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.     

The control of malate dehydrogenase activity by adenine nucleotides in purified potato tuber (Solanum tuberosum L.) mitochondria

The limiting factors of the involvement of malate dehydrogenase in mitochondrial malate oxidation were investigated by using Percoll-purified potato tuber mitochondria. The respective roles of reduced pyridine nucleotides, oxaloacetate, and adenine nucleotides were studied under conditions of high or low phosphorylation potential (Pi + ADP/ATP ratio). Under conditions of high phosphorylation potential, the limitation of malate dehydrogenase activity was caused by the accumulation of oxaloacetate in the medium. In the absence of ADP (phosphorylation potential close to zero), ATP was responsible for the inhibition of malate dehydrogenase activity rather than oxaloacetate or reduced pyridine nucleotides.     

Mechanisms of citrate oxidation by percoll-purified mitochondria from potato tuber

The mechanisms and accurate control of citrate oxidation by Percoll-purified potato (Solanum tuberosum) tuber mitochondria were characterized in various metabolic conditions by recording time course evolution of the citric acid cycle related intermediates and O₂ consumption. Intact potato tuber mitochondria showed good rates of citrate oxidation, provided that nonlimiting amounts of NAD⁺ and thiamine pyrophosphate were present in the matrix space. Addition of ATP increased initial oxidation rates, by activation of the energy-dependent net citrate uptake, and stimulated succinate and malate formation. When the intramitochondrial NADH to NAD⁺ ratio was high, α-ketoglutarate only was excreted from the matrix space. After addition of ADP, aspartate, or oxaloacetate, which decreased the NADH to NAD⁺ ratio, flux rates through the Krebs cycle dehydrogenases were strongly increased and α-ketoglutarate, succinate, and malate accumulated up to steady-state concentrations in the reaction medium. It was concluded that NADH to NAD⁺ ratio could be the primary signal for coordination of fluxes through electron transport chain or malate dehydrogenase and NAD⁺-linked Krebs cycle dehydrogenases. In addition, these results clearly showed that the tricarboxylic acid cycle could serve as an important source of carbon skeletons for extra-mitochondrial synthetic processes, according to supply and demand of metabolites.     

Effect of bicarbonate and oxaloacetate on malate oxidation by spinach leaf mitochondria

Mitochondria isolated from spinach leaves oxidized malate by both a NAD⁺-linked malic enzyme and malate dehydrogenase. In the presence of sodium arsenite the accumulation of oxaloacetate and pyruvate during malate oxidation was strongly dependent on the malate concentration, the pH in the reaction medium and the metabolic state condition.Bicarbonate, especially at alkaline pH, inhibited the decarboxylation of malate by the NAD⁺-linked malic enzyme in vitro and in vivo. Analysis of the reaction products showed that with 15 mM bicarbonate, spinach leaf mitochondria excreted almost exclusively oxaloacetate.The inhibition by oxaloacetate of malate oxidation by spinach leaf mitochondria was strongly dependent on malate concentration, the pH in the reaction medium and on the metabolic state condition.The data were interpreted as indicating that: (a) the concentration of oxaloacetate on both sides of the inner mitochondrial membrane governed the efflux and influx of oxaloacetate; (b) the NAD⁺/NADH ratio played an important role in regulating malate oxidation in plant mitochondria; (c) both enzymes (malate dehydrogenase and NAD⁺-linked malic enzyme) were competing at the level of the pyridine nucleotide pool, and (d) the NAD⁺-linked malic enzyme provided NADH for the reversal of the reaction catalyzed by the malate dehydrogenase.     

Succinate oxidation by coupled potato tuber mitochondria followed by rapid scan spectrometry

Oxidation of succinate by potato tuber mitochondria has been investigated from aerobiosis to complete anuerobiosis. Difference spectra of the various steps were recorded by a rapid scan spectrometer delivering averaged spectra every 3 s in the range 380 to 630 mm. The transitions between state 3 and 4 resulted in large redox changes, essentially for the b cytochromes, and in significant changes in the spectral baseline (light scattering). At anaerobiosis the cytochromes c, c₁ and a were reduced while cytochrome a, remained oxidized. – Addition of uncouplers in aerobiosis induced oxidation of the b cytochromes, and when anaerobiosis occurred cytochromes c, c₁a and a₃ were reduced simultaneously. When uncouplers were added in anaerobiosis a partial oxidation of the b cytochromes and the reduction of cytochrome a₃ were observed. These results are interpreted as the building up of a membrane potential, maximal in state 4 and stable after anaerobiosis. The cytochromes buried in the membrane equilibrate with the membrane potential, and their redox states are sensitive to the changes. Variations of membrane potential also induce changes in the light scattering by the mitochondrial membrane.     

Ability of cytosolic malate dehydrogenase and lactate dehydrogenase to increase the ratio of NADPH to NADH oxidation by cytosolic glycerol-3-phosphate dehydrogenase

At the normal pH of the cytosol (7.0 to 7.1) and in the presence of physiological (1.0 mM) levels of free Mg2+, the Vmax of the NADPH oxidation is only slightly lower than the Vmax of NADH oxidation in the cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) reaction. Under these conditions physiological (30 microM) levels of cytosolic malate dehydrogenase (E.C. 1.1.1.37) inhibited oxidation of 20 microM NADH but had no effect on oxidation of 20 microM NADPH by glycerol-3-phosphate dehydrogenase. Consequently malate dehydrogenase increased the ratio of NADPH to NADH oxidation of glycerol-3-phosphate dehydrogenase. On the basis of the measured KD of complexes between malate dehydrogenase and these reduced pyridine nucleotides, and their Km in the glycerol-3-phosphate dehydrogenase reactions, it could be concluded that malate dehydrogenase would have markedly inhibited NADPH oxidation and inhibited NADH oxidation considerably more than observed if its only effect were to decrease the level of free NADH or NADPH. This indicates that due to the opposite chiral specificity of the two enzymes with respect to reduced pyridine nucleotides, complexes between malate dehydrogenase and NADH or NADPH can function as substrates for glycerol-3-phosphate dehydrogenase, but the complex with NADH is less active than free NADH, while the complex with NADPH is as active as free NADPH. Mg2+ enhanced the interactions between malate dehydrogenase and glycerol-3-phosphate dehydrogenase described above. Lactate dehydrogenase (E.C. 1.1.1.27) had effects similar to those of malate dehydrogenase only in the presence of Mg2+. In the absence of Mg2+, there was no evidence of interaction between lactate dehydrogenase and glycerol-3-phosphate dehydrogenase.     

Glycine metabolism and oxalacetate transport by pea leaf mitochondria

Isolated pea leaf mitochondria oxidatively decarboxylate added glycine. This decarboxylation could be linked to the respiratory chain (in which case it was coupled to three phosphorylations) or to mitochondrial malate dehydrogenase when oxalacetate was supplied. Decarboxylation rates measured as O₂ uptake, or CO₂ and NH₃ release were adequate to account for whole leaf photorespiration. Oxalacetate-supported glycine decarboxylation, measured by linking malate efflux to added malic enzyme, yielded rates considerably less than the electron transport rates. Butylmalonate inhibited malate efflux but not oxalacetate entry; phthalonate inhibited oxalacetate entry but had little effect on malate or α-ketoglutarate oxidation. It is suggested that oxalacetate and malate transport are catalyzed by separate carrier systems of the mitochondrial membrane.     

The carnitine-independent oxidation of palmitate plus malate by moth flight-muscle mitochondria

Mitochondria isolated from the flight muscle of the southern armyworm moth, Prodenia eridania, can oxidize palmitate+malate very rapidly. Added carnitine had no effect on the rate of oxidation of palmitate+malate by flight-muscle mitochondria from two species of moths, and carnitine palmitoyltransferase could not be detected in Prodenia by direct assay. Palmitoylcarnitine was not oxidized by moth mitochondria, but when added in low concentrations it reversibly suppressed the oxidation of palmitate. The evidence indicates that carnitine is not involved in fatty acid degradation by moth flight muscle. Added thiols, including CoA, also suppressed palmitate+malate oxidation. An ATP-dependent fatty acyl-CoA synthetase is present in moth mitochondria.     

Involvement of matrix NADP turnover in the oxidation of NAD-linked substrates by pea leaf mitochondria

The involvement of the internal rotenone-insensitive NADPH dehydrogenase on the inner surface of the inner mitochondrial membrane [ND_in(NADPH)] in the oxidation of strictly NAD⁺-linked substrates by pea ( Pisum sativum L.) leaf mitochondria was measured. As estimated by the inhibition caused by 5 μ M diphenyleneiodonium (DPI) in the presence of rotenone to inhibit complex I, the activity of ND_in(NADPH) during glycine oxidation (measured both as O₂ uptake and as CO₂ release) was 40–50 nmol mg⁻¹ protein min⁻¹. No significant activity of ND_in(NADPH) could be detected during the oxidation of 2-oxoglutarate, another strictly NAD⁺-linked substrate; this was possibly due to its relatively low oxidation rate. Control experiments showed that, even at 125 μ M , DPI had no effect on the activity of glycine decarboxylase complex (GDC) and lipoamide dehydrogenase. The relative activity of complex I, ND_in(NADPH), and ND_in(NADH) during glycine oxidation, estimated using rotenone and DPI, differed depending on the pyridine nucleotide supply in the mitochondrial matrix. This was shown by loading the mitochondria with NAD⁺ and NADP⁺, both of which were taken up by the organelle. We conclude that the involvement of NADP turnover during glycine oxidation is not due to the direct production of NADPH by GDC but is an indirect result of this process. It probably occurs via the interconversion of NADH to NADPH by the two non-energy-linked transhydrogenase activities recently identified in plant 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 O₂ 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.     

The oxidation of malate and exogenous reduced nicotinamide adenine dinucleotide by isolated plant mitochondria

Exogenous NADH oxidation by cauliflower (Brassica oleracea L.) bud mitochondria was sensitive to antimycin A and gave ADP/O ratios of 1.4 to 1.9. In intact mitochondria, NADH-cytochrome c reductase activity was only slightly inhibited by antimycin A. The antimycin-insensitive activity was associated with the outer membrane. Malate oxidation was sensitive to both rotenone and antimycin A and gave ADP/O values of 2.4 to 2.9. However in the presence of added NAD⁺, malate oxidation displayed similar properties to exogenous NADH oxidation. In both the presence and absence of added NAD⁺, malate oxidation was dependent on inorganic phosphate and inhibited by 2-n-butyl malonate.     

Comparative aspects of aminooxyacetate inhibition of glycine oxidation and aminotransferase activity of pea leaf mitochondria

Aminooxyacetate (AOA) was found to inhibit glycine oxidation by pea leaf mitochondria at micromolar levels. The inhibition resulted from an inhibition of both glycine decarboxylase and serine hydroxymethyltransferase (SHMT) activity. Aspartate: 2-oxoglutarate aminotransferase (AsAT) and alanine: 2-oxoglutarate aminotransferase activities of pea leaf mitochondria were also very sensitive to AOA inhibition. Inhibition of both glycine oxidation and aminotransferase activity was likely competitive with respect to the amino group substrate, but also displayed a time-dependent increase in inhibition at constant AOA concentration. In the case of glycine oxidation, this time-dependent component may be related to the rate of penetration of AOA across the inner mitochondrial membrane. Furthermore, the AOA-inhibition of glycine oxidation could be reversed by pyridoxal 5-phosphate (PLP), whereas AOA-inhibited aminotransferase activity was not reversed. The results indicate that the pyridoxal 5-phosphate antagonist, AOA, results in varying types of inhibition depending on the type of enzyme involved.     

Contribution of mitochondria and peroxisomes to palmitate oxidation in pea tissues

Total, mitochondrial and peroxisomal palmitate oxidation capacities were compared in pea, from the dry seed to 14 days after imbibition. Total beta-oxidation varied over the measured time period and showed four peaks of activity at day 2, days 5-6, day 10 and days 12-13. The contribution of peroxisomal and mitochondrial beta-oxidation to this overall beta-oxidation varied. Over the first 48 h of seed germination, peroxisomal beta-oxidation accounted for 80-100% of the total observed beta-oxidation. The larger peaks of beta-oxidation at days 5-6, day 10 and days 12-13 were due primarily to mitochondrial beta-oxidation activity, which accounted for 70-90% of the observed total beta-oxidation at these times. The peaks of activity are related to observed stages in seedling development.     

The permeability of mitochondria to oxaloacetate and malate

1. A spectrophotometric assay of the rates of penetration of oxaloacetate and l-malate into mitochondria is described. The assay is based on the measurement of the oxidation of intramitochondrial NADH by oxaloacetate and of the reduction of intramitochondrial NAD⁺ by malate. 2. The rate of entry of both oxaloacetate and l-malate into mitochondria is restricted, as shown by the fact that disruption of the mitochondrial structure can increase the rate of interaction between the dicarboxylic acids and intramitochondrial NAD⁺ and NADH by between 100- and 1000-fold. 3. The rates of entry of oxaloacetate and malate into liver, kidney and heart mitochondria increased by up to 50-fold on addition of a source of energy, either ascorbate plus NNN′N′-tetramethyl-p-phenylenediamine aerobically, or ATP anaerobically. 4. In the absence of a source of energy the changes in the concentrations of intramitochondrial NAD⁺ and NADH brought about by the addition of l-malate or oxaloacetate were followed by parallel changes in the concentrations of NADP⁺ and NADPH, indicating the presence in the mitochondria of an energy-independent transhydrogenase system. 5. The results are discussed in relation to the hypothesis that malate acts as a carrier of reducing equivalents between mitochondria and cytoplasm.     

Effects of pH, NADH, succinate and malate on the oxidation of glycine in spinach leaf mitochondria

The effect of external pH on several reactions catalyzed by glycine decarboxylase in spinach leaf mitochondria was investigated. Glycine-dependent oxygen consumption showed a pH optimum at 7.6, whereas the release of CO₂ and NH3 from glycine in the presence of oxaloacetate both showed pH maxima at 8.1. Glycine-dependent reduction of 2,6-dichlorophenolindophenol. on the other hand showed a pH optimum at 8.4. It is concluded that these three reactions have different rate-limiting steps. The rate of the glycine-bicarbonate exchange reaction catalyzed by glycine decarboxylase showed no optimum in the pH range investigated, pH 7–9, but increased with decreasing pH. This suggests that CO₂ may be the true substrate in this reaction.
The oxidation of glycine inhibited the oxidation of both malate, succinate and external NADH since the addition of malate, succinate or NADH to mitochondria oxidizing glycine in state 3 resulted in a rate of oxygen consumption which was lower than the sum of the rates when the substrates were oxidized individually. The addition of malate, succinate or NADH did not, however, decrease the rate of CO₂ or NH, release from glycine. It is suggested that the preferred oxidation of glycine by-spinach leaf mitochondria may constitute an important regulatory mechanism for the function of leaf mitochondria during photosynthesis.     

Inhibition of sweet potato glucose 6-phosphate dehydrogenase by NADPH2 and ATP

NADPH₂ and ATP competitively inhibit sweet potato glucose 6-phosphatedehydrogenase with NADP and glucose 6-phosphate (G6P), respectively.At pH 8.0, a Lineweaver-Burk plot of the reciprocal rate againstreciprocal G6P concentration was concave downwards in the presenceand absence of ATP, whereas a double reciprocal plot followedthe Michaelis-Menten relationship at pH 7.0, irrespective ofthe presence of ATP. Many of the other metabolic intermediatestested had no effects on the enzyme reaction. ¹ This paper constitutes Part 96 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. ² Present address: Institute of Applied Microbiology, Universityof Tokyo Bunkyo-ku, Tokyo 113. (Received October 20, 1971; )