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NAD(P) + ‐dependent isocitrate dehydrogenases in mitochondria purified from Picea abies seedlings
Author(s) -
Cornu Sylvie,
Pireaux JeanClaude,
Gerard Joëlle,
Dizengremel Pierre
Publication year - 1996
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1996.tb00219.x
Subject(s) - isocitrate dehydrogenase , nad+ kinase , biochemistry , percoll , respiratory chain , biology , idh1 , dehydrogenase , mitochondrion , succinate dehydrogenase , malate dehydrogenase , glyoxylate cycle , microbiology and biotechnology , enzyme , centrifugation , gene , mutation
Isocitrate dehydrogenase (IDH) activities were measured in mitochondria isolated from aerial parts of 21‐day‐old spruce ( Picea abies L. Karst.) seedlings. Mitochondria were purified by two methods, involving continuous and discontinuous Percoll gradients. Whatever the method of purification, the mitochondrial outer membrane was about 69% intact, and the mitochondria contained very low cytosolic, chloroplastic and peroxisomal contaminations. Nevertheless, as judged by the recovery of fumarase activity, purification on a continuous 28% Percoll gradient gave the best yield in mitochondria, which exhibited a high degree of inner membrane intactness (91%). The purified mitochondria oxidized succinate and malate with good respiratory control and ADP/O ratios. The highest oxidation rate was obtained with succinate as substrate, and malate oxidation was improved (+ 60%) by addition of exogenous NAD + . Experiments using standard respiratory chain inhibitors indicated that, in spruce mitochondria, the alternative pathway was present. Both NAD + ‐isocitrate dehydrogenase (EC 1.1.1.41) and NADP + ‐isocitrate dehydrogenase (EC 1.1.1.42) were present in the mitochondrial matrix fraction, and NAD + ‐IDH activity was about 2‐fold higher than NADP + ‐IDH activity. The NAD + ‐IDH showed sigmoidal kinetics in response to isocitrate and standard Michaelis‐Menten kinetics for NAD + and Mg 2+ . The NADP + ‐IDH, in contrast, displayed lower K m values. For NAD + ‐IDH the pH optimum was at 7.4, whereas NADP + ‐IDH exhibited a broad pH optimum between 8.3 and 9. In addition, NAD + ‐IDH was more thermolabile. Adenine nucleotides and 2‐oxoglutarate were found to inhibit NAD(P) + ‐IDH activities only at high concentrations.