The Purification and Properties of NADP‐Dependent Isocitrate Dehydrogenase from Ox‐Heart Mitochondria

MacFarlane, Neil; Mathews, Brian L.; Dalziel, Keith

doi:10.1111/j.1432-1033.1977.tb11424.x

Cited by 39 publications

(22 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All reagents were analytical grade. NADP-linked isocitrate dehydrogenase was prepared and assayed as described by McFarlane et al [9].…”

Section: Methodsmentioning

confidence: 99%

“…Isocitrate and NADP concentrations were measured using beef heart isocitrate dehydrogenase [6,9]. NADPH concentrations were measured from absorbance at 340 nm using an absorption coefficient of 6.22 x l o 3 M-' cm-' for NADPH [12].…”

Section: Methodsmentioning

confidence: 99%

“…After dialysis for 48 h against three 100-in1 portions and centrifugation, the protein concentration estimated from absorbance measurements at 280 nm and the absorption coefficient (A:;$) of 11.8 cm-' [9] was 3.56 mg ml-', corresponding to a molar concentration of 39.6 pM of the dimeric enzyme, molecular weight 90000 [9]. However, the concentration of active enzyme estimated from the measured specific activity of 18.9 U mg-' and the value of 39 U mg-' for the fully active enzyme [9] was 19.2 yM. The ESR spectra were obtained when 0.25 ml enzyme solution was titrated with five 5 4 and one 10-p1 aliquots of 1 .O mM MnClz.…”

Section: -Oxoglutarate Complexesmentioning

confidence: 99%

See 2 more Smart Citations

Determination of the Stability Constants of Mn²⁺ and Mg²⁺ Complexes of the Components of the NADP‐Linked Isocitrate Dehydrogenase Reaction by Electron Spin Resonance

Kuchel¹,

Reynolds²,

Dalziel³

1980

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

1. The stability constants (K,) of Mn2+ and Mg2+ complexes of isocitrate, 2-oxoglutarate, NADP and NADPH have been estimated by using electron spin resonance to measure free Mn2+ in ligand -metal-ion solutions.2. The values of K, for the Mn2+ complexes at 25 "C, in triethanolamine buffer containing NaCl, pH 7.0 and ionic strength 0.15 M, are 497 M-l for isocitrate, 39 M-' for 2-oxoglutarate, 467 M-' for NADP and 943 M-' for NADPH.3. For the Mg2+ complexes under the same conditions, the Ks values are 357 M-', 25 M-', 133 M-' and 179 M-' respectively. The large difference between the stabilities of the isocitrate and 2-oxoglutarate complexes is thus largely responsible for the observed variation of the apparent equilibrium constant of the NADP-linked isocitrate dehydrogenase reaction with magnesium ion concentration.4. NADP-linked isocitrate dehydrogenase from bovine heart mitochondria binds Mn2 +, and the stability constant of the complex is about 2.2 x lo4 M-'. The formation of this complex may explain the inhibition of the enzyme-catalysed reaction observed with Mn2+ concentrations greater than 0.2 mM in initial rate measurements.Knowledge of the concentrations of ligand-metal complexes in multicomponent enzyme reaction mixtures is of vital importance in studies of the kinetic mechanism of many enzymes. There is evidence that NADP-linked isocitrate dehydrogenase from beef and pig heart mitochondria employs the Mg2+ or Mn2+ complex of isocitrate as preferential substrate [1 -31. However, it is known that the other substrates of this enzyme (NADP, NADPH and 2-oxoglutarate) also bind divalent cations, and the possibility of influence on the reaction rate by metal complexes of NADPH has been suggested [4]. The Mg2+ concentration dependence of the overall equilibrium constant of the reaction catalysed by NADP-linked isocitrate dehydrogenase was shown by Londesborough and Dalziel [5] and used to calculate tentative stability constants for the complexes of isocitrate and 2-0x0- glutarate. The same workers [2,6] later estimated the stability constant of the former complex directly. The aim in the present work was to estimate the stability constants of the Mn2+ and Mg2+ complexes of all four substrates of the enzyme by the same method and under the same conditions, namely in 0.05 M triethanolamine hydrochloride/NaOH buffer pH 7.0, containing 0.1 M NaCl, a medium used in earlier studies of the enzyme [7]. There seem to be no previous reports of the stability constants for the Mg2+ complex of NADPH and the Mn2 + complex of 2-0x0-glutarate, and published values for the stability constants of the other complexes were obtained under diverse conditions.Electron spin resonance is a particularly useful method for direct measurement of some paramagnetic metal ions, such as Mn2+ [8], and was used in the present study. Mg2+ binding was evaluated by competition with Mn", assuming the competition to be purely competitive.

show abstract

“…All reagents were analytical grade. NADP-linked isocitrate dehydrogenase was prepared and assayed as described by McFarlane et al [9].…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: -Oxoglutarate Complexesmentioning

confidence: 99%

See 1 more Smart Citation

Determination of the Stability Constants of Mn²⁺ and Mg²⁺ Complexes of the Components of the NADP‐Linked Isocitrate Dehydrogenase Reaction by Electron Spin Resonance

Kuchel¹,

Reynolds²,

Dalziel³

1980

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…In peas (Pisum sativum L.),the cytosolic ICDH is a 82-kD dimer, and the plastid enzyme is reported to be a 154-kD dimer (2,3). The mitochondrial NADP+-dependent enzyme has not been purified from plants, but in beef (18) and pig heart (4), it is a dimer of 90 to 115 kD. The gene for this enzyme has been cloned from yeast and has a calculated monomer size of 46.6 kD (15).…”

mentioning

confidence: 99%

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

McIntosh

Oliver²

1992

Plant Physiol.

View full text Add to dashboard Cite

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.

show abstract

“…Both enzymes have been purified from various sources and their cDNA sequences determined (Macfarlane et al 1977;Haselbeck et al 1992;Huh et al 1993;Jennings et al 1994;Loftus et al 1994;Yang et al 1996;Nekrutenko et al 1998). ICD1 and ICD2 are homodimers encoded in the nuclear genome (Loftus et al 1994).…”

mentioning

confidence: 99%

Localization of a Mitochondrial Type of NADP-dependent Isocitrate Dehydrogenase in Kidney and Heart of Rat: An Immunocytochemical and Biochemical Study

Haraguchi

Mabuchi

Yokota

2003

J Histochem Cytochem.

View full text Add to dashboard Cite

S U M M A R YWe studied the subcellular localization of the mitochondrial type of NADPdependent isocitrate dehydrogenase (ICD1) in rat was immunofluorescence and immunoelectron microscopy and by biochemical methods, including immunoblotting and Nycodenz gradient centrifugation. Antibodies against a 14-amino-acid peptide at the C-terminus of mouse ICD1 was prepared. Immunoblotting analysis of the Triton X-100 extract of heart and kidney showed that the antibodies developed a single band with molecular mass of 45 kD. ICD1 was highly expressed in heart, kidney, and brown fat but only a low level of ICD1 was expressed in other tissues, including liver. Immunofluorescence staining showed that ICD1 was present mainly in mitochondria and, to a much lesser extent, in nuclei. Low but significant levels of activity and antigen of ICD1 were found in nuclei isolated by equilibrium sedimentation. Immunoblotting analysis of subcellular fractions isolated by Nycodenz gradient centrifugation from rat liver revealed that ICD1 signals were exclusively distributed in mitochondrial fractions in which acyl-CoA dehydrogenase was present. Immunofluorescence staining and postembedding electron microscopy demonstrated that ICD1 was confined almost exclusively to mitochondria and nuclei of rat kidney and heart muscle. The results show that ICD1 is expressed in the nuclei in addition to the mitochondria of rat heart and kidney. In the nuclei, the enzyme is associated with heterochromatin. In kidney, ICD1 distributes differentially in the tubule segments.

show abstract

The Purification and Properties of NADP‐Dependent Isocitrate Dehydrogenase from Ox‐Heart Mitochondria

Cited by 39 publications

References 29 publications

Determination of the Stability Constants of Mn²⁺ and Mg²⁺ Complexes of the Components of the NADP‐Linked Isocitrate Dehydrogenase Reaction by Electron Spin Resonance

Determination of the Stability Constants of Mn²⁺ and Mg²⁺ Complexes of the Components of the NADP‐Linked Isocitrate Dehydrogenase Reaction by Electron Spin Resonance

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

Localization of a Mitochondrial Type of NADP-dependent Isocitrate Dehydrogenase in Kidney and Heart of Rat: An Immunocytochemical and Biochemical Study

Contact Info

Product

Resources

About

The Purification and Properties of NADP‐Dependent Isocitrate Dehydrogenase from Ox‐Heart Mitochondria

Cited by 39 publications

References 29 publications

Determination of the Stability Constants of Mn2+ and Mg2+ Complexes of the Components of the NADP‐Linked Isocitrate Dehydrogenase Reaction by Electron Spin Resonance

Determination of the Stability Constants of Mn2+ and Mg2+ Complexes of the Components of the NADP‐Linked Isocitrate Dehydrogenase Reaction by Electron Spin Resonance

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

Localization of a Mitochondrial Type of NADP-dependent Isocitrate Dehydrogenase in Kidney and Heart of Rat: An Immunocytochemical and Biochemical Study

Contact Info

Product

Resources

About

Determination of the Stability Constants of Mn²⁺ and Mg²⁺ Complexes of the Components of the NADP‐Linked Isocitrate Dehydrogenase Reaction by Electron Spin Resonance

Determination of the Stability Constants of Mn²⁺ and Mg²⁺ Complexes of the Components of the NADP‐Linked Isocitrate Dehydrogenase Reaction by Electron Spin Resonance

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