Membrane-Associated NAD-Dependent Isocitrate Dehydrogenase in Potato Mitochondria

116

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: Nadp Stimulates Malate and Isocitrate Oxidation By Intact MIsupporting

confidence: 83%

NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria

Rasmusson¹,

Møller²

1990

116

“…This has been noted in most studies on the enzyme from a number of sources (5,16,17,22). In an earlier study (7,8), it was suggested that citrate is a potent effector of the pea mitochondrial NAD+ ICDH.…”

Section: Discussionmentioning

confidence: 96%

“…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.

“…NAD (12). Whereas tricarboxylic acid cycle dehydrogenases have most often been assigned to the matrix, Srere (24) and Matlib and O'Brien (15) have made a persuasive case for the association to varying degrees ofputative matrix enzymes to the inner membrane ofmammalian mitochondria.…”

mentioning

confidence: 99%

“…In the presence of exogenous NAD, potato mitochondria show a dual, or dromedary, ICDH isotherm (12 Mitochondria Preparation. Four hundred g of potato tubers were homogenized as described previously (10), except that the The inability of fresh potato slices to oxidize citrate (9), together with the known regulatory characteristics of NAD-linked isocitrate dehydrogenase (1,22), caused us to examine the behavior of ICDH4 in potato mitochondria from fresh slices.…”

mentioning

confidence: 99%

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

Tezuka

Laties²

1983

Self Cite

The isotherm for isocitrate oxidation by potato (Solanum tuberosum L.var. Russet Burbank) mitochondria in the presence of exogenous NAD is characterized by a hyperbolic phase at isocitrate concentrations below 3 millimolar, and a sigmoid, or positively cooperative phase from approximately 3 to 30 mllmlar. The two forms of isocitrate dehydrogenase were separated and characterized following the sonication of mitochondria in 15% glycerol in the absence of buffer, followed by fractionation in a density step gradient to yield inner membrane and matrix components. The membrane-associated isocitrate dehydrogenase was found to have a Hill, or cooperativity, number of 1, while the Hill nmber of the matrix enzyme was 2.5. Upon digitonin extraction the cooperativity number of the membrane enzyme rose to 3.5. The isocitrate Km for the membrane enzyme was calculated to be approximately 5.9 x 10' molar, while the SO.5 for the matrix was 6.9 x 10' molar. The NAD Km for both enzymes was 150 micromolar. Whereas the membrane enzyme proved indifferent to adenine nucleotides, the matrix enzyme was arguably inhibited by AMP and ADP, and inhibited some 25% by 5 millimolar ATP. Both enzymes were negatively responsive to the mole fraction of NADH, the membrane enzyme being 50% inhibited at a mole fraction of 0.26, and the matrix enzyme by a mole fraction of 032. The suggestion is offered that the enzymes in question constitute two forms of a single enzyme, one peripherally associated with the inner membrane, and one soluble in the matrix. It Is proposed that a degree of regulation may be achieved by the apportionment of the enzyme between the bound and free forms. NAD (12). Whereas tricarboxylic acid cycle dehydrogenases have most often been assigned to the matrix, Srere (24) Mitochondria Preparation. Four hundred g of potato tubers were homogenized as described previously (10), except that the The inability of fresh potato slices to oxidize citrate (9), together with the known regulatory characteristics of NAD-linked isocitrate dehydrogenase (1,22), caused us to examine the behavior of ICDH4 in potato mitochondria from fresh slices. In the preceding paper (12), we were led to the view that potato mitochondria contain either two species of ICDH, or a single species in two states, one free in the matrix and another associated with the inner mitochondrial membrane on the matrix side. This deduction stemmed primarily from the disparate behavior of ICDH in intact potato mitochondria in the presence and absence of exogenous '