybean (Glycine max L. cv. Bragg) cotyledons and nodules showed that ADP‐sufficient (state 3) malate plus pyruvate oxidation by mitochondria from 7‐day‐old cotyledons was inhibited 50% by rotenone and state 4 rates were rapid, whereas nodule mitochondria were 80% inhibited by rotenone and had slower state 4 rates of malate plus pyruvate oxidation. Respiration of malate alone (pH 7.6) by cotyledon mitochondria was slow, especially in the absence of ADP; subsequent addition of pyruvate dramatically increased state 4 oxygen uptake concomitant with a rapid rise in mitochondrial NADH (determined by fluorimetry). Rotenone had no effect on this increased rate of state 4 respiration. The rate of malate oxidation by nodule mitochondria was relatively rapid compared with cotyledon mitochondria. The addition of pyruvate in state 4 caused a slow increase in matrix NADH and only a slight stimulation of oxygen uptake. Rotenone inhibited state 4 malate plus pyruvate oxidation by 50% in these mitochondria. From a large number of cotyledon and nodule mitochondrial preparations, a close correlation was found between the rate of state 4 oxygen uptake and rotenone‐resistance. During cotyledon development increased rotenone‐resistance was associated with an increase in the alternative oxidase. Addition of pyruvate to cotyledon mitochondria, during state 4 oxidation of malate in the presence of antimycin A, significantly stimulated O2 uptake and also almost eliminated respiratory control. Such combined operation of the rotenone‐insensitive bypass and the alternative oxidase in vivo will significantly affect the extent to which adenylates control the rate of electron transport.

Subcellular distribution of pyruvate‐degrading enzymes in Chlamydomonas reinhardtii studied by an improved protoplast fractionation procedure

The subcellular distribution of pyruvate‐degrading enzymes has been determined in Chlamydomonas reinhardtii (Dangeard) by protoplast induction with autolysine, dig‐itonin lysis and further fractionation by differential centrifugation using a Percoll cushion. Mitochondrial and plastidic fractions contained intact and physiologically competent organelles ‐ RC 1.7, ADP/O 2.7 and rate of malate oxidation 76 nmol O, (mg protein)‐1min‐1 for mitochondria, CO2; fixation 46.8 μmol (mg Chi)‐1 h‐1 for chloroplasts.Results from protoplast fractionation were further confirmed by the determination of enzyme activities within trypsin‐treated organelles. Mitochondria (formate fermentation) and chloroplasts (chlorofermentation) were shown to possess the capacity for anaerobic pyruvate degradation. Pyruvate dehydrogenase (NAD+, EC 1.2.4.1), pyruvate formate‐lyase (EC 2.3.1.54) and lactate dehydrogenase (NADH, EC 1.1.1.27) showed equal distribution between mitochondria and chloroplasts, whereas activities of phosphotransacetylase (EC 2.3.1.8) and acetate kinase (EC 2.7.2.1) were only detectable in the mitochondrial fraction. NADH‐ and NADPH‐dependent activities of both alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (acylating, EC 1.2.1.10) were localized in the mitochondrial and cytoplasmic or the plastidic and cytoplasmic fractions, respectively, whereas pyruvate decarboxylase (EC 4.1.1.1) was only detected in the cytoplasmic fraction.

Malate Decarboxylation by Mitochondria of the Inducible Crassulacean Acid Metabolism Plant <italic>Mesembryanthemum crystallinum</italic>

Mitochondria isolated from leaves of Mesembryanthemum crystallinum oxidized malate by both NAD malic enzyme and NAD malate dehydrogenase. Rates of malate oxidation were higher in mitochondria from plants grown at 400 mil NaCl in the rooting medium and performing Crassulacean acid metabolism (CAM) than in mitochondria from plants grown at 20 mM NaCl and exhibiting C3-photosynthetic CO2 fixation. The mitochondria isolated from plants both in the CAM and C3 modes were tightly coupled and gave high respiratory control. At optimum pH for malate oxidation (pH 7.0), pyruvate was the major product in mitochondria from CAM-M. crystallinum, whereas mitochondria from C3-M. crystallinum produced predominantly oxaloacetate. Both the extracted NAD malic enzyme in the presence of CoA and the oxidation of malate to pyruvate by the mitochondria from plants in the CAM mode had a pH optimum around 7.0 with activity declining markedly above this pH. The activity of NAD-malic enzyme, expressed on a cytochrome c oxidase activity basis, was much higher in mitochondria from the CAM mode than the C3 mode. The results indicate that mitochondria of this species are adapted to decarboxylate malate at high rates during CAM.

Continuous measurement of oxaloacetate in purified mitochondria from the leaves of Kalanchoë blossfeldiana

A new method for the continuous assay of oxaloacetate released or taken up by plant mitochondria during malate oxidation is described. It is based on the continuous spectrophotometric recording of the reduction level of externally added NAD+ (0.4 mM) to a mitochondrial preparation. In the presence of 20 mM malate and of externally added malate dehydrogenase (EC 1.1.1.37), an equilibrium is reached instantaneously, bringing about a partial reduction of NAD+ and the production of a proportional amount of oxaloacetate (OAA). Owing to the presence of a very active OAA carrier on the inner mitochondrial membrane, the concentration at the equilibrium position of the reactants of the external MDH is permanently displaced by the OAA released or taken up by the mitochondria. Therefore, changes in OAA concentration can be followed from the measurement of the reduction level of the external NAD+. This method appears as sensitive as the classical enzymatic method, but is much more rapid and requires much less mitochondrial protein. The proposed method was applied to Percoll‐purified mitochondria from the leaves of a CAM plant, Kalanchoë blossfeldiana Poelln. cv. Tom Thumb. The simultaneous recording of the change in OAA concentration and of oxygen uptake during malate oxidation emphasizes the major control exerted by OAA on the rate of malate oxidation.

Simultaneous Oxidation of Glycine and Malate by Pea Leaf Mitochondria

Mitochondria isolated from pea leaves (Pisum sativum L.) readily oxidized malate and glycine as substrates. The addition of glycine to mitochondria oxidizing malate in state 3 diminished the rate of malate oxidation. When glycine was added to mitochondria oxidizing malate in state 4, however, the rate of malate oxidation was either unaffected or stimulated. The reason both glycine and malate can be metabolized in state 4 appears to be that malate only used part of the electron transport capacity available in these mitochondria in this state. The remaining electron transport capacity was used by glycine, thus allowing both substrates to be oxidized simultaneously. This can be explained by differential use of two NADH dehydrogenases by glycine and malate and an increase in alternate oxidase activity upon glycine addition. These results help explain why photorespiratory glycine oxidation and its associated demand for NAD do not inhibit citric acid cycle function in leaves.

Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle

After preparation on self-generated Percoll gradients, avocado (Persea americana Mill, var. Fuerte and Hass) mitochondria retain a high proportion of cyanide-insensitive respiration, especially with alpha-ketoglutarate and malate as substrates. Whereas alpha-ketoglutarate oxidation remains unchanged, the rate of malate oxidation increases as ripening advances through the climacteric. An enhancement of mitochondrial malic enzyme activity, measured by the accumulation of pyruvate, closely parallels the increase of malate oxidation. The capacity for cyanide-insensitive respiration is also considerably enhanced while respiratory control decreases (from 3.3 to 1.7), leading to high state 4 rates.Both malate dehydrogenase and malic enzyme are functional in state 3, but malic enzyme appears to predominate before the addition of ADP and after its depletion. In the presence of cyanide, a membrane potential is generated when the alterntive pathway is operating. Cyanide-insensitive malate oxidation can be either coupled to the first phosphorylation site, sensitive to rotenone, or by-pass this site. In the absence of phosphate acceptor, malate oxidation is mainly carried out via malic enzyme and the alternative pathway. Experimental modification of the external mitochondrial environment in vitro (pH, NAD(+), glutamade) results in changes in malate dehydrogenase and malic enzyme activities, which also modify cyanide resistance. It appears that a functional connection exists between malic enzyme and the alternative pathway via a rotenone-insensitive NADH dehydrogenase and that this pathway is responsible, in part, for nonphosphorylating respiratory activity during the climacteric.

The Effect of Rotenone on Respiration in Pea Cotyledon Mitochondria

Respiration utilizing NAD-linked substrates in mitochondria isolated from cotyledons of etiolated peas (Pisum sativum L. var. Homesteader) by sucrose density gradient centrifugation exhibited resistance to rotenone. The inhibited rate of alpha-ketoglutarate oxidation was equivalent to the recovered rate of malate oxidation. (The recovered rate is the rate following the transient inhibition by rotenone.) The inhibitory effect of rotenone on malate oxidation increased with increasing respiratory control ratios as the mitochondria developed. The cyanide-resistant and rotenone-resistant pathways followed different courses of development as cotyledons aged. The rotenone-resistant pathway transferred reducing equivalents to the cyanide-sensitive pathway. Malic enzyme was found to be inhibited competitively with respect to NAD by rotenone concentrations as low as 1.67 micromolar. In pea cotyledon mitochondria, rotenone was transformed into elliptone. This reduced its inhibitory effect on intact mitochondria. Malate dehydrogenase was not affected by rotenone or elliptone. However, elliptone inhibited malic enzyme to the same extent that rotenone did when NAD was the cofactor. The products of malate oxidation reflected the interaction between malic enzyme and malate dehydrogenase. Rotenone also inhibited the NADH dehydrogenase associated with malate dehydrogenase. Thus, rotenone seemed to exert its inhibitory effect on two enzymes of the electron transport chain of pea cotyledon mitochondria.

Formate Oxidation and Oxygen Reduction by Leaf Mitochondria

Mitochondria isolated from the leaves of several plant species were investigated for the presence of NAD-linked formate dehydrogenase. The NADH produced was oxidized by the electron transport sequence and was coupled to ATP synthesis. The amounts of formate dehydrogenase, and, thereby, the capacity for formate-dependent O(2) uptake, varied greatly among species. While no activity was detectable in mitochondria from soybean leaves, the rate of formate oxidation by spinach mitochondria was about one-half the rate of malate oxidation. In spinach, only mitochondria from green tissues oxidized formate. These last two observations raise questions as to the role of this reaction and the possible sources of the formate metabolized.

On methods for the isolation of mitochondria from etiolated corn shoots

Mitochondria were isolated from etiolated corn shoots by two methods, one involving gradient purification and the other by centrifuging through a 0.6 M sucrose “cushion”. There were no major differences in State 3 or State 4 malate oxidation rates, respiratory control ratios, ADP:O ratios, outer membrane integrity, or swelling rates in salt solutions. The slightly superior quality of cushion mitochondria probably reflects the shorter time required for isolation. Corn mitochondria isolated by both methods showed passive swelling in KCl solutions but this does not appear to reflect anything abnormal about membrane integrity.

Effect of Phosphate and Uncouplers on Substrate Transport and Oxidation by Isolated Corn Mitochondria

A study was made to determine conditions under which malate oxidation rates in corn (Zea mays L.) mitochondria are limited by transport processes. In the absence of added ADP, inorganic phosphate increased malate oxidation rates by processes inhibited by mersalyl and oligomycin, but phosphate did not stimulate uncoupled respiration. However, the uncoupled oxidation rates were inhibited by butylmalonate and mersalyl. When uncoupler was added prior to substrate, subsequent O(2) uptake rates were reduced when malate and succinate, but not exogenous NADH, were used. Uncoupler and butylmalonate also inhibited swelling in malate solutions and malate accumulation by these mitochondria, which were found to have a high endogenous phosphate content. Addition of uncoupler after malate or succinate produced an initial rapid oxidation which declined as the mitochondria lost solute and contracted. This decline was not affected by addition of ADP or AMP, and was not observed when exogenous NADH was substrate. Increasing K(+) permeability with valinomycin increased the P-trifluoromethoxy (carboxylcyanide)phenyl hydrazone inhibition. Kinetic studies showed the slow rate of malate oxidation in the presence of uncoupler to be characterized by a high Km and a low V(max), probably reflecting a diffusion-limited process.The results indicate that rapid malate and succinate oxidation require the operation of both the phosphate and dicarboxylate transporters, which in turn depend on maintenance of a proton motive force across the inner membrane. In addition, phosphate can stimulate acceptorless malate oxidation by reaction with the coupling mechanism, and in uncoupled mitochondria which are depleted of substrate there is a slow rate of oxidation which appears to be limited by diffusive entry.

Dietary Lipid and Postnatal Development. II. Palmityl Coenzyme A Oxidation in Heart and Liver

There is only indirect evidence to suggest that the sudden postpartum appearance of dietary lipid regulates the perinatal development of the enzyme pathways required for fatty acid oxidation. To test this idea directly, rabbit pups were maintained on diets containing lipid to equal either 14.2% (LF) or 77.6% (HF) of the total caloric intake. Palmityl coenzyme A oxidation rates in the presence of excess ADP and carnitine were measured polarographically in heart and liver homogenates. No significant difference in oxidation rates between HF and LF groups was observed even at 10 days of age. Palmityl coenzyme A oxidation in both groups was carnitine dependent and was in general the same as that of mother-fed animals. Similarly, an evaluation of cytochrome oxidase activity and glutamate + malate-supported respiration in heart and liver homogenates revealed no difference attributable to diet. To consider the possibility that fatty acid oxidation might be specifically increased or decreased over other mitochondrial respiratory activity as a function of diet, palmityl coenzyme A oxidation rates were normalized with respect to glutamate + malate oxidation rates. A similar comparison was made relative to cytochrome oxidase activity. Still no differences were observed between HF and LF groups. By studying the maximum rate of oxygen utilization in the presence of excess carnitine and palmityl-coenzyme A we would have detected any change in a rate-limiting step for fatty acid oxidation beyond acyl activation. We must conclude, therefore, that large differences in the proportion of postnatal dietary lipid do no influence the cellular capacity to oxidize palmityl coenzyme A.