Expression and characterization of recombinant cytosolicNAD+-dependent malate dehydrogenase from Mesembyanthemum crystallinum

An NAD⁺-dependent cytosolic malate dehydrogenase (MDH, EC 1.1.1.37) from leaves of Mesembryanthemum crystallinum in the Crassulacean Acid Metabolism (CAM) mode was cloned, expressed in E. coli and characterized. The recombinant enzyme had a subunit molecular mass of 39.5 kDa and was recognized by antibodies raised against the cytosolic MDH from Ananas comosus . Its activity showed a maximum in the pH range of 7.5–9.5. The purified MDH is highly but not completely specific for oxaloacetate, as indicated by a low activity using various other α-ketoacids as substrates. The sequence data, subunit mass and immunoreactivity suggest that the MDH that has been cloned and characterized corresponds to the cytosolic isoform. Yet, the biochemistry of this enzyme comparative with the only other CAM plant cytosolic MDH characterized so far (that of pineapple) hints at a distinct isoform being expressed in M. crystallinum leaves.     

Purification and characterization of an NAD-dependent malate dehydrogenase from leaves of the crassulacean acid metabolism plant Aptenia cordifolia

An NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37) was purified and characterized from leaves of Aptenia cordifolia L. f. (Schwant). This plant performs crassulacean acid metabolism (CAM), as indicated by: (a) elevated levels of phosphoenolpyruvate carboxylase (PEPC) and NAD(P) malic enzyme; (b) regulation of PEPC compatible with its function during the night; (c) characteristic day/night changes in titratable acidity; and (d) gas exchange profile consistent with that shown by CAM plants. These features remained unchanged by water availability or salt stress, suggesting constitutive CAM. The purified MDH showed a subunit molecular mass of 39.4 kDa, a native mass of 83 kDa (dimer) and a pI of 5.8. It cross-reacted with antibodies against cytosolic malate dehydrogenase (cMDH) from pineapple. Maximum activities for oxaloacetate (OAA) reduction or malate oxidation were observed at pH 7.0 and between pH 7.2 and 8.4, respectively. The enzyme was inhibited by excess OAA, in a pH-dependent manner. A discontinuity was observed in Arrhenius plots at 33 °C, with an activation energy twice as high below this temperature. Although immunologically related, some physical and kinetic dissimilarities between the A. cordifolia and pineapple enzymes suggest that diverse CAM metabolic subtypes may require different MDH isozymes to carry out OAA reduction.     

Purification and characterization of a novel mannitol dehydrogenase from Lactobacillus intermedius

Mannitol 2-dehydrogenase (MDH) catalyzes the pyridine nucleotide dependent reduction of fructose to mannitol. Lactobacillus intermedius (NRRL B-3693), a heterofermentative lactic acid bacterium (LAB), was found to be an excellent producer of mannitol. The MDH from this bacterium was purified from the cell extract to homogeneity by DEAE Bio-Gel column chromatography, gel filtration on Bio-Gel A-0.5m gel, octyl-Sepharose hydrophobic interaction chromatography, and Bio-Gel Hydroxyapatite HTP column chromatography. The purified enzyme (specific activity, 331 U/mg protein) was a heterotetrameric protein with a native molecular weight (MW) of about 170 000 and subunit MWs of 43 000 and 34 500. The isoelectric point of the enzyme was at pH 4.7. Both subunits had the same N-terminal amino acid sequence. The optimum temperature for the reductive action of the purified MDH was at 35 degrees C with 44% activity at 50 degrees C and only 15% activity at 60 degrees C. The enzyme was optimally active at pH 5.5 with 50% activity at pH 6.5 and only 35% activity at pH 5.0 for reduction of fructose. The optimum pH for the oxidation of mannitol to fructose was 7.0. The purified enzyme was quite stable at pH 4.5-8.0 and temperature up to 35 degrees C. The K(m) and V(max) values of the enzyme for the reduction of fructose to mannitol were 20 mM and 396 micromol/min/mg protein, respectively. It did not have any reductive activity on glucose, xylose, and arabinose. The activity of the enzyme on fructose was 4.27 times greater with NADPH than NADH as cofactor. This is the first highly NADPH-dependent MDH (EC 1.1.1.138) from a LAB. Comparative properties of the enzyme with other microbial MDHs are presented.     

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.     

Phosphoenolpyruvate Carboxylase During Development of Crassulacean Acid Metabolism and During a Diurnal Cycle in Mesembryanthemum Crystallinum

Crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinumwas induced by transfer of plants from 100 to 400 mM NaCl. Diurnalmalate fluctuations developed slowly; maximum rates of net malatesynthesis in the dark were reached only on the 10th day afterNaCl was increased to 400 mM. In contrast, phosphoenolpyruvatecarboxylase (PEPC) activity, assayed at optimum pH of 8–0,had nearly reached its maximum on the 5th day after plants weretransferred to 400 mM NaCl. Characteristics of PEPC changedduring the first 12 d of exposure of plants to 400 mM NaCl.There were increases in the ratio of PEPC activity at pH 7 0/PEPCactivity at pH 8.0, and decreases in the Km for PEP measuredat pH 7.0, and possibly in the degree of malate inhibition.All further measurements were made once CAM was well established.In vivo rates of malate synthesis were 14–18 times smallerthan PEPC activity at 2 mM PEP, both processes being measuredat 15 °C. It is suggested that the high PEPC levels favourrapid, preferential flow of carbon to malate, by maintainingvery low PEP levels in the cytoplasm. PEPC changed in characteristicsduring the diurnal cycle. During the first few minutes afterisolation, extracts made during the first hours of the day,when malate was consumed, showed very low PEPC activity at pH7.0 but high activity at pH 8.0. The activity of PEPC at pH7.0 rose gradually during storage of the extracts at 0 °C,usually reaching the activity at pH 8.0 after about 30–50min. In contrast, extracts obtained during the first hours ofthe night, when malate was synthesized, showed high PEPC activityat both pH 7.0 and 8–0 within 30–50 s after extraction.The results indicate that PEPC of M. crystallinum, performingdistinct CAM, may exist in two states. One state would favourrapid malate synthesis and transport to the vacuoles and wouldfunction during the night. The second state, with little activitybelow pH 7.5, would occur during the day, thus preventing complicationsof continued synthesis of malate while it is converted to carbohydrates.     

Partial purification and characterization of pyruvate kinase from the plant fraction of soybean root nodules

Pyruvate kinase (PK, EC 2.7.1.40) was partially purified from the plant cytosolic fraction of N₂-fixing soybean ( Glycine max [L.] Merr.) root nodules. The partially purified PK preparation was completely free of contamination by phospho enol pyruvate carboxylase (PEPC, EC 4.1.1.31), the other major phospho enol pyruvate (PEP)-utilizing enzyme in legume root nodules. Latency experiments with sonicated nodule extracts showed that Bradyrhizobium japonicum bacteroids do not express either PK or PEPC activity in symbiosis. In contrast, free-living B. japonicum bacteria expressed PK activity, but not PEPC activity. Antibodies specific for the cytosolic isoform of PK from castor bean endosperm cross-reacted with a 52-kDa polypeptide in the partially purified PK preparation. At the optimal assay pH (pH 8.0 for PEPC and pH 6.9 for PK) and in the absence of malate, PEPC activity in crude nodule extracts was 2.6 times the corresponding PK activity. This would tend to favour PEP metabolism by PEPC over PEP metabolism by PK. However, at pH 7.0 in the presence of 5 m M malate, PEPC activity was strongly inhibited, but PK activity was unaffected. Thus, we propose that PK and PEPC activity in legume root nodules may be coordinately regulated by fluctuations in malate concentration in the plant cytosolic fraction of the bacteroid-containing cells. Reduced uptake of malate by the bacteroids, as a result of reduced rates of N₂ fixation, may favour PEP metabolism by PK over PEP metabolism by PEPC.     

Methods to extract NAD+-malate dehydrogenase efficiently from white spruce needles

The spectrophotometrically-determined activity of NAD⁺-malate dehydrogenase (MDH, EC 1.1.1.37) from white spruce [ Picea glauca (Moench) Voss] needles was assayed with NADH and oxaloacetate. Activity was very low when extracted with only acetate buffer (pH 5.4), phosphate buffer (pH 6.8), or Tris-HCl buffer (pH 8.0). However, activity increased from 1 to over 200 μmol (g dry weight)^-1 min^-1 with the addition of polymers such as polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG) and the detergents, Tween 80, Tergitol 15-S-9 and Triton X-100. Best activity was observed when extracted in a buffer at pH 6.8 and with 1% (v/v) for the three detergents and PEG, and 6% (w/v) for PVP.
MDH activity decreased with age of the needles on the tree. Six-year-old needles contained only about one-fifth of the activity of current year, fully-expanded needles. The main decrease in enzyme activity was observed in one-year-old needles. Protein content obtained from needles extracted with just phosphate buffer (pH 6.8) was very low, but increased greatly when the above chemicals were added to the buffer. In contrast with needles, extracts of vegetative buds contained much higher levels of MDH and protein when extracted with only phosphate buffer (pH 6.8). Although MDH activity in needle extracts declined with storage of the extracts at 4°C in the dark for 6 days, the decrease was least for buffers containing a combination of different protective agents.     

Purification and properties of microbody malate dehydrogenase from Spinacia oleracea leaf tissue

The microbody isoenzyme of malate dehydrogenase (EC 1.1.1.37) from leaves of Spinacia oleracea was purified to a specific activity of 3000 units/mg protein and examined for a number of physical, kinetic, and immunological properties. The purified enzyme has a molecular weight of approximately 70,000 and an isoelectric point of 5.65. Thermal inactivation first order rate constants were 0.068 (35 °C), 0.354 (45 °C), and 2.11 (55 °C) for irreversible denaturation. Apparent millimolar Michaelis constants are 0.34 (NAD, pH 8.5) 0.16 (NADH, pH 7.5), 3.33 (malate, pH 8.5), 0.07 (OAA, pH 6.0), 0.06 (OAA, pH 7.5), and 0.50 (OAA, pH 9.0). The enzyme is stablized by 20% glycerol and can be stored for several months at 4 °C without detectable loss of activity. The purified enzyme is sensitive to the ionic strength of the assay medium exhibiting a pH optimum of 5.65 at high ionic strength and 7.00 at low ionic strength. Rabbit antiserum prepared against the purified microbody MDH shows a single precipitin band on immunodiffusion analysis. Immunological studies indicate that rabbit antiserum prepared against the purified microbody enzyme cross reacts approximately 10% with the mitochondrial isoenzyme of MDH. No cross reaction was shown with the soluble isoenzyme. In general, the data presented in this report tend to support the notion of organelle specific isoenzymes of malate dehydrogenase in higher plant tissues and uniqueness of the microbody form of malate dehydrogenase in particular.     

Isolation, nucleotide sequence analysis, and disruption of the MDH2 gene from Saccharomyces cerevisiae: evidence for three isozymes of yeast malate dehydrogenase.

The major nonmitochondrial isozyme of malate dehydrogenase (MDH2) in Saccharomyces cerevisiae cells grown with acetate as a carbon source was purified and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a subunit molecular weight of approximately 42,000. Enzyme assays and an antiserum prepared against the purified protein were used to screen a collection of acetate-nonutilizing (acetate-) yeast mutants, resulting in identification of mutants in one complementation group that lack active or immunoreactive MDH2. Transformation and complementation of the acetate- growth phenotype was used to isolate a plasmid carrying the MDH2 gene from a yeast genomic DNA library. The amino acid sequence derived from complete nucleotide sequence analysis of the isolated gene was found to be extremely similar (49% residue identity) to that of yeast mitochondrial malate dehydrogenase (molecular weight, 33,500) despite the difference in sizes of the two proteins. Disruption of the MDH2 gene in a haploid yeast strain produced a mutant unable to grow on minimal medium with acetate or ethanol as a carbon source. Disruption of the MDH2 gene in a haploid strain also containing a disruption in the chromosomal MDH1 gene encoding the mitochondrial isozyme produced a strain unable to grow with acetate but capable of growth on rich medium with glycerol as a carbon source. The detection of residual malate dehydrogenase activity in the latter strain confirmed the existence of at least three isozymes in yeast cells.     

Overexpression of malate dehydrogenase in transgenic tobacco leaves: enhanced malate synthesis and augmented Al-resistance

Numerous studies with transgenic plants have demonstrated that overexpression of enzymes related to organic acid metabolism under the control of CaMV 35S promoter increased organic acid exudation and Al-resistance. The synthesis of organic acids requires a large carbon skeleton supply from leaf photosynthesis. Thus, we produced transgenic tobacco overexpressing cytosolic malate dehydrogenase (MDH) cDNA from Arabidopsis thaliana (amdh) and the MDH gene from Escherichia coli (emdh), respectively, under the control of a leaf-specific light-inducible promoter (Rubisco small subunit promoter, PrbcS) in the present study. Our data indicated that an increase (120–130%) in MDH-specific activity in leaves led to an increase in malate content in the transgenic tobacco leaves and roots as well as a significant increase in root malate exudation compared with the WT plants under the acidic (pH 4.5) conditions irrespective of 300 μM Al³⁺ stress absence or presence. After being exposed to 25 μM Al³⁺ in a hydroponic solution, the transgenic plants exhibited stronger Al-tolerance than WT plants and the degree of A1 tolerance in the transgenic plants corresponded with the amount of malate secretion. When grown in an Al-stress perlite medium, the transgenic tobacco lines showed better growth than the WT plants. The results suggested that overexpression of MDH driven by the PrbcS promoter in transgenic plant leaves enhanced malate synthesis and improved Al-resistance.     

Regulation of phosphoenolpyruvate carboxylase from Crassula by interconversion of oligomeric forms

Using size-exclusion high-performance liquid chromatography, it is shown that phosphoenolpyruvate carboxylase from Crassula argentea, a crassulacean acid metabolism (CAM) plant, exists primarily in the form of a tetramer of a 100-kDa subunit at night and as a dimer of the same subunit during the day. The tetrameric enzyme from night leaves is not inhibited by malate, while the dimeric form from day leaves can be completely inhibited by malate. The purified day, or dimer, form of the enzyme can be converted to the tetramer by concentration and exposure to Mg2+. When thus converted, the tetramer is insensitive to malate inhibition, and is more strongly activated by glucose 6-phosphate than the dimer. The purified night, or tetramer, form is converted to the dimer by incubation for 60 min at pH 8.2. This enzyme may also be converted to the dimer by adding 1.5 mM malate to the elution buffer, but preincubation for 15 min with phosphoenolpyruvate prevents disaggregation when chromatographed with buffer containing malate. Preincubation with 1mM EDTA and subsequent chromatography with buffer containing malate shows a progressive dissociation of the tetrameric form with increasing time of preincubation. The implications of these observations for the diurnal regulation of phosphoenolpyruvate carboxylase in CAM metabolism are discussed.     

Effect of NaCl on the in vitro Activity of Malate Dehydrogenase in Salt Marsh Halophytes of the U.S.

The in vitro effect of NaCl on NAD-malate dehydrogenase (E.C. 1.1.1.37; MDH) from desalted extracts of roots and leaves of six salt marsh halophytes was investigated. The plants, all native and important constituents of the salt marshes of the east coast of the U.S., included Spartina alterniflora Loisel., Spartina patens (Aiton) Muhl., Distichlis spicata (L.) Greene, Juncus roemerianus Schleele, Salicornia virginica L., and Borrichia frutescens (L.) DC. In the leaf extracts of all species except Borrichia frutescens, the MDH activity was slightly stimulated by NaCl at concentrations around 0.05 M at optimal pH (8.0–8.5) and was reduced by NaCl in higher concentrations. MDH activity in the leaf extract of Borrichia frutescens was more salt-tolerant and maximal activity occurred around 0.25 M NaCl at optimal pH (7.0). Even though similar pH optimums for activity were exhibited in the root and leaf extracts of each species, the MDH activity in the root extract was more salt-tolerant than that in the leaf extract. NaCl at concentrations up to 0.1 M stimulated the MDH activity in the root extracts of all species except that of Borrichia frutescens, which had an optimal activity in 0.5 M NaCl. In the root and leaf extracts of Borrichia frutescens, the activity of cytosol MDH was much more salt-tolerant than that of the mitochondrial MDH. A shift of the optimal pH to more acidic values with increasing concentrations of NaCl was noted in the extracts of all the species except Borrichia frutescens. The action of NaCl on MDH activity appeared to be a general ionic effect as judged by the response of the enzyme activity in the presence of iso-ionic concentrations of other salts and isoosmotic mannitol. Thus, the response of the MDH from five of the salt marsh plants to NaCl is similar to that of glycophytes. However, Borrichia frutescens possesses a salt-tolerant MDH that has optimal activity in a salt concentration as high as that of the environment.     

Activation of the plastid isozyme of phosphofructokinase from developing endosperm of Ricinuscommunis by fructose 2,6-bisphosphate

The plastid isozyme of phosphofructokinase from developing castor oil seeds is stimulated by low concentrations of fructose 2,6-bisphosphate when assayed at pH 7.0. The stimulation involves a shift in fructose 6-phosphate kinetics from sigmoidal to near hyperbolic. The plastid isozyme is unaffected by fructose 2,6-bisphosphate when assayed at pH 8.0, and the cytosolic isozyme is unaffected at either pH 7.0 or 8.0. There is no interaction between fructose 2,6-bisphosphate and the other regulators of the $\text{Ricinus}$ phosphofructokinases; phosphoenolpyruvate, 2-phosphoglycerate, 3-phosphoglycerate and inorganic phosphate.     

Purification and characterization of adenylate kinase isozymes from rat muscle and liver

Isozymes of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) were purified from skeletal muscle and liver of rats to essentially homogeneous states by acrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. The isozyme from muscle was purified by acidification to pH 5.0, and column chromatography on phosphocellulose, Sephadex G-75 and Blue Sepharose CL-6B, while that from liver was purified by column chromatography on Blue Sepharose CL-6B, Sephadex G-75 and carboxymethyl cellulose. By these procedures the muscle isozyme was purified about 530-fold in 29% yield, and the liver isozyme about 3600-fold in 27% yield from the respective tissue extracts. The molecular weights of the muscle and liver isozymes were estimated as about 23 500 and 30 500, respectively, by both sodium dodecyl sulfate gel electrophoresis and molecular sieve chromatography, and no subunit of either isozyme was detected. The isoelectric points of the muscle and liver isozymes were 7.0 and 8.1, respectively. The Km values of the respective enzymes for ATP and ADP were similar, but the Km(AMP) of the liver isozyme was about one-fifth of that of the muscle isozyme. Immunological studies with rabbit antiserum against the rat muscle isozyme showed that the muscle isozyme was abundant in muscle, heart and brain, while the liver isozyme was abundant in liver and kidney.     

The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms

Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.     

Isolation and characterization of the yeast gene encoding the MDH3 isozyme of malate dehydrogenase.

The MDH3 isozyme of Saccharomyces cerevisiae was purified from a haploid strain containing disruptions in genomic loci encoding the mitochondrial MDH1 and nonmitochondrial MDH2 isozymes. Partial amino acid sequence analysis of the purified enzyme was conducted and used to plan polymerase chain reaction techniques to clone the MDH3 gene. The isolated gene was found to encode a 343-residue polypeptide with a molecular weight of 37,200. The deduced amino acid sequence was closely related to those of MDH1 (50% residue identity) and of MDH2 (43% residue identity). The MDH3 sequence was found to contain a carboxyl-terminal SKL tripeptide, characteristic of many peroxisomal enzymes, and immunochemical analysis was used to confirm organellar localization of the MDH3 isozyme. Levels of MDH3 were determined to be elevated in cells grown with acetate as a carbon source, and under these conditions, MDH3 contributed approximately 10% of the total cellular malate dehydrogenase activity. Disruption of the chromosomal MDH3 locus produced a reduction in cellular growth rates on acetate, consistent with the presumed function of this isozyme in the glyoxylate pathway of yeast. Combined disruption of MDH1, MDH2, and MDH3 loci in a haploid strain resulted in the absence of detectable cellular malate dehydrogenase activity.     

In vitro dissociation-recombination of malate dehydrogenase subunits in Corydalis solida

Summary Two allelic forms of NAD specific malate dehydrogenase were found in samples of a wild population of Corydalis solida. The dimeric nature and the origin of the heterodimeric form has been demonstrated by in vitro dissociation and recombination of the subunits detected by subsequent electrophoresis. The method is applicable for polyacrylamide gel electrophoresis of crude leaf extracts of individual MDH isozyme forms.     

Functional interrelations between isozymes of dehydrogenases in the intact and denervated rabbit muscles]

A correlation is shown to exist between malate dehydrogenase (MDH), lactate dehydrogenase (LDH) and glycerol-3-phosphate dehydrogenase (glycerol-3-PDH activity values, lactate/pyruvate and malate/oxaloacetate coefficients, MDH and LDH isozyme spectra and kinetic properties of LDH isozymes in soluble fractions of cytoplasm from intact rabbit m. soleus (red), m. gastrocnemius (mixed) and m. quadratus lumborum (white). In denervated soleus and gastrocnemius the cytoplasmic MDH/LDH, mitochondrial MDH/LDH, MDH mitochondrial/MDH cytoplasmic activity ratios, concentrations of substrates and isozyme spectra of MDH and LDH tend to equalize. The obtained results indicate the importance of isozyme composition and total activity ratios of the dehydrogenases for regulation of pyruvate and NADH metabolic pathways.     

Changes in oxidative properties of Kalanchoe blossfeldiana leaf mitochondria during development of Crassulacean acid metabolism

Kalanchoe blossfeldiana plants grown under long days (16 h light) exhibit a C₃-type photosynthetic metabolism. Switching to short days (9 h light) leads to a gradual development of Crassulacean acid metabolism (CAM). Under the latter conditions, dark CO₂ fixation produces large amounts of malate. During the first hours of the day, malate is rapidly decarboxylated into pyruvate through the action of a cytosolic NADP⁺-or a mitochondrial NAD⁺-dependent malic enzyme. Mitochondria were isolated from leaves of plants grown under long days or after treatment by an increasing number of short days. Tricarboxylic acid cycle intermediates as well as exogenous NADH and NADPH were readily oxidized by mitochondria isolated from the two types of plants. Glycine, known to be oxidized by C₃-plant mitochondria, was still oxidized after CAM establishment. The experiments showed a marked parallelism in the increase of CAM level and the increase in substrate-oxidation capacity of the isolated mitochondria, particularly the capacity to oxidize malate in the presence of cyanide. These simultaneous variations in CAM level and in mitochondrial properties indicate that the mitochondrial NAD⁺-malic enzyme could account at least for a part of the oxidation of malate. The studies of whole-leaf respiration establish that mitochondria are implicated in malate degradation in vivo. Moreover, an increase in cyanide resistance of the leaf respiration has been observed during the first daylight hours, when malate was oxidized to pyruvate by cytosolic and mitochondrial malic enzymes.Abbreviations CAM Crassulacean acid metabolism - MDH malate dehydrogenase - ME malic enzyme     

Oxidations of various substrates and effects of the inhibitors on purified mitochondria isolated from <Emphasis Type="Italic">Kalanchoë pinnata</Emphasis>

Kalanchoë 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.