Malate Metabolism in Leaf Mitochondria from the Crassulacean Acid Metabolism Plant Kalanchoë blossfeldiana Poelln

The mechanisms and the controlling factors of malate oxidation by mitochondria from leaves of Kalanchoë blossfeldiana Poelln. plants performing Crassulacean acid metabolism were investigated using Percollpurified mitochondria. The effects of pH and of various cofactors (ATP, NAD⁺, coenzyme A) on malate dehydrogenase (EC 1.1.1.37) and malic enzyme (EC 1.1.1.39) solubilized from these mitochondria were examined. The crucial role of cofactor concentrations in the mitochondrial matrix on the pathways of malate oxidation is shown. The distribution of the electrons originating from malate between the different electron transport pathways and its consequence on the phosphorylation yield was studied. It was found that, depending on the electron transport pathway used, malate oxidation could yield from 3 to 0 ATP. Assayed under conditions of high reducing power and high energy charge, the ability of malic enzyme to feed electrons to the cyanide-resistant nonphosphorylating alternative pathway was found to be higher than that of other dehydrogenases linked to the functioning of the Krebs cycle (pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase). The physiological significance of such a functional relationship between malic enzyme activity and the nonphosphorylating alternative pathway is discussed in relation to Crassulacean acid metabolism.     

Temperature Effects on Phosphoenolpyruvate Carboxylase from a CAM and a C(4) Plant : A Comparative Study

The effect of temperature in the range from 10 to 35°C on various characteristics of phosphoenolpyruvate carboxylase from the leaves of a CAM plant, Crassula argentea and a C₄ plant Zea mays shows a number of different effects related to the environment in which these distinct types of metabolic specialization normally operate. The Arrhenius plot of V_max for the two enzyme forms shows that the CAM enzyme has a linear increase with temperature while the C₄ enzyme has an inflection at 27°C implying a conformational or aggregational change in the enzyme or a shift in reaction mechanism to one requiring a lower activation energy. The Arrhenius plot of K_m for the two enzymes reveals the startling fact that at temperatures above 20°C an increasing temperature causes an increase in K_mPEP for the CAM enzyme while the C₄ enzyme displays a decreased K_m as the temperature increases. The inhibitory effect of 5 millimolar malate also shows opposite trends for the two enzymes. For the CAM enzyme the percent inhibition by malate increases from essentially none at 15°C to 70% at 35°C. For the C₄ enzyme the percent inhibition drops from about 60% at 20°C to 2% at 30°C. Similar opposite behavior of the two enzymes is found with the K_i for malate. Pretreatment at high temperatures for periods up to 2 hours was found to result in differences similar to those described above if the treated enzyme were subsequently assayed at 25°C.     

The influence of temperature on malic Acid metabolism in grape berries: I. Enzyme responses

Phosphoenolpyruvate (PEP) carboxylase activity in immature `Carignane' grape berries (Vitis vinifera L.) had a temperature optimum of about 38 C, whereas malic enzyme activity rose with increasing temperature between 10 and 46 C. In vitro temperature inactivation rates for the PEP carboxylase were markedly greater than for the malic enzyme activity. From the simultaneous action of malic acid-producing enzymes (PEP carboxylase and malic dehydrogenase) and malic acid-degradating enzyme (malic enzyme) systems at different temperatures, the greatest tendency for malic acid accumulation in immature grape berries was at 20 to 25 C. Time-course measurements of enzymic activity from heated, intact berries revealed greater in vivo temperature stability for the malic enzyme activity than for the PEP carboxylase activity.     

Malate oxidation, rotenone-resistance, and alternative path activity in plant mitochondria

The effect of cyanide and rotenone on malate (pH 6.8), malate plus glutamate (pH 7.8), citrate, α-ketoglutarate, and succinate oxidation by cauliflower (Brassica oleracea L.) bud, sweet potato (Ipomoea batatis L.) tuber, and spinach (Spinacia oleracea and Kalanchoë daigremontiana leaf mitochondria was investigated. Cyanide inhibited all substrates equally with the exception of malate plus glutamate; in this case, inhibition of O₂ uptake was more severe due to an effect of cyanide on aspartate aminotransferase. Azide and antimycin A gave similar inhibitions with all substrates. Subsequent addition of NAD had no effect with any substrate. Providing that oxalacetate accumulation was prevented, rotenone inhibited all NAD-linked substrates equally and caused ADP:O ratios to decrease by one-third. Addition of succinate to mitochondria oxidizing malate stimulated oxygen uptake, but adding citrate and α-ketoglutarate did not. These results indicate that there is no direct link between malic enzyme and the rotenone- and cyanide-resistant respiratory pathways, and that there is no need to postulate separate compartmentation of malic enzyme and the other NAD-linked enzymes in the matrix.     

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 α-ketoglutarate and malate as substrates. Whereas α-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 inhibition of β-lactamases from Gram-negative bacteria by clavulanic acid

The β-lactamase from Klebsiella pneumoniae E70 behaved in a similar fashion to the TEM-2 plasmid mediated enzyme on reaction with clavulanic acid. Both enzymes produced two types of enzyme–clavulanate complex, a transiently stable species (t_½=4min at pH7.3 and 37°C) and irreversibly inhibited enzyme. In the initial rapid reaction (2.5min) the enzymes partitioned between the transient and irreversible complexes in the ratios 3:1 for TEM-2 β-lactamase and 1:1 for Klebsiella β-lactamase. Biphasic inactivation was observed for both enzymes and the slower second phase was rate limited by the decay of the transiently stable complex. This decay released free enzyme for further reaction with fresh clavulanic acid, the products again partitioning between transiently stable and irreversibly inhibited enzyme. This cycle continued until all the enzyme had been irreversibly inhibited. A 115 molar excess of inhibitor was required to achieve complete inactivation of TEM-2 β-lactamase. Hydrolysis of clavulanic acid with product release appeared to occur with the inhibition reaction, which explained this degree of clavulanic acid turnover. The stoichiometry of the interaction with Klebsiella β-lactamase was not examined. The penicillinase from Proteus mirabilis C889 was rapidly inhibited by low concentrations of clavulanic acid. The major product was a moderately stable complex (t_½=40min at pH7.3 and 37°C); the proportion of the enzyme that was irreversibly inactivated was small. The cephalosporinase from Enterobacter cloacae P99 had low affinity for the inhibitor and only reacted with high concentrations of clavulanic acid (k=4.0m⁻¹·s⁻¹) to produce a relatively stable complex (t_½=180min at pH7.3 and 37°C). No irreversible inactivation of this enzyme was detected. The rates of decay of the clavulanate–enzyme complexes produced in reactions with Proteus and Enterobacter enzymes were markedly increased at acid pH.     

Further observations on the production of amino acids by rat-liver mitochondria and other subcellular fractions

1. Rat-liver mitochondria showed a decrease in amino acid production after preparation in 0·25m-sucrose containing EDTA (1mm), but an increase in water content. When EDTA was replaced by Mn²⁺ (1mm) or succinate (1mm), both amino acid production and water content were lowered, whereas preparation in 0·9% potassium chloride caused an increase in both. 2. Amino acid production by rat-liver homogenates prepared in 0·9% potassium chloride or 0·25m-sucrose was similar (q_{amino acid} 0·047 and 0·042 respectively aerobically). After freezing-and-thawing q_{amino acid} values were approximately doubled, and approached that of a homogenate prepared in water. 3. All cations tested inhibited amino acid production by mitochondria, Hg²⁺ and Zn²⁺ being the most effective in tris–hydrochloric acid buffer. In phosphate buffer Mg²⁺ and Mn²⁺ had no effect. Of the anions tested only pyrophosphate and arsenate had any inhibitory effect at final concn. 1mm. 4. Iodosobenzoate (1mm) and p-chloromercuribenzenesulphonate (1mm) inhibited mitochondrial amino acid production by 70–80%, whereas soya-bean trypsin inhibitor, EDTA and di-isopropyl phosphorofluoridate inhibited by a maximum of 30%. Respiratory inhibitors had no effect. 5. Rat-liver homogenate and subcellular fractions each showed an individual pattern of inhibition when a series of inhibitors was tested. 6. Amino acid production by mitochondria was decreased by up to 50% in the presence of oxidizable substrate, apart from α-glycerophosphate and palmitate, which had no effect. CoA stimulated amino acid production in tris–hydrochloric acid but not in phosphate buffer, α-oxoglutarate abolishing the stimulation. 7. Cysteine and glutathione stimulated amino acid production by whole mitochondria by 30%, but only reduced glutathione stimulated production in broken mitochondria. 8. Adrenocorticotrophic hormone and growth hormone stimulated mitochondrial amino acid production by 21–24%, whereas insulin inhibited production by 25%. 9. Coupled oxidative phosphorylation increased amino acid production by up to 154% at 25° and 40°. The increase was abolished by 2,4-dinitrophenol. 10. Amino acid incorporation in mitochondria was accompanied by an increase in amino acid production, both being decreased by chloramphenicol. 11. Mitochondrial production of ninhydrin-positive material was increased in the presence of albumin. The biggest increase was noted for the soluble fraction of broken mitochondria. No increase was found in the presence of ¹⁴C-labelled algal protein or denatured mitochondrial protein.     

Moderately High Temperatures Inhibit Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase-Mediated Activation of Rubisco

We tested the hypothesis that light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inhibited by moderately elevated temperature through an effect on Rubisco activase. When cotton (Gossypium hirsutum L.) or wheat (Triticum aestivum L.) leaf tissue was exposed to increasing temperatures in the light, activation of Rubisco was inhibited above 35 and 30°C, respectively, and the relative inhibition was greater for wheat than for cotton. The temperature-induced inhibition of Rubisco activation was fully reversible at temperatures below 40°C. In contrast to activation state, total Rubisco activity was not affected by temperatures as high as 45°C. Nonphotochemical fluorescence quenching increased at temperatures that inhibited Rubisco activation, consistent with inhibition of Calvin cycle activity. Initial and maximal chlorophyll fluorescence were not significantly altered until temperatures exceeded 40°C. Thus, electron transport, as measured by Chl fluorescence, appeared to be more stable to moderately elevated temperatures than Rubisco activation. Western-blot analysis revealed the formation of high-molecular-weight aggregates of activase at temperatures above 40°C for both wheat and cotton when inhibition of Rubisco activation was irreversible. Physical perturbation of other soluble stromal enzymes, including Rubisco, phosphoribulokinase, and glutamine synthetase, was not detected at the elevated temperatures. Our evidence indicates that moderately elevated temperatures inhibit light activation of Rubisco via a direct effect on Rubisco activase.     

High Temperature Inhibits Ascorbate Recycling and Light Stimulation of the Ascorbate Pool in Tomato despite Increased Expression of Biosynthesis Genes

Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31°C) and irradiance regimes (darkness or 150 µmol m^-2 s^-1). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27°C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12°C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31°C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12°C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling.     

Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures

Respiration rates of Zea mays L. seedling tissues grown at 30 and 14°C were measured at 25°C at different stages of seedling growth. Accumulation of heat units was used to define the developmental stages to compare respiration between the two temperatures. At both temperatures, respiration rates of most tissues were highest at the youngest stages, then declined with age. Respiration rates of mesocotyl tissue were the most responsive to temperature, being nearly twofold higher when grown at 14 compared to 30°C. Alternative pathway respiration increased concomitantly with respiration and was higher in mesocotyls grown in the cold. When seedlings were started at 30 then transferred to 14°C, the increase in alternative pathway respiration due to cold was not observed unless the seedlings were transferred before 2 days of growth. Seedlings transferred to 14°C after growth at 30°C for 2 days had the same alternative oxidase capacity as seedlings grown at 30°C. Seedlings grown at 14°C for 10 to 12 days, then transferred to 30°C, lost alternative pathway respiratory capacity over a period of 2 to 3 days. Western blots of mitochondrial proteins indicated that this loss of capacity was due to a loss of the alternative oxidase protein. Some in vitro characteristics of mitochondria were determined. The temperature optimum for measurement of alternative oxidase capacity was 15 to 20°C. At 41°C, very little alternative oxidase was measured, i.e., the mitochondrial oxygen uptake was almost completely sensitive to cyanide. This inactivation at 41°C was reversible. After incubation at 41°C, the alternative oxidase capacity measured at 25°C was the similar to when it was measured at that temperature directly. Isolated mitochondria lost alternative oxidase capacity at the same rate when incubated at 41°C as they did when incubated at 25°C. Increasing the supply of electrons to isolated mitochondria increased the degree of engagement of the alternative pathway, whereas lower temperature decreased the degree of engagement. Lower temperatures did not increase the degree of engagement of the pathway in intact tissues. We interpret these observations to indicate that the greater capacity of alternative oxidase in cold-grown seedlings is a consequence of development at these low temperatures which results in elevated respiration rates. Low temperature itself does not cause greater capacity or engagement of the alternative oxidase in mitochondria that have developed under warm temperatures. Our hypothesis would be that the low growth temperatures require the seedlings to have a higher respiration rate for some reason, e.g., to prevent the accumulation of a toxic metabolite, and that the alternative pathway functions in that respiration.     

Some Properties of Thiosulfate-Oxidizing Enzyme from Marine Heterotroph 16B

Thiosulfate-oxidizing enzyme has been demonstrated in cell-free extracts of the marine, thiosulfate-oxidizing pseudomonad strain 16B. The enzyme, partially purified by ion-exchange chromatography and calcium phosphate gel treatment, catalyzed the oxidation of thiosulfate to tetrathionate with the concomitant reduction of ferricyanide. Native but not mammalian cytochrome c was also reduced by the enzyme in the presence of thiosulfate. The enzyme was located exclusively in the supernatant of ultracentrifuged cell extracts. The most purified enzyme preparation, like intact cells, exhibited a temperature optimum of 30 to 31°C. However, it exhibited no definite pH optimum. At pH 6.1 to 6.3 and 30°C, the K_m for thiosulfate was 1.57 mM. At lower temperatures, the apparent K_m for thiosulfate increased, but the apparent maximum velocity remained virtually unchanged. Thiosulfate oxidation in intact cells exhibited an increase in the pH optimum at lower temperatures. The thiosulfate-oxidizing enzyme of marine heterotroph 16B is compared with thiosulfate-oxidizing enzymes from other bacteria, and the effect of temperature on the relationship between pH and thiosulfate oxidation is discussed with reference to the natural habitat of the bacterium.     

Changes in the Enzymes for Fatty Acid Synthesis and Desaturation during Acclimation of Developing Soybean Seeds to Altered Growth Temperature

Temperature-induced changes in the enzymes for fatty acid synthesis and desaturation were studied in developing soybean seeds (Glycine max L. var Williams 82). Changes were induced by culture of the seed pods for 20 hours in liquid media at 20, 25, or 35°C. Linoleoyl and oleoyl desaturases were 94 and 10 times as active, respectively, in seeds cultured at 20°C as those cultured at 25°C. Both desaturases had negligible activity in seeds cultured at 35°C compared to seeds cultured at 20°C. Though less dramatic, other enzymes also showed differences in activity after 20 hours in culture at 20, 25, or 35°C. Stearoyl-acyl carrier protein (ACP) desaturase and CDP-choline:diacylglycerol phosphorylcholine transferase were most active in preparations from 20°C cultures. Activities were twofold lower at 25°C and a further threefold lower in 35°C cultures. Cultures from 25 and 35°C had 60 and 40%, respectively, of the phosphorylcholine:CTP cytidylyl transferase activity present in cultures grown at 20°C. Fatty acid synthetase, malonyl-coenzyme A:ACP transacylase, palmitoyl-ACP elongation, and choline kinase were not significantly altered by culture temperature. These data suggest that the enzymes for fatty acid desaturation and phosphatidylcholine synthesis can be rapidly modulated in response to altered growth temperatures, while the enzymes for fatty acid synthesis and elongation are not.     

Effects of Temperature on Electron Transport in Arum maculatum Mitochondria

The effects of temperature upon the respiratory pathways of Arum maculatum mitochondria have been studied. The alternate oxidase sustained a greater proportion of the total respiration at low temperatures than at higher temperatures. Arrhenius plots of respiratory activities show two discontinuities, one at 14°C and one at 21°C. The lower temperature discontinuity was associated with electron transport from succinate dehydrogenase to the alternative oxidase, enzymes that face the inner side of the membrane while the higher temperature discontinuity was associated with electron transport from the external NADH dehydrogenase to cytochrome c oxidase, which face the outer side of the membrane. Both discontinuities resulted in a decrease in the activation energy for electron transport on one side of the membrane. Arrhenius plots of transmembrane electron transport showed discontinuities at both 14° and 21°C but the upper discontinuity resulted in an increase in the activation energy. Activation energies determined for the respiratory activities show that above 21°C the exogenous NADH-cytochrome pathway and the succinate-alternative oxidase pathway were lower than those for the NADH-alternative pathway or the succinate cytochrome pathway.     

Temperature Dependence of Carbon Isotope Fractionation in CAM Plants

The carbon isotope fractionation associated with nocturnal malic acid synthesis in Kalanchoë daigremontiana and Bryophyllum tubiflorum was calculated from the isotopic composition of carbon-4 of malic acid, after appropriate corrections. In the lowest temperature treatment (17°C nights, 23°C days), the isotope fractionation for both plants is −4‰ (that is, malate is enriched in ¹³C relative to the atmosphere). For K. daigremontiana, the isotope fractionation decreases with increasing temperature, becoming approximately 0‰ at 27°C/33°C. Detailed analysis of temperature effects on the isotope fractionation indicates that stomatal aperture decreases with increasing temperature and carboxylation capacity increases. For B. tubiflorum, the temperature dependence of the isotope fractionation is smaller and is principally attributed to the normal temperature dependences of the rates of diffusion and carboxylation steps. The small change in the isotopic composition of remaining malic acid in both species which is observed during deacidification indicates that malate release, rather than decarboxylation, is rate limiting in the deacidification process.     

Synthesis of Extracellular Proteinase by Pseudomonas fluorescens Under Conditions of Limiting Carbon, Nitrogen, and Phosphate

The influence of carbon, nitrogen, and phosphate concentrations on growth and proteinase production by Pseudomonas fluorescens 32A was examined. In mineral salts medium containing dialyzed skim milk supernatant as an inducer, maximum growth was obtained at 1.0 and 2.5 mM orthophosphate at 20 and 5°C, respectively. At both temperatures, 5 mM orthophosphate was required for maximum proteinase production, whereas significant inhibition was found at 10 mM. Orthophosphate was the only phosphate compound able to support growth. With sodium pyruvate as the carbon source, maximum enzyme synthesis was at 100 mM carbon at both temperatures. At both 20 and 5°C maximum growth and enzyme production was found with 10 mM NH₄Cl. A bioassay for available phosphate based on the growth of P. fluorescens 32A in phosphate-limited mineral salts medium showed that skim milk and skim milk supernatant contained 50 and 10 mM orthophosphate, respectively. Proteinase production in skim milk was 2.6- and 12-fold greater than that in optimal mineral salts medium at 20 and 5°C, respectively. These results suggest that proteinase production in milk does not occur as a result of nutrient limitation and may be regulated in part by milk phosphates.     

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 α-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.     

Influence of growth temperature on respiratory characteristics of mitochondria from callus-forming potato tuber discs

The uninhibited respiration of mitochondria, isolated from potato tuber discs (Solanum tuberosum L. cv. Bintje) incubated on a callus-inducing medium at 28°C, is higher than that of mitochondria from tissue incubated at 8°C. This respiration is composed of a CN-sensitive and a CN-resistant part. The capacity of the CN-resistant alternative oxidase pathway is larger in mitochondria from 28°C tissue than in mitochondria from 8°C tissue (35% and 8% of uninhibited respiration, respectively). The alternative pathway is operative both in mitochondria from 28°C tissue and 8°C tissue.

The observed difference in uninhibited respiration, is not only caused by lower values of respiration via the alternative pathway in mitochondria from 8°C tissue, but also by lower values of respiration via the cytochrome pathway.

A positive correlation has been demonstrated between the incubation temperature (ranging from 4-37°C) and the relative capacity of respiration via alternative pathway in the mitochondria. Induction of alternative pathway is not directly correlated with growth (in terms of increase in fresh weight) of the potato tuber discs.

     

The localization of proteolytic activity in rat liver mitochondria and its relation to mitochondrial swelling and aging

1. On storage of rat liver mitochondria at 0°, water content, total amino acid content and leakage of protein all rose steadily over a 72hr. period. The initial ratio of intramitochondrial to extramitochondrial amino acid concentration lay between 18 and 24. Initially this rose, but it then fell to 1·9 at the end of storage. The concentration gradient between internal and external amino acids was relatively constant throughout the period. These processes were accentuated at 22° and 40°, the concentration gradient reaching 70μmoles/ml., water content rising to 8·3mg./mg. dry wt. and protein leakage reaching 42% of total mitochondrial protein. `Swelling agents' produced no correlated changes in amino acid production and swelling. 2. Added glutamate was not concentrated within the pellet of whole or disrupted mitochondria. Endogenous amino acids were distributed evenly between the pellet and the supernatant of disrupted mitochondria. It is concluded that amino acids are produced within mitochondria and that adsorption and uptake from the medium do not contribute significantly to amino acids in the pellet. 3. β-Glycerophosphate, a lysosome protectant, increased amino acid production by rat liver mitochondria. Treatment with Triton X-100 and disruption by freezing and thawing showed that 56% of proteolytic activity was `free' in whole mitochondria, whereas only 11% of acid phosphatase activity, a lysosomal enzyme, was `free'. 4. `Light' mitochondria contained 30% more neutral proteolytic activity but 300% more acid phosphatase activity than `heavy' mitochondria. 5. Electron micrographs of mitochondrial preparations showed less than one particle in 500 that could be identified as a lysosome. Treatment with Triton X-100 disrupted the structure of roughly 50% of the mitochondria; the rest appeared to retain their membrane, cristae and ground substance. Freezing and thawing caused gross swelling and loss of ground substance and rupture of external membranes. 6. Of the recovered proteolytic activity, 81% at pH7·4 and 70% at pH5·8 were found in the high-speed supernatant of broken mitochondria. A further fivefold increase in specific activity was found in the first protein fraction obtained by Sephadex G-50 gel filtration. 7. Between 60 and 80% of proteolytic activity was found in the 40–60%-saturated ammonium sulphate precipitate. Almost all of the soluble-fraction proteolytic activity could be recovered in a pH5·0 supernatant. 8. The results give no support to the view that mitochondrial neutral proteolytic activity reflects lysosomal content. 9. The possible role of intramitochondrial amino acid production and the proteolysis of internal barriers in passive swelling of mitochondria is discussed.     

Temperature Effects on Mitochondrial Respiration in Phaseolus acutifolius A. Gray and Phaseolus vulgaris L

Electron transport, using succinate as a substrate, was measured polarographically in mitochondria isolated from Phaseolus vulgaris and P. acutifolius plants at 25°C and 32°C. Mitochondria isolated from P. vulgaris plants grown at 32°C had reduced electron transport and were substantially uncoupled. Growth at 32°C had no effect on electron transport or oxidative phosphorylation in P. acutifolius compared to 25°C grown plants. Mitochondria isolated from 25°C grown P. vulgaris plants measured at 42°C were completely uncoupled. Similarly treated P. acutifolius mitochondria remained coupled. The uncoupling of P. vulgaris was due to increased proton permeability of inner mitochondrial membrane. The alternative pathway was more sensitive to heat than the regular cytochrome pathway. At 42°C, no alternative pathway activity was detected. The substantially greater heat tolerance of P. acutifollus compared to P. vulgaris mitochondrial electron transport suggests that mitochondrial sensitivity to elevated temperatures is a major limitation to growth of P. vulgaris at high temperatures and is an important characteristic conveying tolerance in P. acutifolius.