Isolation and Characterization of Inner Membrane-Associated and Matrix NAD-Specific Isocitrate Dehydrogenase in Potato Mitochondria

117

Purified potato tuber (Solanum tuberosum L. cv Bintje) mitochondria contain soluble, highly latent NAD+-and NADP+-isocitrate dehydrogenases, NAD+-and NADP+-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160,25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP+-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP -malate dehydrogenase activity is probably due to unspecificity of the NAD+-malate dehydrogenase. NADP -specific isocitrate dehydrogenase had much lower Kms for NADP+ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD -specific enzyme (101 micromolar for NAD and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP+-specific isocitrate dehydrogenase whereas the NAD+-specific enzyme had a sharp optimum at pH 7.8. Extemally added NADP+ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where extemal NADPH oxidation was inhibited. This shows that (a) NADP+ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP+-reducing activities of malate dehydrogenase and the NADP+-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.Plant mitochondria are generally held to utilize only NAD(H) as the reducing equivalent for its respiratory processes, whereas NADP(H) is considered to be associated mainly with the bio-and photosynthetic reactions of the cytoplasm and chloroplasts (10). NADP(H) also takes part in maintenance, as the reduced form is the substrate of GR2, reducing GSSG to GSH. GSH functions as an antioxidant in the plant cell. In chloroplasts it interacts with dehydroascorbate reductase and ascorbate peroxidase to remove H202 formed in the light reaction, but in the rest of the cell it is controversial whether GSH is oxidized by that mechanism or by GSH peroxidase when scavenging H202 (27).NADPH-specific GR has recently been found and characterized in mitochondria from pea leaves (7 generated by autooxidation of redox components in the mitochondrial electron transport chain by 02 (9), either directly or through the superoxide dismutase with superoxide radicals as intermediates; GR might participate in the removal of the H202 formed.Another NADP(H)-utilizing enzyme, the nicotinamide nucleotide transhydrogenase, has also been reported to be present in plant mitochondria (3). The transhydrogenase in animals is an energetically coupled membrane-bound enzyme catalyzing the conversion of NADH + NADP+ into NAD+ + NADPH and vice versa (12). This reaction can also be accomplished without proton...

Section: Comparison Of the Properties Of Nadp -And Nad -Icdhsupporting

confidence: 77%

“…The co-operativity number was determined to be 3.9 using Hill plots. Co-operativity of NAD+-ICDH for isocitrate has previously been reported for pea (4) and potato (28) mitochondria, but with a Hill number of about 2.5.…”

Section: Comparison Of the Properties Of Nadp -And Nad -Icdhmentioning

confidence: 99%

NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria

Rasmusson¹,

Møller²

1990

117

“…On the other hand, inasmuch as freezing and thawing elicit a change akin to that caused by a drop in pH in the presence of NAD, while the isotherm remains sigmoid in the absence of NAD (data not shown), it would seem that it is the proposed membrane-bound ICDH that responds to the drop in pH and to freezing and thawing, respectively. The kinetics of the isolated membrane-associated enzyme reenforces this suggestion (23).…”

mentioning

confidence: 61%

“…The change in state being suggested is to be distinguished from the condensation of oligomeric subunits described by Hayman and Colman for NAD-linked ICDH (13). The isolation and characterization of the two potato enzymes have shown that they are indistinguishable when extracted at modest salt concentrations, but distinct, as in intact mitochondria, when extracted in the near absence of salt (23). Accordingly, the condition brought to mind is allotopy, wherein the characteristics of an enzyme are profoundly affected by the association of the enzyme with a membrane (19).…”

mentioning

confidence: 88%

Membrane-Associated NAD-Dependent Isocitrate Dehydrogenase in Potato Mitochondria

Laties

1983

Self Cite

The oxidation isotherms for citrate and isocitrate by potato (Solanm tuberosum var. Russet Burbank) mitochondria in the presence of NAD differ markedly. Citrate oxidation shows positively cooperative kinetics with a sigmoid isotherm, whereas isocitrate oxidation shows MichaelisMenten kinetics at concentrations up to 3 millimolar, and cooperative kinetics thereafter up to 30 millimolar. In the absence of exogenous NAD, the isocitrate isotherm is sigmoid throughout. The dual isotherm for isocitrate oxidation in the presence of exogenous NAD reflects the operation of two forms of isocitrate dehydrogenase, one in the matrix and one associated with the inner mitochondrial membrane. Whereas in intact mitochondria the activity of the membrane-bound enzyme is insensitive to rotenone, and to butylmalonate, an inhibitor of organic acid transport, isocitrate oxidation by the soluble matrix enzyme is inhibited by both. The membrane-bound isocitrate dehydrogenase does not operate through the NADH dehydrogenase on the outer face of the inner mitochondrial membrane, and is thus considered to face inward. The regulatory potential of isocitrate dehydrogenase in potato mitochondria may be realized by the apportionment of the enzyme between its soluble and bound forms.In an early study, fresh potato slices were found to lack full' tricarboxylic acid cycle activity in consequence of their inability to oxidize citrate to a-ketoglutarate (15). At the same time NADlinked isocitrate dehydrogenase was shown to exhibit positively cooperative kinetics (12,22), and accordingly, ICDH2 was proposed as a potential regulator ofthe tricarboxylic acid cycle subject to modulation and control by an array of cofactors and metabolic intermediates (1,6,8,9,12). Subsequently, Ribereau-Gayon and Laties (20,21) demonstrated the sigmoidicity of the isotherm for isocitrate oxidation in potato mitochondria together with the socalled activating effect of citrate, which both lowered the SO.5 for isocitrate and seemingly converted the isotherm from sigmoid to hyperbolic (see below). During this period, the details of di-and tricarboxylic acid transport across the inner mitochondrial membrane received exhaustive attention, and it was established that carrier-mediated tricarboxylic acid uptake into the mitochondrion was dependent upon concomitant outward transport of dicarboxylic acid-malate in particular-the latter, in turn, being taken up from the milieu in exchange for intramitochondrial phosphate (3

“…The NAD+-specific ICDH has not been purified to homogeneity from a plant source, although some work has been done on the enzyme from peas (6-9, 11, 12) and potato (Solanum tuberosum) (17,20,21). Cox and Davies (7) man-Plant Physiol.…”

mentioning

confidence: 99%

NAD⁺-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

McIntosh

Oliver²

1992

The NAD'-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP'-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD' and NADP'-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD -dependent isocitrate dehydrogenase showed sigmoidal kinetics in response to isocitrate (So.s = 0.3 mM). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD+ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD+ (Km = 0.2 mM). NADH was a competitive inhibitor (Ki = 0.2 mM) and, unexpectedly, NADPH was a noncompetitive inhibitor (Ki = 0.3 mM). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.