Characteristics of external and internal NAD(P)H dehydrogenases in Hoya carnosa mitochondria.

This study aims at characterizing NAD(P)H dehydrogenases on the inside and outside of the inner membrane of mitochondria of one phosphoenolpyruvate carboxykinase-crassulacean acid metabolism plant, Hoya carnosa. In crassulacean acid metabolism plants, NADH is produced by malate decarboxylation inside and outside mitochondria. The relative importance of mitochondrial alternative NADH dehydrogenases and their association was determined in intact-and alamethicin-permeabilized mitochondria of H. carnosa to discriminate between internal and external activities. The major findings in H. carnosa mitochondria are: (i) external NADPH oxidation is totally inhibited by DPI and totally dependent on Ca(2+), (ii) external NADH oxidation is partially inhibited by DPI and mainly dependent on Ca(2+), (iii) total NADH oxidation measured in permeabilized mitochondria is partially inhibited by rotenone and also by DPI, (iv) total NADPH oxidation measured in permeabilized mitochondria is partially dependent on Ca(2+) and totally inhibited by DPI. The results suggest that complex I, external NAD(P)H dehydrogenases, and internal NAD(P)H dehydrogenases are all linked to the electron transport chain. Also, the total measurable NAD(P)H dehydrogenases activity was less than the total measurable complex I activity, and both of these enzymes could donate their electrons not only to the cytochrome pathway but also to the alternative pathway. The finding indicated that the H. carnosa mitochondrial electron transport chain is operating in a classical way, partitioning to both Complex I and alternative Alt. NAD(P)H dehydrogenases.

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.

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.